Rapid, sensitive, and highly accurate detection of COVID-19 is essential to slow the pandemic. However, current methods are limited by a high incidence of false negative results that may contribute to further spread as patients harboring the virus go undetected. This highlights the urgent need for a more sensitive methodology. Addressing this requirement, a new COVID-19 RT-Digital PCR Detection Kit from Gnomegen LLC has recently been granted emergency use authorization (EUA) by the FDA.
Developed for use with Gnomegen’s novel digital real-time PCR instrument, the COVID-19 RT-Digital PCR Detection Kit achieves timely, precise quantification of nucleic acid from the SARS-CoV-2 virus in nasal, nasopharyngeal, and oropharyngeal swab specimens collected from individuals suspected of having COVID-19 by their healthcare provider. Compared to its use with other qPCR-based systems, using the COVID-19 RT-Digital PCR Detection Kit for digital real-time PCR affords higher detection rates and a four-fold greater sensitivity than existing qPCR-based methods. It also demonstrates a superior linear range than end-point digital PCR.
Digital real-time PCR combines the ability of digital PCR to deliver highly sensitive, absolute quantification of sample material with the capacity of real-time PCR to produce a dynamic readout. By bringing these features together in one platform, Gnomegen has surpassed the sensitivity and quantification accuracy of existing technologies, as well as added the capability to multiplex. This approach has already proven highly successful in China, where Gnomegen’s HER2 Amplification Detection Kit has been CFDA approved to monitor breast cancer/gastric cancer patients.
With digital real-time PCR, researchers can analyze up to five targets simultaneously in a single reaction. This reduces the use of both sample material and reagents in comparison to established qPCR methods that require four individual samples per test (3 targets and 1 reference control). Not only does this allow for repeated, high volume analysis of similar targets at increased throughput, but the decreased sample requirement also reduces the likelihood of cross-contamination.
Gnomegen’s COVID-19 RT-Digital PCR Detection Kit consists of primers and TaqMan probes for the nucleocapsid gene (N-gene) specific to the COVID-19 virus, as per the recommendations of the Centers for Disease Control and Prevention (CDC). Used with Gnomegen’s digital real-time PCR instrument, the kit provides rapid analysis of patient sample material to detect even very low levels of the virus.
Briefly, SARS-CoV-2 nucleic acid is first extracted, isolated, and purified from nasal, nasopharyngeal, and oropharyngeal swab specimens using authorized extraction methods, before being reverse transcribed into cDNA using real-time RT-digital PCR in a single microwell chip. PCR amplification and detection testing are then performed with an authorized real-time PCR instrument. In comparison to the widely employed real time PCR instrument that is currently approved for this clinical use, Gnomegen’s digital real-time PCR instrument offers several advantages.
Using three sample types (cDNA, inactivated virus, and viral whole genome) as reference material, Gnomegen has generated analytical data using the COVID-19 RT-Digital PCR Detection Kit with their own digital real-time PCR platform and using current qPCR methods. Results indicate that, when used in conjunction with Gnomegen’s digital real-time PCR instrument, the COVID-19 RT-Digital PCR Detection Kit achieves fourfold greater sensitivity, with a limit of detection of just 2.5 copies per reaction compared to 10 copies per reaction for qPCR.
Figure 1. Comparison of % positive target amplification using dPCR and qPCR. At 95% positive amplification, the limit of detection is just 2.5 copies per reaction for dPCR compared to 10 copies per reaction for qPCR.
Importantly, using the COVID-19 RT-Digital PCR Detection Kit with the digital real-time PCR instrument, detection rates are increased from 62.5% to 95% where copy numbers are low. Although both systems can detect the virus when input is high, digital real-time PCR offers a clear benefit when it comes to detecting low level infection. This demonstrates the power of Gnomegen’s technology platform to detect COVID-19 infected patients that might otherwise be missed.
In addition to greater sensitivity and improved detection rates compared to qPCR, Gnomegen’s digital real-time PCR platform also benefits from a superior linear range at higher inputs than that of end-point digital PCR. With an input of 100ng RNA, digital real-time PCR exhibits an R2 value of 0.9999, whereas this is just 0.3344 for the end-point digital PCR method.
Figure 2. Comparison of linearity for digital real-time PCR and end-point digital PCR.
Tackling the COVID-19 pandemic relies on rapid, sensitive, highly accurate detection of the virus in patient sample material. Gnomegen’s COVID-19 RT-Digital PCR Detection Kit and digital real-time PCR instrument are delivering on this need.
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