RTPCR for All

Real-time polymerase chain reaction (RTPCR or sometimes qPCR with “q” for quantitative) is shorthand for a sensitive, specific, and rapid analytical method for identifying genetic sequences of interest (and hence the microbes possessing those sequences) in environmental or biological samples.  While the details of RTPCR overwhelm me, my simple understanding is that the sample DNA is labeled with a probe of the known sequence (e.g., a sequence specific to the organism one is looking for) and amplified by copying until it can be detected and measured (for more, see a Wikipedia article).  The technique has been widely used in research for more than 20 years and is slowly finding commercial applications in areas such as forensics, clinical diagnostics, surveillance for biological threats, and food and water testing (e.g., Biofire Diagnostics Products ).  In the global health world, RTPCR is attractive because it is highly specific and sensitive and doesn’t require time-consuming culturing to increase the number of microbes to be detected, but its expense, complexity, and need for trained personnel and consistent utilities are barriers to its use in low-resource, high-disease-burden countries.

That being said, RTPCR diagnostics, and other DNA-based tests, are being developed for global diseases, most notably for one of the Big Three diseases, tuberculosis (TB), where early detection and differentiation of drug-resistant strains are sorely needed.  In the 2013 TB Diagnostics Pipeline Report (2013 Pipeline Report), twelve of the 21 tests listed as under development or in testing for wider use are DNA/PCR-based.  In two of my previous posts (“ReDux” and “ReDux Part II”), I wrote about the deployment of one RTPCR test, the Xpert MTB/RF assay, developed by the US company, Cepheid, with help from the Gates Foundation and the National Institute for Allergy and Infectious Disease.  Endorsed by WHO in 2010, the test is slowly being adopted in high-TB-burden countries for confirmatory testing (not initial diagnosis), in part due to its cost which is about $15,000 per machine and $17 per test although several organizations now provide subsidies that bring down the per test cost to less than $10 (Cepheid press release).  The impact of the Xpert assay on TB care is still in question; the authors of recent study of the use of the test concluded “clinical and programmatic effects and cost-effectiveness remain to be defined” and a point-of-care, initial assay was still “urgently needed” (Lawn et al. 2013).  This got me wondering what efforts there may be to develop a RTPCR assay system that could be cheap, robust, and easily-used and -deployed for global health.

An RTPCR assay has two parts.  First, a kit of reagents to prepare the sample and label the unique sequences is needed.  Coincidently, I know of a company that is doing that, Co-Diagnostics HBDC (for high-burden developing countries; Co-Dx HBDC) which is a division of Cooperative Diagnostics, a company started in 2007 by Brent Satterfield whom I met in 2008.  Co-Dx HBDC has a TB kit on sale and is working on others for HIV, HCV, and malaria (Co-Dx Products).  I haven’t spoken with Brent in years so I don’t know the price of kits or how sales are going.  The second RTPCR part is a box to amplify the DNA and read the resulting products.  I found a four PCR machines that I presume are useable for RTPCR and will fit any budget:

  • The Gene Machine is a cheap, do-it-yourself (DIY) PCR designed and built by Russell Durrett using PVC pipes, a 150-watt light bulb, a computer fan, and a Arduino Uno microcontroller (Popular Science article).  If you don’t have space at your kitchen table to build your Gene Machine and live near Boston, you can undertake a similar project at Boston’s Open Source Science Laboratory (BOSSLAB project).
  • Another DIY PCR was developed at ATX Hackerspace and is said to cost $85 in parts and a few hours in time according to a nice description at Instructables (DYI PCR).  I’m not sure if the PBR can in the first photo is a required or optional part.
  • The OpenPCR is for those who would rather build from a kit.  It is priced at $649 and has a nice wood housing with DNA-inspired decoration (OpenPCR).
  • The Palm PCR is a hand-held PCR by the Korean company, Ahram Bio, that debuted in 2010 as I noted in my post “BIO-Bits”.  The company came out with a low-end model that is available in white only from Zymogen Life Sciences (India) for introductory price of $3500 (to fit any budget Palm PCR).  I’m not sure if this device is RTPCR-ready but it sure is cute.

I also found two academic projects that have resulted in prototypes designed to be inexpensive to buy and operate.  A team at the Stanford School of Medicine recently published results of an epidemiological study in which their RTPCR system using inexpensive non-fluorescent probes identified toxic strains of E. coli (GenomeWeb article).  Even more interesting is the RTPCR system brought to prototype stage by a group at Cal Tech consisting of professors Axel Scherer and David Baltimore (he of the Nobel Prize and my MIT undergrad advisor) and senior scientist, George Maltezos.  According to a February 2013 CalTech News article, the project was funded by two Gates grants and the device is “small enough to stow in a backpack and is as simple to operate as a DVD player.”  What is not mentioned is that the team formed a company called s2a Molecular, Inc. (“From sample to answer”) this year.  Unfortunately, the s2a website is under development.  I wonder if the company has data on human samples, has done any field tests, and has lined up any corporate partners?

 

I don’t often correct my posts (because I don’t fact check and no one catches me) but I wrote last week that the two biosimilars were recommended for European approval.  Actually, it was one.  Celltrion’s Remsima will be sold as by Hospira as Inflectra (FierceBiotech article).

 

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