Sniffing Out Prostate Cancer

  • Current Prostate-Specific Antigen (PSA) Tests Are Less than 25% Accurate
  • Trained “Sniffer” Dogs Detect Prostate Cancer-Specific VOCs in Urine with 98% Accuracy
  • Researchers Hope to Analyze VOCs using Gas Chromatography and Validate Results with Canine Studies to Develop Highly Accurate, Non-Invasive Tests for Prostate Cancer

We’re all familiar with news events involving dogs specifically trained to sniff out fugitives, explosives, or cocaine (among other things). This remarkable canine ability is due to some fascinating olfactory factoids. A dog’s sense of smell is a thousand times more sensitive than that of humans, and a dog has more than 220 million olfactory receptors in its nose, while humans have only 5 million.

Taken from finearts. com

Interestingly—if not amazingly—canine sniffing sensitivity has been investigated as a novel means of detecting cancer by simply smelling urine, or more accurately, smelling volatile organic compounds (VOCs) emitted from a urine sample. The following sections provide synopses of some notable publications dealing with new and improved methods for detection of prostate cancer. While reading this post, keep in mind that the principle here is analogous to humans being able to smell a distinctive VOC in their urine after eating asparagus, which is due to formation of volatile asparagusic acid. But I digress…

Dogs Sniff Out Prostate Cancer

The Problem: Prostate cancer represents the fifth most frequent cancer in the world, and according to current CDC statistics is still the number one killer of men in the US, followed by lung and colon cancers, as shown in the chart below.

Taken from

Prostate-specific antigen (PSA) testing is currently used for detection of prostate cancer. Details of the testing process can be read elsewhere, but I’ll briefly describe the steps of the exemplary assay depicted below. In the first incubation phase, specific autoantibodies (present in the sample, calibrators or controls) bind to the immobilized antigen. In the second incubation phase, the dimethyl acridinium ester (DMAE) conjugate reacts with the coated magnetic particle-autoantibody complexes. Non-bound material is washed away after every incubation step, and chemiluminescence is activated by the addition of “trigger” solutions (hydrogen peroxide and an alkali) resulting in oxidation of the ester to a photo-excited form. Return to a stable state is accompanied by the emission of light, which is measured and expressed in Relative Light Units (RLU). A direct relationship exists between the amount of total PSA in the sample and the RLUs detected.

Taken from

Since PSA testing is a great tool for the detection of prostate cancer, there is a strong need for more accurate tests. According to an NIH fact sheet, approximately 75% of men who have prostate biopsies due to elevated PSA levels DO NOT have prostate cancer. In fact, over 1 million unnecessary prostate biopsies will be performed in the US this year alone. Reported costs for this biopsy procedure range from $1,500-$6,000, resulting in billions of wasted dollars each year. Moreover, this huge false positive PSA rate exposes millions of men worldwide to an invasive procedure that has risks including sepsis and death.

Taken from pinterest. com

A Canine Solution? In 1989, Williams & Pembroke provided the first evidence for sniffer dogs that could detect VOCs from melanoma cancer in human urine samples. Fast forwarding to 2014, Taverna et al. reported that the olfactory system of highly trained dogs detects prostate cancer in urine samples. Two 3-year-old female German Shepherd explosion-detection dogs were trained to identify prostate cancer-specific VOCs in urine samples from 362 patients with prostate cancer (low-risk to metastatic) and on 540 healthy controls free of any kind of cancer.

Amazingly, dog 1 sensitivity was 100% and specificity was 98.7%, while for dog 2 sensitivity was 98.6% and specificity was 97.6%. Analysis of the few false-positive cases revealed no consistent pattern in participant demographics or tumor characteristics. It was concluded that “[a] trained canine olfactory system can detect prostate cancer specific VOCs in urine samples with high estimated sensitivity and specificity. Further studies are needed to investigate the potential predictive value of this procedure to identify prostate cancer.” While I did not find any such confirmatory follow-up studies with trained dogs, I did find the following investigations of non-canine alternatives to PSA. Interestingly, one of these studies is based on the knowledge that dogs can accurately detect VOCs in urine and the study plans to validate its results with canine studies.

Taken from

A Chromatographic Solution? As a chemist, its seemed reasonable to me to assume that state-of-the-art separation technology could be applied to VOC analysis to develop a more practical and reproducible replacement of PSA tests. I was gratified to find one such report in 2016 by a British research team that used gas chromatography (GC), depicted below, which is commonly employed for separation and detection of smallish, volatile molecules such as VOCs.

Dubbed “Odoreader,” the GC system was developed by a team led by Chris Probert from the University of Liverpool’s Institute of Translational Medicine and Norman Ratcliffe from the University of the West of England in Bristol. The researchers tested the Odoreader on 155 men presenting to urology clinics, of which 58 were diagnosed with prostate cancer, 24 with bladder cancer and 73 with hematuria or weak urine stream without cancer.

For prostate cancer diagnosis, this GC equipped with an automated data analysis system classified samples with 95% sensitivity and 96% specificity, while for bladder cancer diagnosis, the system had 96% sensitivity and 100% specificity. It was concluded that the results of this pilot study “indicate that the GC system is able to successfully identify patterns that allow classification of urine samples from patients with urological cancers,” adding that “larger cohort studies are planned to investigate the potential of this system.”

Not surprisingly, these very promising results have prompted others to investigate analogous GC methods capable of elucidating the structures of key molecules in the mixture of VOCs associated with prostate cancer. Mangilal Agrawal at Indiana University, together with his postdoc, Amanda Siegel, are doing so by coupling the power of GC to separate molecules and the power of mass spectrometry to identify molecules. Then they plan to validate these biomarkers with canine studies much like the one discussed above. Once validated, they will use the biomarkers to develop a non-invasive ‘strip sensor’ or dipstick test that can be used at doctors’ offices to detect the presence of prostate-specific VOCs. They presented preliminary findings using this GC/MS technology at the 2017 National American Chemical Society Meeting in a 15-minute press release video session with Q&A that I watched with interest.

In conclusion, my hope is that these GC based methods, in combination with continued canine studies, will soon lead to much more accurate strip sensor tests to replace PSA testing. These more accurate tests will benefit millions of men around the world by avoiding unnecessary prostate biopsies, and reduce health care costs.

As usual, your comments are welcomed.


Taken from Deng et al.

Aptamers (which I’ve blogged about previously and can be prepared from randomized oligonucleotide libraries from TriLink), are also being extensively investigated as potentially more specific prostate cancer detection tests than antibody-based immunoassays. One recent example reported by Deng et al. is depicted below. Basically, a three-layer core–shell nanostructure consisting of a silver core, a silica spacer, and a fluorescent dye RuBpy-doped outer silica layer was fabricated, and allows metal-enhanced fluorescence (MEF). A target-triggered MEF ‘turn-on’ strategy based on the optimized composite nanoparticles was successfully constructed for quantitative detection of prostate specific antigen (PSA), by using RuBpy as the energy donor and BHQ-2 as the acceptor. The hybridization of the complementary DNA of PSA-aptamer immobilized on the surface of the MEF nanoparticles with PSA-aptamer modified with BHQ-2, brought BHQ-2 in close proximity to RuBpy-doped silica shell and resulted in the decrease of fluorescence. In the presence of target PSA molecules, the BHQ-PSA aptamer is dissociated from the surface of the nanoparticles with the fluorescence switched on.




Highlights from the 2017 AgBio Innovation Showcase Held by UC Davis

  • An Inconvenient Truth About Unsustainable Global Food Supply
  • Agricultural Biotechnology (AgBio) is Providing Transformative Solutions
  • Highlights from the Inaugural AgBio Innovation Showcase

Taken from the

With expected global population to reach 8.3 billion in 2030, it’s clear that excessive exploitation of food resources is no longer sustainable and the problem will simply worsen with environmental problems and effects of climate change. This ominous outlook by food experts is reminiscent of former Vice President Al Gore’s dire vision for global warming in an award winning documentary film in 2016 titled An Inconvenient Truth.

This very real challenge of achieving adequate and sustainable food supplies—globally, not just for developed countries—has been, and continues to be, addressed by nucleic acid-based agricultural biotechnology (aka AgBio). At the forefront of this battle is development of genetically modified foods (aka genetically engineered foods or bioengineered foods), which are foods produced from organisms that have had changes introduced into their DNA using the methods of genetic engineering. Genetic engineering techniques allow for the introduction of new traits, as well as greater control over traits than was possible with previous methods such as selective breeding and mutation breeding.

Taken from

Last year, I published a blog about genetically modified organisms (GMOs) in which I made fairly general comments about complex government regulatory issues related to “science vs. semantics” and varying degrees of country/consumer acceptance and rejection. This blog is somewhat of a follow-up to that post, and I will share specifics from the 2017 AgBio Innovation Showcase held by the University of California Davis at its World Food Center, which include GMO and non-GMO technologies. The Center was founded in 2013 as an institute aimed at “bridging agriculture, food science, nutrition, veterinary medicine, public health and policy in new and transformational ways.”

2017 AgBio Innovation Showcase

Taken from

This inaugural event was held on May 8-9 and featured the most promising AgBio and AgTech startups and research projects. The showcase featured solutions in high-value, nutritious agriculture and food from across the globe. The four major showcase themes were Automation & Robotics, Boosting Nutrition & Sensory Value, Innovation in the Livestock & Dairy Sectors, and Water Management. I’ve selected several highlights that are summarized below. Takeaways from panel discussions about the future of agriculture can be read elsewhere.

Ag Biotech

  • Afingen – This biotech start-up was spun out of Lawrence Berkeley National Laboratory (LBNL) in 2014 and features technology based on proprietary cisgenesis. Cisgenesis involves modification of a recipient plant with a natural gene from a crossable plant. Importantly, cisgenic plants can harbor one or more cisgenes, but they do not contain any transgenes and therefore yield new, improved plant varieties that are classified non-GMO.
  • Taken from

    Bee Vectoring Technology – How this Canadian company cleverly turns bees into delivery agents that deposit biological products on crops for pest management is best understood by watching this video (details for which may be read in a patent). In brief, powder-form biologics to be delivered are placed in commercially-reared bee. The biologics stick to the bees’ feet and are released when the bee collects pollen from the targeted crop.

    Taken from

  • MiraculeX – A unique West African plant protein called miraculin (named for its “miracle” ability to transform sour foods into sweet treats), makes it possible to bite into a lemon and taste nothing but sweet lemonade. MiraculeX reportedly inserts the protein’s DNA into the genetic code of ordinary lettuce, which is grown hydroponically and in less than a week is ready to be harvested for processing.
  • Trace Genomics – This startup service in San Francisco provides advice to growers based on analysis of their soil. Growers simply provide a soil sample, from which TraceGenomics extracts DNA from the organisms in the soil and prepares a sequencing library to analyze the soil microbial community. Interpreted results are provided along with information about soil health, nutritional status, and corresponding recommendations for how to improve crop yield and quality.


  • AstRoNa Biotechnologies – This UC Davis startup aims to commercialize an easy-to-use, hand-held pathogen detection device to quickly monitor food contamination by bacteria, viruses, and fungi. It’s basically “farm-to-table” analysis. The team reportedly developed a disposable test kit to capture and amplify RNA of pathogens, focusing on coli O157:H7. A fully automated handheld instrument is under development and will feature sample multiplexing, quantitative detection, and software to create a traceable record of safety—recording time, location, user, and results in real time.
  • SnapDNA – This startup has an R&D agreement with the US Department of Agriculture to develop rapid, highly specific tests for foodborne pathogens, including Salmonella enterica and human noroviruses (the latter of which is featured in an earlier blog). I was unable to find many details, but a board member states that SnapDNA is “a semiconductor-based bio-chip and multiplexed DNA detection platform.” Adding that “[a]major differentiator of SnapDNA is the specificity to detect DNA at the level of microbial strains in a fast, low cost test, major pain points in current systems.”

Food Science & Animal Health

  • Taken from

    Bonumose – This startup in Virginia is scaling up enzymatic production of tagatose (pictured below), which—unlike sucrose and high fructose corn syrup—does not raise blood sugar levels, is low-calorie, and does not cause tooth decay. Beyond not being harmful to health, tagatose provides positive health benefits: it is an effective prebiotic (good for gut health), blocks adsorption of sucrose and starch, is clinically-proven to reduce blood sugar levels in diabetics, contributes to a feeling of satiety, and breaks up dental biofilm. Even better, tagatose is nearly indistinguishable from sucrose in terms of taste and food functionality. And it blends very well with high intensity sweeteners such as stevia. I want some asap!

  • Resilient Biotics – This El Cerrito, California startup utilizes deep sequencing to characterize host genotypes, commensal microbial communities, and pathogen strain variants for microbiome resolution to rapidly identify important genetic elements and key microbial strains that influence states of health and disease. Heuristic search methods can rapidly pinpoint diagnostic biomarkers for pathogen identification and risk prediction. Resilient Biotics is actively developing live biotherapeutics to address major infectious diseases of the respiratory tract in production animal systems.

AgBio Meets CRISPR

As if the 2017 AgBio Innovation Showcase wasn’t stimulating enough, I was thrilled to discover another upcoming meeting that combines two of my favorite topics: agbio and CRISPR. Devoted readers of my blogs will recall numerous past postings on CRISPR for gene editing and other useful manipulations of genomic DNA. My search of Google Scholar indicated no AgBio CRISPR publications to date, but that will likely change, as evidenced by the upcoming conference.

Interested readers can register at the link above and download a detailed agenda and list of confirmed speakers. In doing so, it is apparent that this conference will comprehensively cover the newest topics and the regulatory status related to CRISPR/Cas9 technology.

I look forward to reading about these developments, and posting comments in a future blog titled AgBio Meets CRISPR. If you happen to be attending this conference, please share details about what you learned in the comments section below.

As usual, your comments are welcomed.



Advances in Aptamer Applications – Part 2

  • Top Cited Aptamer Publications Over the Past Three Years
  • Jerry’s Picks for Top 3 Aptamer Publications So Far This Year
  • TriLink Products Cited in Numerous Aptamer Publications

Aptamers are highly structured nucleic acids that bind to a specific target molecule. RNA or DNA aptamers are usually selected from a very large pool (aka library) of random sequences, and can be comprised of either natural and/or chemically modified nucleotides. My first blog on aptamers was titled Aptamers: Chemistry Bests Mother Nature’s Antibodies. This purposefully provocative claim was intended to emphasize the growing body of evidence that collectively indicates aptamers can perform better than antibodies in many applications.

NMR-derived structures of aptamers binding to either a large protein or small molecule. Taken from genelink .com

Because it has been nearly four years since that boastful blog in 2013, I thought it was time to survey aptamer applications published since then to comment on what has been trending or is otherwise notable. I found more than 1,500 articles in PubMed for 2014 through 2017 (estimate) that have the search term “aptamer” in the title or abstract. Given this huge number of publications, I used Google Scholar citation frequency as a numerical indicator of interest, importance and/or impact for these publications in each year. I also decided to focus on original publications that, by definition, excludes review articles. 

Top 3 Cited Publications in 2014

  1. Activatable fluorescence/MRI bimodal platform for tumor cell imaging via MnO2 nanosheet–aptamer nanoprobe (109 citations)

This Chinese team of researchers led by uber-prolific Weihong Tan, about whom I’ve previously blogged, designed a novel methodology for imaging tumor cells using quenched-fluorescent aptamers. In the presence of target cells, the binding of these “dark” aptamers to cell surface markers weakens the adsorption of aptamers on MnO2 nanosheets causing partial fluorescence recovery (i.e., unquenching), thus illuminating the target cells, as well as facilitating endocytosis into target cells. After endocytosis, reduction of MnO2 nanosheets by glutathione further activates the fluorescence signals and generates large amounts of Mn2+ ions as a contrast agent for magnetic resonance imaging (MRI).

Taken from

  1. A phase II trial of the nucleolin-targeted DNA aptamer AS1411 in metastatic refractory renal cell carcinoma (88 citations)

Taken from

The anticancer mechanism of action for DNA aptamer AS1411, which has multiple G-quadruplex moieties that disrupt cancer cell replication following nucleolin-mediated uptake, is depicted below and detailed elsewhere. In this clinical study, it was found that AS1411 appears to have limited activity in patients with metastatic renal cell carcinoma. However, rare, dramatic and durable responses can be observed and toxicity is low. Further studies with AS1411 and other nucleolin-targeted compounds may benefit from efforts to discover predictive biomarkers for response.

  1. An aptamer-based dipstick assay for the rapid and simple detection of aflatoxin B1 (61 citations)

Aflatoxin B₁ structure. Taken from

Aflatoxin B₁ (AFB1) produced by Aspergillus flavus and A. parasiticus is considered the most toxic aflatoxin and it is highly implicated in hepatocellular carcinoma in humans. In this work by Korean researchers, a rapid and simple dipstick assay based on an aptamer has been developed for determination of AFB1 contamination in food. The dipstick assay format was based on a competitive reaction of a biotin-modified aptamer specific to AFB1 between target and Cy5-modified DNA probes. Streptavidin and anti-Cy5 antibody as capture reagents were immobilized at test and control lines on a membrane of the dipstick assay. The method was confirmed to be specific to AFB1, and the entire process of the assay can be completed within 30 min.

Top 3 Cited Publications in 2015

  1. Aptamer-conjugated silver nanoparticles for electrochemical dual-aptamer-based sandwich detection of staphylococcus aureus (63 citations)

Taken from sciencedirect .com

Staphylococcus aureus (S. aureus) is one of the most important human pathogens and causes numerous illnesses. This report by Iranian researchers describes a sensitive and highly selective dual-aptamer-based sandwich immunosensor for the detection of S. aureus. As depicted below, a biotinylated primary anti-S.aureus aptamer was immobilized on streptavidin coated magnetic beads (MB), which serves as a capture probe. A secondary anti-S.aureus aptamer was conjugated to silver (Ag) nanoparticles such that, in the presence of target bacterium, a sandwich complex is formed on the MB surface and the electrochemical signal of Ag is measured by anodic stripping voltammetry.

  1. Aptamer-based fluorescence biosensor for chloramphenicol determination using upconversion nanoparticles (59 citations)

Chloramphenicol. Taken from Wikipedia .com

Chloramphenicol (CAP) shown below is a naturally occurring antibiotic that is artificially manufactured for use in veterinary and human medicine. Due to its adverse effects in humans, use of the antibiotic is restricted and, in Europe, ‘zero tolerance’ for CAP in food products has been legislated. In this report by Chinese researchers, detection of CAP uses aptamer-conjugated magnetic nanoparticles for both recognition and concentration, together with upconversion nanoparticles for detection. The method was validated for measurement of CAP in milk vs. a commercially available enzyme-linked immunosorbent assay (ELISA) method.

  1. A new aptamer/graphene interdigitated gold electrode piezoelectric sensor for rapid and specific detection of Staphylococcus aureus (48 citations)

Taken from mdpi .com

This work by Chinese investigators describes a novel aptamer/graphene interdigitated gold electrode piezoelectric sensor for detecting S. aureus by binding to the aptamer, which is immobilized on the graphene via the π–π stacking of DNA bases, as depicted below. When S. aureus is present, aptamer dissociates from the graphene and thus leads to change of oscillator frequency of the piezoelectric sensor.

Top 3 Cited Publications in 2016

  1. Aptamer–MIP hybrid receptor for highly sensitive electrochemical detection of prostate specific antigen (38 citations)

This study in the UK uses a thiolated DNA aptamer for prostate specific antigen (PSA) immobilized on the surface of a gold electrode. Controlled electropolymerization of dopamine around the complex served to create an imprint of the complex following removal of PSA. This molecularly imprinted polymer (MIP) cavity was found to act synergistically with the embedded aptamer to provide recognition properties superior to that of aptamer alone. A generalized depiction for producing a MIP is shown below.

Taken from sigmaaldrich .com

  1. Aptamer-functionalized nanoparticles for surface immobilization-free electrochemical detection of cortisol in a microfluidic device (34 citations)

Taken from

Monitoring the periodic diurnal variations in cortisol (aka hydrocortisone, show below) from small volume samples of serum or saliva is of great interest, due to the regulatory role of cortisol within various physiological functions and stress symptoms. This publication from China reports use of aptamer-functionalized gold nanoparticles pre-bound with electro-active triamcinolone for detection of cortisol based on its competitive binding to the aptamer by monitoring a signal from the displaced triamcinolone using square wave voltammetry at graphene-modified electrodes. The assay was benchmarked vs. ELISA and radioimmunoassays.

  1. Multifunctional aptamer-based nanoparticles for targeted drug delivery to circumvent cancer resistance (32 citations)

Taken from Liu et al. Biomaterials (2016)

In yet another publication from China, Liu et al. report use of a G-quadruplex nanostructure to target cancer cells by binding with nucleolin, in a manner analogous to that mentioned above. A second component is double-stranded DNA (dsDNA), which is rich in GC base pairs that can be applied for self-assembly with doxorubicin (Dox) for delivery to resistant cancer cells. These nanoparticles were found to effectively inhibit tumor growth with less cardiotoxicity.

Jerry’s Top 3 Publication Picks for 2017-to-Date

Here are my Top 3 “fav” aptamer articles published during the first half of 2017, and my reasons for these aptamer selections—pun intended. Interested readers can consult the original publication for technical details.

  1. Targeted delivery of CRISPR/Cas9 to prostate cancer by modified gRNA using a flexible aptamer-cationic liposome

CRISPR/Cas9 is unquestionably—in my opinion—the hottest topic in nucleic acid-based R&D these days, as I have previously blogged about. Off-target effects of CRISPR/Cas9 can be problematic, so using targeted delivery to cells of interest is an important approach for mitigating this problem. In this study, an aptamer-liposome-CRISPR/Cas9 chimera was designed to combine efficient delivery with adaptability to other situations. The chimera incorporated an RNA aptamer that specifically binds prostate cancer cells expressing the prostate-specific membrane antigen as a ligand, and the approach “provides a universal means of cell type-specific CRISPR/Cas9 delivery, which is a critical goal for the widespread therapeutic applicability of CRISPR/Cas9 or other nucleic acid drugs.”

  1. A cooperative-binding split aptamer assay for rapid, specific and ultra-sensitive fluorescence detection of cocaine in saliva

This report claims the first ever development of a split aptamer that achieves enhanced target-binding affinity through cooperative binding. In this instance, a split cocaine-binding aptamer incorporates two binding domains, such that target binding at one domain greatly increases the affinity of the second domain. This system afforded specific, ultra-sensitive, one-step fluorescence detection of cocaine in saliva without signal amplification. This limit of detection meets the standards recommended by the European Union’s Driving under the Influence of Drugs, Alcohol and Medicines program.

  1. Detection of organophosphorus pesticide–Malathion in environmental samples using peptide and aptamer based nanoprobes

Environmental contamination with pesticide residues has necessitated the development of rapid, easy and highly sensitive approaches for the detection of pesticides such as malathion, a toxic organophosphorus pesticide, widely used in agricultural fields. These Indian investigators employed an aptamer, cationic peptide and unmodified gold nanoparticles. The peptide, when linked to the aptamer renders the gold nanoparticles free and therefore, red in color. When the aptamer is associated with malathion, however, the peptide remains available to cause the aggregation of the nanoparticles and turn the suspension blue. The sensitivity was tested in real samples and the results implied the high practicability of the method.

Aptamer Publications in 2014-Present Citing TriLink Products

I was pleasantly surprised to find more than 250 publications on aptamers in Google Scholar citing the use of TriLink products since 2014. This volume of literature is way too large to summarize succinctly, so I decided to do a quick scan to select the following items that provide an indication of the broad diversity of applications partially enabled by TriLink products:

2’-F-UTP. Taken from trilinkbiotech .com

In closing, I should first mention that, while scanning the aptamer/TriLink publications mentioned above, it was evident that the most frequently cited TriLink products were 2’-F-CTP and 2’-F-UTP, which are incorporated into aptamers to impart nuclease resistance, as discussed on a TriLink webpage.

My second and last comment is that, as you may have noticed, there seems to be a high proportion of aptamer publications coming out of China and/or coauthored by Chinese investigators collaborating with researchers in other countries. This despite the fact that Chinese publications in Life Sciences are ~6-times fewer that those from the US, according to reliable statistics. I have no idea why this is so, but thought it’s an intriguing factoid.

As usual, your comments are welcomed.








Studying Telomeres in Space

  • Telomeres are DNA Biomarkers for Your Biological “Age”
  • Telomere Shortening Due to Stress was Expected During Spaceflight, but Exactly the Opposite has Been Found
  • Raising New Questions to Answer

After you read this blog about studying telomeres in space, I think you will agree with my opinion that scientific advances can sometimes occur amazingly fast. Telomeres (which are peculiar DNA structures that I’ll explain below) went from esoteric Nobel Prize subject matter in 2009 to the focus of spaceflight science in just six short years. Now, telomeres are being investigated by PCR on the International Space Station (ISS)! With a wink and a nod to Star Trek, this is indeed “warp speed” progress!

Taken from

What are telomeres?

A telomere is a region of repetitive nucleotide sequences at each end of a chromosome, which protects the end of the chromosome from deterioration or from fusion with neighboring chromosomes. For vertebrates like us, the repetitive sequence of nucleotides in telomeres is TTAGGG, with the reverse complementary DNA strand being AATCCC, as depicted below. This sequence of TTAGGG is repeated ~2,500 times in humans.

During chromosome replication, the enzymes that duplicate DNA cannot continue their duplication all the way to the end of a chromosome, causing the end of the chromosome to be shortened in each replication. The telomeres are thus disposable “buffers” at the ends of chromosomes which are truncated during cell division, as depicted below.

Taken from

Telomers, however, are replenished by an enzyme named telomerase. This peculiar enzyme has an embedded RNA template and incorporates DNA nucleotides, as depicted below, and is therefore a special kind of reverse transcriptase. In people, it has been found that telomeres shorten with age in all replicating somatic cells that have been examined. In fact, average telomere length declines from about 11 kilobases at birth to less than 4 kilobases in old age, with the average rate of decline being greater in men than in women. Thus, telomere length can serve as a biomarker of a cell’s biological (versus chronological) “age” or potential for further cell division.

Taken from

‘Houston, we have a problem’

This now famous phrase, which was used in the past tense by the crew of the Apollo 13 moon flight to report a major technical problem back to their Houston base, echoed in mind when I learned that space flight might lead to telomere shortening. Yikes! This molecular-level change could indeed be a serious problem, and was first suggested by findings from laboratory microgravity simulations reported in 2008 by Chinese researchers. Since it was known that space flight leads to bone loss, they cultured bone stem cells (BSCs) under simulated microgravity in a rotary cell culture system.

This led to significantly decreased activity of telomerase. It was postulated that reduced bone formation in space flight may partly be due to the altered potential differentiation of BSCs associated with telomerase activity, which plays a key role in regulating the lifespan of cell proliferation and differentiation. Additionally, telomerase activation or telomerase replacement may act as a potential countermeasure for microgravity-induced bone loss.

Taken from

If you’re thinking that these “potential countermeasures” are fanciful, you’d better think again. I recently came across a patent that was published last year on methods and compositions for increasing telomerase activity in cells, including pharmaceutical formulations. Moreover, there are now various commercially available supplements claiming to promote telomerase activity, such as that picture below. I hasten to add that I do not advocate use of any such supplement, and that interested readers should consult their primary care physician or certified nutritionist.

Twins and telomeres

Although identical twins are almost the same genetically, differences in environment, diet and other outside factors can affect their health in different ways. Consequently, identical twins have been enrolled in various studies that require deciphering effects due to “nature vs. nurture” (i.e. intrinsic genetics vs. external factors). Part of the Twins Study supported by NASA was aimed at examining the effects of space travel on one of a pair of twins: astronaut Scott Kelly, who stayed on the ISS for one year, while his twin brother, Mark, remained on Earth. In brief, Prof. Susan Bailey at Colorado State University is exploring differences between the twins’ telomeres to determine if telomeres respond differently to spaceflight and then how such changes relate to the various medical endpoints studied by other Twins Study investigators.

Scott Kelly (left) and his identical twin brother Mark in 2015 prior to Scott’s one-year mission to the ISS. Taken from

Preliminary research results for this part of the NASA Twins Study (reported at NASA’s annual Investigators’ Workshop earlier this year) were a quite surprising because they were opposite of what was expected, thus raising more questions than providing answers. It had been theorized that exposure to microgravity and stress during prolonged spaceflight would shorten telomeres, but instead Bailey’s team found telomeres in Scott’s white blood cells increased in length while in space! This finding was rationalized as being due to increased exercise and reduced caloric intake during the space mission. Upon his return to Earth, however, these telomeres began to shorten again.

This is yet another example of a biomedical phenomenon being far more complex that first theorized, and one that becomes less understood as more and more data are obtained. We’ll all have to patiently stay tuned for how this telomeres-in-space story evolves. The good news, as I’ll explore in the next and final section of this blog, is that there are exciting plans to use PCR to measure telomere length extraterrestrially! This is very “far out” science—pun intended.

Studying telomeres in space by PCR

Taken from

The aforementioned Twins Study involved taking blood samples from an astronaut during spaceflight for lab analysis upon return to Earth. To obtain much more data, and to do so in real time while in space, NASA launched the Genes in Space-2 mission in April 2017. The goal is to determine whether astronauts aboard the ISS can analyze telomeres by PCR reactions in a small thermal cycling device (miniPCR system) and thus measure and monitor telomere changes during spaceflight.

In addition to testing the miniPCR system, the Genes in Space-2 mission has a secondary goal to test the feasibility of techniques used to measure telomere length. Currently, Single Telomere Length Analysis (STELA) is the only suitable technique for use on the ISS due to technical requirements. The Genes in Space-2 mission will also be testing the feasibility of a loop-mediated isothermal amplification (LAMP) colorimetric assay for detection of amplification aboard the ISS. Please stay tuned for updates on the outcome of these very important feasibility experiments.

Scheme for STELA procedure. Taken from Xing et al. (2009)

Julian Rubenfein. Taken from

As a side note, the Genes in Space competition for 2017 selected this experiment on telomere amplification in microgravity from 375 submissions by nearly 850 students in grades 7 to 12 from across the US. This telomere experiment was proposed by 17-year old Julian Rubinfien from Stuyvesant High School in New York City, who is pictured below. I encourage you to read this interesting, although lengthy interview about his background and the experimental rationale. What’s even more interesting is this short video of Julian at the launch and his comments—very impressive!

I strongly encourage you to read more about all the award-winning experiments in this exciting round of competition among young, highly motivated, advanced students, who I’m sure will be successful in whatever they do in the future.

Your thoughts or comments here are welcomed.


Profs. Elizabeth Blackburn and Carol Greiner—who received a Nobel Prize in 2009 for seminal work on telomeres—co-founded Telome Health Inc. (THI) in 2010 to leverage the predictive power of telomere-length assays to help assess health status, disease and mortality risk, and response to specific therapies. THI subsequently announced TeloTest™ as a diagnostic test that measures average telomere length by qPCR. TeloTest™ was the first saliva-based telomere test available on the market, and is currently offered by a company named TeloYears.

The clinical utility of testing telomere length in a saliva-based test was recently reported from an independent, large clinical study sponsored jointly by Kaiser Permanente, University of California, San Francisco (UCSF), and National Institutes of Health. In the study, the average telomere length of 100,000 Kaiser patients was measured and analyzed relative to other health domains and clinical outcomes.

My recently obtained TeloTest™ results from TeloYears indicated that my biological age is 4 years older than my chronological age. Naturally, I was hoping to learn that my telomere-based age would be less than my actual age. Alas, the results are what they are, so I’ll be following diet, exercise, sleep, and stress-management recommendations you can read about at TeloYears Learning Center.



CRISPR-C2c2 Update: Powerful New Diagnostic Method Using CRISPR

  • CRISPR Craze Continues Led in Part by Wunderkind Feng Zhang
  • Nonspecific CRISPR-C2c2 “Collateral” Cutting Channeled into Diagnostics
  • Turning Biochemical “Lemons to Lemonade”

This blog post is about a new and powerful diagnostic approach based on CRISPR, which I’ll get to below. But first I’d like to point out several reasons why this is an especially interesting development:

  • Taken from

    Any new method using CRISPR is more “sizzle” for this “super-hot” technology

  • Feng Zhang, the 32-year-old author of this publication, is regarded as a wunderkind
  • My past blog post on CRISPR-C2c2 “collateral” cutting noted possibilities for turning “lemons into lemonade”
  • Such “lemonade” has emerged as a diagnostic cleverly acronymized as SHERLOCK

So, without further ado, here’s a brief recap of CRISPR-C2c2 as an intro to SHERLOCK (think Holmes), followed by why this acronym is apropos for a diagnostic method that magnifies detection, in this case Zika virus, about which I’ve previously commented. 

What’s CRISPR-C2c2?

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (CRISPR-Cas) adaptive immune systems in microbes contain endonucleases that can be leveraged for CRISPR-based gene editing using targeted CRISPR RNAs (crRNAs), as I’ve outlined in a previous blog post. While such Cas enzymes target DNA, others target RNA and function as RNA-guided RNases, which was reported by Feng Zhang and collaborators in Science last year in an article titled C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector.

My blog post about that C2c2 publication provides more information, but the main point related to the present blog post about diagnostics is that after recognition of its RNA target, activated C2c2 engages in nonspecific (aka “collateral”) cleavage of nearby non-targeted RNAs. This post-cleavage non-specificity can be transformed, if you will, from something “bad” to something “good” in terms of mechanisms, which I described as being akin to converting ‘lemons into lemonade’. You’ll now see how SHERLOCK is such ‘lemonade’ concocted by Zhang and coworkers.


Taken from US 20140199245 A1

Before answering this question, and not to confuse you, I need to first mention that the nuclease originally called C2c2 was renamed Cas13a to be more systematic, and will be referred to herein as such. Having said that, Cas13a can be reprogrammed with crRNAs to provide a platform for specific RNA sensing. Molecular recognition of an intended RNA target by crRNA in vitro serves as a sequence-specific “trigger” to activate Cas13a’s nonspecific, “collateral” cleavage of labeled RNA reporters. Cleavage of these fluorescently labeled, internally quenched RNAs leads to unquenching and fluorescent light emission for detection, as depicted below for a generic RNase.

Now that you have a sense of how CRISPR-Cas13a can generate a signal, you’re probably asking ‘What is SHERLOCK?’ It’s an acronym coined by a large team lead by CRISPR genome-editing pioneer Feng Zhang and bioengineer James Collins, both from the Broad Institute. SHERLOCK stands for High-sensitivity Enzymatic Reporter UnLOCKing, which is a recently published new method for sequence-specific detection of DNA or RNA in any sample of interest. This general utility represents a major advance in CRISPR-based diagnostics.

Feng Zhang. Taken from // James Collins. Taken from

As depicted below, sample prep workflows allow for input of either double-stranded DNA (dsDNA) or RNA, which are first amplified by recombinase polymerase amplification (RPA) directly or as cDNA, after reverse transcription (RT-RPA), respectively. In either case, further amplification utilizes T7 transcription into RNA (blue = target RNA; yellow = non-target RNA). Following cleavage of target RNA by Cas13a-crRNA, a commercially available “cleavage reporter” (i.e. internally quenched) RNA undergoes “collateral” cleavage to generate a reporter signal (i.e. fluorescence; pictured below) for real-time detection of the target.

Taken from Feng Zhang, Collins & coworkers Science (2017)

An advantage of RPA is that it is performed isothermally, which allows simplification of the detection device by eliminating the need for a thermal cycler, thus lending to incorporation into small, portable point-of-care (POC) systems (about which I previously commented several times). The abovementioned RPA/T7 sample prep methodology was shown to have attomolar (aM, 10−18 mol/L) sensitivity in model systems, and was next investigated for sensitivity and specificity of virus detection, as well as simplification of reagent format.

Zhang, Collins & coworkers constructed lentiviruses harboring genome fragments of either Zika virus (ZIKV) or the related flavivirus Dengue (DENV), and showed that SHERLOCK detected viral particles down to 2 aM could discriminate between ZIKV and DENV. To explore the potential for POC use of SHERLOCK with paper-spotting and lyophilization (aka freeze-drying), they first demonstrated that Cas13a-crRNA complexes lyophilized and subsequently rehydrated could detect 20 femtomolar (fM, 10−15 mol/L) non-amplified RNA, and that target detection was also possible on glass fiber paper.

The other components of SHERLOCK, namely the RPA reagents and T7 polymerase, were already known to be amenable to freeze-drying and storage at ambient temperatures. In combination, freeze-drying and paper-spotting the Cas13a detection reaction resulted in comparable levels of sensitive detection of RNA as aqueous reactions. Although paper-spotting and lyophilization slightly reduced the absolute signal of the readout, SHERLOCK could readily detect mock ZIKV virus at concentrations as low as 20 aM. Most importantly, SHERLOCK detected ZIKV in clinical isolates (ZIKV RNA extracted from patient serum or urine samples and reverse transcribed into cDNA) could be detected at concentrations down to 1.25 × 103 copies/mL (2.1 aM), as verified by qPCR.

Concluding Comments

If you reflect upon the schematic for SHERLOCK, you’ll note that the input can be either DNA or RNA, which get amplified to produce many copies of RNA that serve as substrates for cleavage by Cas13a-crRNA, thus inducing collateral cleavage of reporter RNA to produce a detectable signal. Lest you think SHERLOCK is too costly to be practical, its developers provide a detailed cost accounting that estimates $0.61 per test, which you’re welcome to compare with your cost of conventional qPCR. I’m quite sure that will lead you to concur with me that $0.61 per test is relatively inexpensive.

If you’re questioning whether SHERLOCK is generally applicable, I urge you to read Zhang, Collins & coworkers in its entirety to learn more about SHERLOCK’s proven ability to detect and distinguish (1) various bacterial pathogens, (2) single-base cancer mutations in cell-free DNA, and (3) health-related single-nucleotide polymorphisms (SNPs) benchmarked against 23andMe genotyping data as the gold standard of these SNPs.

In an article in Science about SHERLOCK, Harvard University’s George Church (who I’ve proclaimed is The Most Interesting Scientist in the World and is the co-founded of a CRISPR therapeutics company), sums up his reaction in one word: ‘Wow.’ I agree!

The article concludes with Collins saying the Broad is now ‘aggressively exploring’ how to commercialize SHERLOCK and may launch a startup company. But before a diagnostic comes to market, it must pass muster at regulatory agencies such as the U.S. Food and Drug Administration. I’m betting is does.

I welcome your sharing any thoughts or comments about this new CRISPR-based diagnostic method.

Lab-on-a-Drone and Other Innovative Point-of-Care Devices

  • Lab-in-a-Box…Think Bento
  • Lab-on-a-Robot…Rolls Along 
  • Lab-on-a-Drone…PCR-on-the-Fly

Honey! I shrunk the lab! 

Taken from

Researchers have long dreamed of a “lab-on-chip” (LOC) wherein common laboratory procedures have been miniaturized and integrated in various formats using microfluidics—small, interconnected channels resembling electronic circuits on a chip—that provide low-cost assays for “point-of-care” (POC) applications. The cartoon to the right humorously but concisely depicts the general concept of LOC, for which there are virtually an infinite number of specific embodiments made possible by continuing development of many clever fabrication and microfluidic technologies for “shrinking” lab procedures.

Importantly, lab personnel are thus freed-up from slavish, repetitive tasks to instead carry out discovery and development work. Testament to the significance of LOC is evident from the astounding—to me—130,000 items I found in Google Scholar by searching LOC as an exact-word phrase. There is also a LOC Wikipedia site and a journal for LOC specialists named—appropriately—Lab on a Chip, which is already in its 15th year.

What follows is my take on some of the conceptual morphing, so to speak, of LOC-enabled devices that can be packed for portability, driven by remote control, or flown-“in-and-out” for all manner of unconventional, but critically important POC situations needing nucleic acid-based tests.


In archived blogs I’ve previously commented on examples of commercially available portable POC devices that are variations of a lab-in-a-box that can be easily carried in luggage or a back pack. By way of updates, here are some new applications for these systems illustrating wide diversity of use and location:

  • Ubiquitome’s hand-held qPCR system for molecular testing in New Zealand forests aimed at protecting indigenous Kauri trees—the oldest tree species in the world.
  • Amplyus’ miniPCR system for combating Ebola in villages deep in Sierra Leone, Africa.
  • Oxford Nanopore’s thumb-drive size DNA sequencer to identify organisms in the Canadian high Arctic.

Taken from @WhyteLab

RAZOR system by BioFire Defense. Taken from

In the above examples, sample prep workflow is still in need of automation with appropriate LOC technology. However, progress in this regard is being made. One example is the RAZOR system developed by BioFire Defense (pictured below) that features a qPCR lab-in-a-box with ready-to-use, freeze-dried reagent pouches for the detection and identification of pathogens and bio-threat agents. While the progress is impressive, there is still work to be done. A dramatized video for RAZOR usage revealed that much manual manipulation and dexterity with syringes are still required, which suggests the need for complete LOC automation in the future.

Another example of facilitating POC sample prep is the Bento Lab, which is named to be word-play on Bento Box—a complete Japanese lunch in a small, partitioned box-like plate. This portable DNA laboratory created by Bethan Wolfenden and Philipp Boeing at University College London is small enough to fit into a laptop bag, weighs only 6.6 pounds, and can now be preordered for ~$1,000 as a “must have” accessory for so-called “citizen scientists,” some of whom have had early access and have posted their personal Bento Lab stories.

The Bento Lab. Taken from Bento Lab


Biohazard accidents happen, as do bio-threat acts of terrorism. In these seriously scary situations, it may be safer or necessary for first-responders to deploy an Autonomous Vehicle as a self-navigating/driving lab-on-a-robot. Sounds far out, but the first example of a mobile lab-on-a-robot was demonstrated in 2008 by Berg et al., and is pictured below.

Taken from Berg et al. (2008)

This particular lab-on-a-robot is able to autonomously navigate by GPS, acquire an air sample, perform multi-step analysis [i.e. injection, capillary electrophoretic separation, and electrochemical (EC) detection], and send data (electropherogram) to a remote station without exposing an analyst to the testing environment. It’s easy to imagine adapting this kind of robot for carrying out qPCR with EC or fluorescence detection, or nanopore sequencing, for rapidly identifying pathogens.


A logical variation of lab-on-a-robot is to attach the lab part to Unmanned Aerial Systems, (more commonly called drones), thus affording a means for “fly-in, fly-out” applications that require speed to and from a location, or for deployment to otherwise inaccessible locations. This biotech version of drone delivery was initially demonstrated for drone pick-up to aerially transport blood samples from patients to central testing labs, as reported by Amukele et al.

Victor Ugaz. Taken from

The much more difficult task of attaching a lab testing module to a drone has been recently demonstrated by Prof. Victor Ugaz and coworkers at Texas A&M University. Their pioneering 2016 publication titled Lab-on-a-drone: toward pinpoint deployment of smartphone-enabled nucleic acid-based diagnostics for mobile health care is loaded with details, and is a “must read” for technophiles. What follows is my extraction of some unique highlights of that work, as well as information I learned by contacting Prof. Ugaz, who incidentally has received numerous awards and honors.

The basic idea investigated by these researchers was to design a drone-compatible system that could perform what I call “qPCR-on-the-fly.” The drone would require low power consumption and use a smartphone for both fluorescence detection—via its camera—and data analysis via radio transmission of results on-the-fly.

To reduce power consumption by conventional PCR using thermal cycling, which uses power for both heating and cooling during each cycle of amplification, the Texas team invented a radically different approach to achieve isothermal PCR. As depicted below, this new method called convective thermocycling operates isothermally at 95oC and involves movement of reactants upward, away from the heater, through progressively cooler regions and then traveling downward to repeat heating, etc. in a cyclic manner.

Taken from Ugaz & coworkers (2016)

They mimicked POC for an Ebola virus epidemic, which required on-site sample prep and then reverse transcription of viral RNA into cDNA prior to hot start qPCR that is incompatible with convective PCR. The sample prep step was very cleverly achieved using centrifuge adapters that connect to the drone in place of propellers. These centrifuges in turn—pun intended—were fabricated using state-of-the-art 3D printing, and are pictured below.

Taken from Ugaz & coworkers (2016)

The in-flight lab-on-a-drone is pictured below. While in-flight, smartphone-enabled qPCR (as depicted above) takes place during the return trip to home base in order to save time for re-equipping the drone to return to another site, thus increasing overall patient analysis throughput per drone.

Taken from Ugaz & coworkers (2016)

I contacted Prof. Ugaz to ask whether the reverse transcription (RT)-PCR could also be carried out in flight to further automate and increase drone throughput. He replied as follows:

“Many thanks for your interest in our work!  For the purposes of these proof of concept studies, we performed the RT and hot start steps off-device in a conventional thermocycler. However, these steps could straightforwardly be embedded in the portable device.  In principle it should be just a matter of either programming the heater to run through these additional steps (in which case we need to consider the thermal transient between steps, since we are trying to keep the device as simple as possible), or possibly have multiple separate heating zones on the device and have the user physically move the reactor from one to another for each step.  There are multiple possibilities to achieve this that can be explored, and the ‘best’ choice is likely related to the specific application that is envisioned.  But to answer your question, yes this is possible as a relatively straightforward extension of the current design…I have a student who will be working on this during the summer.” 

To my surprise and delight, Prof. Ugaz also informed me of his interest in investigating TriLink’s CleanAmp™ technologies for CleanAmp™ hot start PCR and CleanAmp™ hot start RT-PCR. He said that “[w]e are looking forward to testing this soon and will keep you posted!”

This work by Prof. Ugaz will hopefully lead to encouraging results, and provide a great example of how TriLink CleanAmp™ technologies are enabling both scientific advancement as well as an amazingly interesting, new application such as that in this lab-on-a-drone story.

As always, your comments here are welcomed.

Update on Zika Virus Detection by RT-PCR

  • Various RT-PCR Assays for Zika Virus Have now Received Emergency Use Authorization (EUA) from the FDA
  • Quest Diagnostics’ Assay Approved by FDA for General Use as a Zika Test
  • Troubled Theranos Touts New “miniLab” for Zika EUA
  • Vaccine Development Progressing Albeit Relatively Slowly
Aedes aegypti mosquito. Taken from

Aedes aegypti mosquito. Taken from

In January 2016, I posted a blog about the then emerging public awareness of Zika virus (ZIKV), which is spread by the bite of infected mosquitos—primarily the Aedes aegypti mosquito. Sadly, ZIKV can be passed from a ZIKV-infected pregnant woman to her fetus leading to development of a brain defect (microcephaly) and/or other malformities. Moreover, ZIKV is now associated with sexual transmission and blood transfusion. This is scary news.

This update was prompted by the additional fact that ZIKV is on the rise and there is still no vaccine, according to the Centers for Disease Control and Prevention (CDC)—my “go to” source for loads of authoritative information about this infectious disease. Weekly updated ZIKV-infection “case counts” in US States and US Territories were given as ~1,200 and ~6,500, respectively on Aug 10th, and increasing to ~3,400 and ~20,000 on Sep 21st —only 6 weeks later!

As I pointed out in my previous blog on ZIKV, absent an anti-ZIKV vaccine, there is considerable interest in mosquito abatement as well as early detection, notably by reverse-transcription PCR (RT-PCR) of this RNA Flavivirus. Now that Zika infection by mosquitos in Florida has been found, leading to several CDC warnings, I thought it would be both apropos and technically interesting to provide the following update on ZIKV structure and RT-PCR.

ZIKV Genome and Structure

As of last month, my PubMed search of “Zika [Title/Abstract] AND RT-PCR [Anywhere]” gave 55 publications. Incidentally, there are now a number of Zika genome sequencing publications, such as this lead reference. It’s worth noting that ZIKV genome sequencing enables monitoring the potential evolution of new genomic variants that might foil existing RT-PCR assays and guide the selection of new primers for RT-PCR.

ZIKV genome. Taken from with permission from SIB Swiss Institute of Bioinformatics, ViralZone

ZIKV genome. Taken from with permission from SIB Swiss Institute of Bioinformatics, ViralZone

It’s also worth pointing out that Zika’s overall molecular capsid structure is enveloped, spherical, and has a diameter of ~40 nm in diameter, as depicted below in schematic form, and pictured in the accompanying electron microscopic image. The surface proteins are arranged in an icosahedral-like symmetry.

ZIKV capsid structure. Taken from with permission from SIB Swiss Institute of Bioinformatics, ViralZone

ZIKV capsid structure. Taken from with permission from SIB Swiss Institute of Bioinformatics, ViralZone

This is a transmission electron micrograph (TEM) of ZIKV, which is a member of the family Flaviviridae. Virus particles are ~40 nm in diameter, with an outer envelope, and an inner dense core (see above). The arrow identifies a single virus particle. Taken from

This is a transmission electron micrograph (TEM) of ZIKV, which is a member of the family Flaviviridae. Virus particles are ~40 nm in diameter, with an outer envelope, and an inner dense core (see above). The arrow identifies a single virus particle. Taken from


In the interest of giving explicit credit to various investigative groups who have developed RT-PCR assays for ZIKV, here are links and short snippets I’ve selected from publications, in chronological order, found in the aforementioned PubMed search. Interested readers are encouraged to check out the original publication for details.

The first report of an RT-PCR assay for ZIKV appears to be by Faye et al. in 2008 at the Institut Pasteur de Dakar in Senegal, who targeted the envelope protein coding region and tested ZIKV isolates previously collected over a 40-year period from various African countries and hosts. The assay’s detection limit and repeatability were respectively 7.7pfu/reaction and 100% in serum; none of 19 other Flaviviruses tested were detected. Faye et al. in 2013 extended this work to include quantitative RT-PCR detection of ZIKV and evaluation with samples from field-caught mosquitoes.

Kinetics of ZIKV detection in urine compared to serum from 6 patients was described by Gourinat et al. in 2015 at the Institut Pasteur, Noumea, New Caledonia using RT-PCR primers and probes previously reported by others. Urine samples were positive for ZIKV more than 10 days after onset of disease, which was a notably longer period than 2-3 days for serum samples. These researchers concluded that urine samples are useful for diagnosis of ZIKV infections, and are preferred to serum wherein virus titer diminishes more rapidly.

Musso et al. in 2015 working in Tahiti, French Polynesia with 1,067 samples collected from 855 patients presenting symptoms of Zika fever found that analysis of saliva samples increased the rate of detection of ZIKV at the acute phase of the disease compared to serum samples. They noted that saliva was of particular interest when blood was difficult to collect, especially for children and neonates.

Most recently, Xu et al. in 2016 in China reported the development of a SYBR Green (intercalator dye)-based qRT-PCR assay for detection of ZIKV. Although their results indicate that the assay is specific, it’s important to note that SYBR-type detection can be subject to nonspecific artifacts, for which TriLink’s proprietary CleanAmp™ Primers can be investigated to potentially ameliorate such problems, as discussed in this downloadable pdf publication by TriLink researchers.

ZIKV In Vitro Diagnostic Assays

As detailed elsewhere, the Secretary of Health and Human Services (HHS) earlier this year determined that “there is a significant potential for a public health emergency that has a significant potential to affect national security or the health and security of United States citizens living abroad and that involves Zika virus.” The Secretary of HHS further declared that “circumstances exist justifying the authorization of the emergency use of in vitro diagnostics for detection of Zika virus…”.

The following RNA-based assays and suppliers are currently listed by the FDA for this Emergency Use Authorization (EUA):

  • xMAP® MultiFLEX™ Zika RNA Assay (Luminex Corporation)
  • VERSANT® Zika RNA 1.0 Assay (kPCR) Kit (Siemens Healthcare Diagnostics Inc.)
  • Zika Virus Real-time RT-PCR Test (Viracor-IBT Laboratories, Inc.)
  • Aptima® Zika Virus Assay (Hologic, Inc.)
  • RealStar® Zika Virus RT-PCR Kit U.S. (Altona Diagnostics)
  • Zika Virus RNA Qualitative Real-Time RT-PCR (Focus Diagnostics)
  • Trioplex Real-time RT-PCR Assay (CDC)

Among these, two are particularly notable—in my opinion. Trioplex Real-time RT-PCR Assay is a multiplexed laboratory test developed by the CDC to simultaneously detect ZIKA, dengue virus, and chikungunya virus RNA, each of which can be transmitted primarily by Aedes aegypti mosquitos to cause infections with similar symptoms. Consequently, it is useful to have a single test that can detect each of these viruses in the same sample. Full details for the Trioplex assay are provided in a 40-page downloadable pdf from the CDC at this link.

In brief, this CDC-developed test uses virus-specific primer pairs and fluorogenic hydrolysis probes each differentially dual-labeled with fluorescent reporter and quencher dyes for in vitro detection of complementary DNA (cDNA). The detection follows reverse-transcription of RNA isolated from clinical specimens including serum, cerebral spinal fluid, urine, and amniotic fluid. It’s worth pointing out that I subsequently found a publication in 2016 by several collaborating academic groups regarding an analogous triplex RT-PCR assay for the same three viruses.

The other notable assay is Zika Virus RNA Qualitative Real-Time RT-PCR developed by Focus Diagnostics, which in April 2016 was the first of the aforementioned ZIKV tests to receive authorization by the FDA for use by qualified labs to detect ZIKV RNA in blood samples of those meeting CDC clinical criteria or of people who may have lived in or traveled to an affected location or had other exposure to the virus. Quest Diagnostics, the parent company of Focus Diagnostics, announced it would make the test broadly available to physicians, including those in Puerto Rico in May 2016.

ZIKV EUA Sought by Theranos for Its new “miniLab”

Theranos, which has been in the news regarding troubles over its stealthy proprietary system for finger-stick blood tests, appears to be pivoting its strategic plans. It announced at the August 2016 American Association of Clinical Chemistry Meeting its R&D for a new, fully automated miniLab system, including analytical and method comparison results of its ZIKV nucleic acid-amplification-based assay.

According to its press release, the company collected finger-stick samples from subjects, including people in the ZIKV-infested Dominican Republic, and shipped those to Palo Alto, California to run on the miniLab. Although I was unable to obtain these particular results, I did find the following figure at the TechCrunch website showing functional components of the miniLab along with an article by Sarah Buhr that’s worth a quick read, in my opinion.

Taken from

Taken from

According to the aforementioned Theranos press release, the company has submitted assay validation data for this Zika assay to the FDA for an EUA. The company also states that it is unaware of any currently available capillary (i.e. finger-stick) test for ZIKV.

I’ll stay tuned for future general information about the miniLab, as well as information specifically related to ZIKV. If I hear of anything, I’ll add as a comment here or in a new post with technical details about its nucleic acid assays.

ZIKV Vaccine Status

I hope this blog has convinced you that RT-PCR of ZIKV has provided improved molecular diagnostics. I’m guessing, that like me, you find it unfortunate that there isn’t a proven anti-ZIKA vaccine as of yet. This is especially frustrating given the fact that Zika disease has been known for more than 50 years, and that it is evidently on the rise globally, including in the USA according to regularly updated CDC statistics.

In February 2016, the Obama administration requested $1.9 billion in funding for the NIH to develop a ZIKV vaccine. The US Congress continues to be deadlocked by partisan politics despite the fact that Florida state and local officials are scrambling to contain the ZIKV outbreak in Miami Beach. This outbreak poses a serious threat to the health of residents, as well to visitors who drive the region’s $24 billion-a-year tourism industry.

Nevertheless, some progress has been reported for US studies in monkeys, and US-based Inovio says it’s received FDA approval to begin studies in humans. Outside the US, Sanofi Pasteur in France is said to be poised for initiating its trials in humans, and French biotech company Valneva is reported to have succeeded in generating a ‘highly purified inactivated vaccine candidate’ using the same technical approach it used for its encephalitis vaccine that is marketed in the United States and Europe.

Lagging—dare I say “glacially slow”—action against ZIKV by the World Health Organization is quite disappointing to me, and is reminiscent of what I’ve commented on in an earlier blog concerning this bureaucracy’s ineffective response to the Ebola virus. If I had my druthers, TriLink’s previously announced engagement by Battelle in development of Ebola mRNA vaccine would somehow materialize for a Zika mRNA vaccine. In this regard, GlaxoSmithKline is reported to be preparing research studies alongside the NIH’s Vaccine Research Center to test a self-amplifying mRNA vaccine technology for Zika. Interested readers can check out this link to a fascinating PNAS publication by Geall et al. on biosynthetic self-amplifying mRNA vaccines delivered in lipid nanoparticles.

As always, your comments are welcomed.


Taken from Fleming et al. (2016) ACS Infectious Diseases

Taken from Fleming et al. (2016) ACS Infectious Diseases

One of my previous posts featured recent elucidation of biologically functional G-quadraplexes in living cells. Consequently, it’s apropos to mention here that Fleming et al. at the University of Utah have just now published the first analysis of potential G-quadruplex sequences (PQS) in the RNA genome of ZIKV. As depicted in this artistic cartoon, several PQS were found, with the most stable located near the end of the 3’ untranslated region (3′-UTR). Importantly, these investigators propose a rationale for screening G-quadruplex-binding compounds as a completely new class of anti-ZIKV drug candidates. In my opinion, this is a great example of how basic biochemical research can lead to new strategies for much needed antiviral drugs.

Spartan Cube—The World’s Smallest Molecular Diagnostic Device

  • Records Are Meant to be Broken—Including Those for PCR Diagnostics
  • Spartan Bioscience Claims It’s Cube is World’s Smallest Mol Dx Device 
  • The Cube was Launched at the Recent AACC Clinical Lab Expo 


[22]-annulene. Taken from

[22]-annulene. Taken from

In the spirit of the recent Olympic games, and as the saying goes—records are meant to be broken. When I was in grammar school, a big buzz in sports was who would be the first to break the 4-minute mile; answer: Roger Bannister in 3 minutes and 59.4 seconds in 1954—now 17 sec faster. In my college craze days, it was how many persons can fit into a Volkswagen Beetle; answer: I couldn’t find the first feat, but the current record is 20 crammed into an old style Beetle in 2010. Then during my graduate organic chemistry studies, there was interest in synthesizing increasingly larger annulenes—completely conjugated CnHn monocyclic hydrocarbons akin to benzene (n=6); answer: [22]-annulene with n=22 (see below) synthesized by F. Sondheimer. But I digress…

Our collective fascination with records—and beating them—also applies to all sorts of instruments for health-related sciences, such as the most powerful MRI imaging systems (currently from GE) or longest-DNA-sequencing system (currently from PacBio). Due to the seemingly endless utility of PCR, there is a continual stream of claims for the fastest PCR system (currently from BJS Biotechnologies) or—more to point herein—smallest PCR system.

To wit, regular readers of my blog will recall an April 2016 post titled World’s Smallest Real-Time PCR Device, which referred to a hand-held system reported by Ahrberg et al. for real-time, quantitative PCR (qPCR). That system is pictured below next to the original system commercialized by ABI in the 1990s that weighed 350 pounds and was 7 feet long!

Left: World’s smallest real-time PCR device. (Taken from Ahrberg et al). Right: Applied Biosystems 7700 real-time PCR system. (Taken from

Left: World’s smallest real-time PCR device. (Taken from Ahrberg et al). Right: Applied Biosystems 7700 real-time PCR system. (Taken from

It seems that PCR records fall as easily as those in the Olympics. Not even a year later, Ahrberg’s claim is being challenged by Canadian company Spartan Bioscience, which recently introduced its Cube device at the 2016 AACC Annual Scientific Meeting & Clinical Lab Expo. Following are some technical details that I thought were worth sharing.

Taken from

Taken from

Cube Facts

Given its amazingly small size of only 4 x 4 x 4 inches, there apparently has been some remarkable engineering achievements to be able to squeeze-in what’s needed for the rapid heating and cooling required for PCR thermal cycling. Ditto for the optics required to enable fluorescence detection. Like other relatively small devices intended for emerging Point-of-Care (POC) applications in a doctor’s office or clinic, there is wireless connectivity to a laptop that serves as the user interface for operation and data analysis, as well as a power source for the Cube via a USB cable.

You’re likely wondering by now how much the Cube will sell for. Unfortunately, I was not able to obtain a list price from Spartan’s CEO at this time, so we’ll all have to wait and see.  I’ll post the answer as a comment to this blog as soon as I find out the price.

Inside the Cube—Perhaps 

I actually don’t know exactly what’s inside the Cube, but some possibilities of what might be are as follows. I’ve based these educated guesses on a Spartan Bioscience patent (US pat. no. 8,945,880) by Paul Lem and others entitled Thermal cycling by positioning relative to fixed-temperature heat source. As depicted below, a hot block provides a heat source at a fixed temperature to thermally cycle PCR reaction vessels that can be precisely moved by a micrometer to and from the hot block in a repeated manner.

Taken from US patent no. 8,945,880

Taken from US patent no. 8,945,880

As for how fluorescence might be measured to monitor each PCR reaction in real-time, one possibility is depicted below. Basically, each reaction tube is proximate to excitation light from an LED, and has a slit at the bottom for emitted light that is collected and processed into a typical real-time PCR curve.

Taken from US patent no. 8,945,880

Taken from US patent no. 8,945,880

Before the Cube

Prior to launching the Cube, Spartan Bioscience has been selling an FDA-Cleared in vitro diagnostic product called Spartan RX, which is also relatively compact, and carries out fully automated—“cheek swab-to-result”—PCR analysis of certain Cytochrome P450 2C19 (CYP2C19) genotypes. Roughly 1-in-3 people carry CYP2C19 mutations that can impair metabolism of a wide variety of commonly used drugs. Consequently, these PCR-based results are a valuable aid to clinicians in determining strategies for therapeutics that are metabolized by the Cytochrome P450 2C19.

I was favorably impressed by the fact that this CYP2C19 assay qualifies for reimbursement from Medicare and most insurers, according to the company’s website, which adds that there is an ongoing 6,000-patient clinical trial entitled Tailored Antiplatelet Initiation to Lessen Outcomes due to Clopidogrel Resistance after Percutaneous Coronary Interventions (TAILOR PCI).

The Spartan RX cheek swab POC results have been recently compared to centralized genotyping with a TaqMan® allelic discrimination assay (Life Technologies) using qPCR and with the GenID® reverse dot-blot hybridization assay (Autoimmun Diagnostika GmbH). Published results indicate excellent agreement, and led to the following conclusions by the authors: “Compared to both laboratory-based genotyping assays, the POC assay is accurate and reliable, provides rapid results, can process single samples, is portable and more operator-friendly, however the tests are more expensive.”

I look forward to finding out more about the Cube and the PCR results it can obtain. I’ll post more information on my blog as it becomes available. As usual, your comments are welcomed.


To me, there’s something visually intriguing about a cube, perhaps because it’s one of the so-called Platonic Solids, which have been known since antiquity and studied extensively by the ancient Greeks.

Taken from

Taken from

Platonic solids such as the cube have also fascinated chemists, as evidenced by there being a substantial amount of published literature on the synthesis and physical properties of platonic solids. For example, cubane (C8H8) is a synthetic hydrocarbon molecule that consists of eight carbon atoms arranged at the corners of a cube, with one hydrogen atom attached to each carbon atom. A solid crystalline substance, cubane was first synthesized in 1964 by Philip Eaton and Thomas Cole. Prior to this work, researchers believed that cubic carbon-based molecules would be too unstable to exist.

Taken from

Taken from

On the fun side, the cube was morphed—so to speak—into what became an amazingly popular game, or should I say, object of competition. Rubik’s Cube is a 3-D combination puzzle invented in 1974 by Hungarian sculptor and professor of architecture Ernő Rubik. If you’re wondering about the world’s record for solving this puzzle, it’s currently a mind-boggling 4.9 sec, according to a list (with video links) of this and past records that appear to have been broken regularly, just as I opined at the beginning of this blog. But I digress…yet again.

Point-of-Care PCR 2.0

  • Ubiquitome Quickens Pace of POC Apps for Its Freedom4
  • Cepheid Unveils its POC Diagnostics System
  • Hopkins Crew Brews “Coffee Mug-Sized” Gizmo for Fully Automated Chlamydia Testing
Kiwi Dr. Jo-Ann Stanton holding Ubiquitome’s Freedom4 at Tri-Con 2015

Kiwi Dr. Jo-Ann Stanton holding Ubiquitome’s Freedom4 at Tri-Con 2015

Regular readers of this blog will recall a recent byline exclaiming “Honey I Shrunk the qPCR Machine”, which spotlighted the unveiling of startup company Ubiquitome’s first point-of-care (POC) product—Freedom4—developed in New Zealand. Up until then, this far away—for me—exotic island country brought to mind folks fondly nicknamed Kiwi—after the native flightless bird, not Chinese fruit. Mightily impressed by this tiny but powerful qPCR device, I vowed to thereafter keep an eye on these Kiwis’ democratized POC apps enabled by its nifty handheld 4-sample high-performance qPCR device.

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Pseudouridine Biomarker for Breast Cancer

As you are probably aware, October is National Breast Cancer Awareness month. Everyone from the NFL to Yoplait yogurt seems to be engaged in campaigns for fundraising and awareness. I think it’s great to see the extensive community support for this worthy cause. Since breast cancer awareness is top of mind this month, I thought I’d follow up my latest blog about pseurdouridine with a ‘mini blog’ highlighting some interesting research involving a pseudouridine biomarker for breast cancer.

I’d also like to mention that TriLink is participating in Breast Cancer Awareness month. For every order placed in October, TriLink will dontate $5 to Susan G Komen to support the upcoming 3-Day Walk being held November 20-22 in San Diego.


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