Digital PCR: A More Accurate Method For Covid-19 Detection

At the time of writing this article at 10.04 hours GMT, 10 October 2020, there are 37,152,125 active SARS-COV-2 cases globally. The need for sophisticated testing technology remains paramount. 

The clinical need for technology that can reduce the number of false negatives is particularly critical. False negatives delay the correct diagnosis and rapid treatment initiation. That hampers breaking the transmission chain. 

Academic publications in Emerging Microbes & Infections, Plos One, Technology Networks, and more have been reporting on the success of dPCR technology in eliminating false-negative results.    

PCR Technology: A Historical Snapshot

Kary Mullis discovered Polymerase Chain Reaction (PCR) in 1983. Experts hailed it as a groundbreaking step forward in radically expanding the scope of detecting and manipulating nucleic acid. 

Thermal cyclers, the machine needed to perform PCR hit the market in 1987. Since then, PCR has become an invaluable tool in clinical genetics and molecular biology. 

The Core Principle Of PCR

PCR is a technique to amplify DNA (Deoxyribonucleic Acid) so that it becomes possible to generate many millions of copies of a particular segment of DNA. Only a tiny amount of starting material is necessary. 

The specificity of PCR depends on hybridization, while enzyme-based amplification determines its sensitivity. A series of temperature cycles repeated 20-40 times constitute the basic PCR methodology. 

The denaturation of DNA duplexes and the hybridization of two DNA primers (technically, oligonucleotides) accompanying the target sequence happens in each cycle. Each thermal cycle also includes the elongation of the DNA primers by a DNA polymerase. 

A DNA polymerase is an enzyme that assembles nucleotides to produce DNA molecules. Nucleotides, as we know, are the building blocks of DNA. 

The figure below represents the basic PCR methodology.

dna

Source

The Evolution of PCR

The advantages of the PCR technology over traditional DNA cloning technology made it the hot favorite of clinical geneticists and molecular biologists alike. Experiments to further improve the method continued alongside. Several variations of PCR technology have evolved as a result. 

Hot Start PCR, Reverse Transcription PCR (RT-PCR), Quantitative Real-Time PCR (qPCR), and the combined technique of RT-PCR and qPCR are all versions of the basic PCR. RT-PCR is also known as quantitative real-time PCR.

Among these, the qPCR technology continues to be one of the most favored methods for determining cDNA (complementary DNA) and gDNA (genomic DNA) levels. 

One of the major problems with the qPCR technique is the high variability of the resulting data. The non-comparability and non-reproducibility of data in the absence of validation and verification of both samples and primers add to the problem. 

Experts have identified inadequate dilution of residual protein and chemical contaminants as the root cause of this problem. These factors variably inhibit Taq polymerase and primer annealing, resulting in data diversity.

Samples containing low abundant targets with small-expression variations of two-fold or lower are the most vulnerable to this snag. Digital polymerase chain reaction (dPCR) is a more sophisticated technique with several advantages over qPCR. 

Digital PCR (dPCR) 

Bert Vogelstein and Kenneth W. Kinzler first introduced the digital PCR (dPCR) technique in August 1999 in an article published in the PNAS (Proceedings in the National Academy of Sciences in the U.S.A). 

The article draws from the authors’ successful application of this method to detect a mutant ras oncogene in the stool of patients suffering from colorectal cancer. They describe the technique as one that can convert the analogue and exponential nature of the PCR to a linear digital signal.  

The method is to isolate single molecules by dilution to then individually amplify them with PCR. The dilution makes it possible to separately analyze each product using fluorescent probes. That facilitates checking the presence of mutations. That was the beginning of dPCR.

Fluidigm launched the BiomarkTM, the first commercial dPCR platform, in 2006. The 12.765 Digital Array, a chip of 12 panels, formed the basis of this system. Each panel is divided into 765 6-nL chambers. 

The number of individual partitions of each sample, the enhanced hands-on time, and the volume of the reactions limited the digital PCR technology on the BiomarkTM platform. The most problematic issue, however, was its cost-intensity. 

The cost for a single reaction in the qPCR system was a little over one dollar. In comparison, the dPCR method needed several 100 dollars for a single reaction. Read more about the Digital PCR at Stilla Technologies

dPCR For COVID-19 Detection

The quantitative real-time PCR (RT-PCR) has been the most widely used method for testing COVID-19. One of the problems with this method is the generation of false negatives in specimens with low viral loads. 

A study conducted in the Renmin Hospital of Wuhan University, China, established dPCR as a more accurate method for COVID-19 detection. The researchers tested 77 samples by both RT-PCR and dPCR to be able to generate evidence of the comparable accuracy of the two methods. 

The dPCR technique identified 26 patients as COVID positive though they had been detected as negative by the RT-PCR method. Each of these samples had a low viral load.

These findings are consistent with the earlier characterization of dPCR as a more sensitive technique. Most COVID tests use the inherently complex nasopharyngeal swabs. Low viral load in the early phase of the infection is a typical feature. 

The dPCR technique is particularly efficient in quantifying nucleic acids from complicated backgrounds. That makes dPCR more accurate for COVID testing than RT-PCR.    

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