Next Generation Direct PCR for Molecular Diagnostics

The COVID-19 pandemic has highlighted the important role that polymerase chain reaction (PCR) can play in molecular diagnostics. PCR-based testing quickly became the predominant method used to identify infected individuals and stem chains of transmission. However, traditional PCR technologies depend on RNA or DNA extraction before PCR testing, which adds time and costs to the workflow.

Technology Networks spoke to Dr. George Huang, chief financial officer at Entopsis, to learn how these steps can be omitted using direct PCR technology. In this interview, Dr. Huang also discusses the advantages of this approach, why uptake has so far not been greater and what sets next generation direct PCR reagents apart.

Anna MacDonald (AM): What is direct PCR, how does it differ from traditional PCR?

George Huang (GH): Direct PCR / extraction-free PCR / direct amplification refers to PCR performed directly from a sample, without isolating or purifying RNA or DNA beforehand. In contrast, RNA or DNA is isolated in a series of steps and only the purified nucleic acids are used for PCR in traditional PCR. As such, direct PCR does not require columns or beads for binding to RNA or DNA, nor does it require a series of reagents for lysing viruses and cells, washes and eluting nucleic acids. These additional testing components add cost, waste time, require specialized equipment for automation and open the door for multiple testing bottlenecks, as seen during the pandemic.

AM: What are the advantages of this approach and what applications is it most suited towards?

GH: Direct PCR offers many advantages over PCR utilizing traditional RNA/DNA extraction. Below are the top three categories.

1.       Cost- and time-savings. Some labs can save > 50% with direct PCR and may test an extra batch of samples in the same 8-hour shift, leading to higher profits and/or faster turnaround times. These savings extend beyond the cost of direct PCR kits, and include savings on consumables, expensive equipment and additional personnel to scale-up testing.

2.       No specialized equipment or consumables required. With direct PCR labs are not dependent on vendor-specific platforms and obligated to purchase expensive consumables. Moreover, testing labs are minimally affected when these vendors have shortages of consumables resulting in testing bottlenecks, as was seen during the COVID-19 pandemic.

3.       Doesn’t bias the microRNA profile. A growing body of literature is showing that traditional RNA extraction approaches that use “capture and release” technology (e.g., silica columns, magnetic beads, etc.) bias the microRNA profile. Direct PCR, since it maintains the integrity of the sample, is not expected to have this problem.

AM: Given the benefits why has uptake not been greater?

GH: There are a number of rational reasons why direct PCR has been adopted at a slower than expected pace, given the many benefits it offers. For decades, traditional RNA and DNA extraction preceded PCR testing and this was taken as a necessary step because there were few or no alternatives. As such, numerous life science companies built businesses around traditional RNA and DNA extraction. They created nucleic acid binding columns and magnetic beads, various adapters, specialized equipment and associated consumables which keep consumers dependent on their platform.

To some extent, late adopters have hindered adoption of direct PCR technologies. We have encountered clinical testing laboratories unwilling to update their technology, despite numerous advantages. Part of the problem is a lack of familiarity with new technology and a limited track record with new products.

Another key barrier to widespread adoption of direct PCR technologies was poor performance compared to RNA/DNA extraction and the requirement for alterations to PCR testing kits. Frankly speaking, traditional direct PCR technologies often resulted in high Ct values or no Ct values for various applications. In addition, many of these products force labs to add additional components to your qPCR/RT-qPCR master mix, which may affect historical results and force labs to extensively re-validate commercial tests. The ideal direct PCR product is one that offers results comparable to RNA/DNA extraction and allows you to use PCR detection kits, without altering the test kit.

Nonetheless, direct PCR is increasingly being adopted in the USA, Latin America and elsewhere with the launch of PCRopsis™ next-generation direct PCR products that overcome problems with traditional (generation 1) direct PCR technologies. We see this with an increasing number of commercial PCR tests incorporating direct PCR approaches into their kits. We are taking our time and educating select, cutting-edge testing laboratories with how best to use PCRopsis™ technologies to outcompete labs using traditional RNA/DNA extraction with a focus on costs/time savings and performance. I believe that market forces are the best driver of new technologies and will favor technologies that save time and improve profit, while offering competitive performance.

AM: You recently demonstrated that Entopsis’ PCRopsis™ reagent outperformed other direct PCR reagents, how is this achieved?

GH: At its core, Entopsis is a research and development company that solves industry agnostic problems in unique and impactful ways. One of Entopsis’ internally developed platforms is a nanotechnology-based, machine learning driven screening approach that allows for the fast identification of materials with desired properties. This platform has been used for the development of cancer diagnostics, in-line removal of toxic components from insulin pumps, equipment-free precipitation of select blood cells, equipment-free concentration of clinical samples, improvement of isothermal PCR tests and others.

Our teams, including a diverse investor base, were surprised and irritated when COVID-19 tests could not be performed at scale due to bottlenecks in testing supplies, namely viral RNA isolation kits. This is when we decided to invest heavily in addressing this dangerous shortcoming in the PCR testing industry. We repurposed our in-house technologies and used our distinct know-how to develop direct PCR reagents that overcome limitations found in direct PCR products on the market and offer results comparable to traditional RNA and DNA isolation. Our line of PCRopsis™ next-generation direct PCR reagents accomplish this goal by simultaneously performing five tasks in a single reagent: 1) lyse virus or cell 2) disrupt nucleic acid structure 3) dissociate bound proteins 4) stabilize nucleic acids 5) sequester PCR inhibitors.

PCRopsis™ next-generation direct PCR reagents offer unique advantages when compared to traditional direct PCR kits:

1) stability at room temperature, offering savings in transportation and handling costs and added convenience.

2) products like PCRopsis™ RVD-E and PCRopsis™ Buccal, allow you to extract RNA and DNA directly from swabs, thereby negating the need for transport mediums, which also presented a bottleneck during the pandemic.

3) robust performance, results comparable to RNA and DNA extraction.

4) simple protocol that does not require alterations to your PCR mixture (simply incubate your sample with PCRopsis™ reagents and use resulting sample directly into your desired PCR mixture).

AM: How can Entopsis’ PCRopsis™ reagent be applied to COVID testing?

GH: Entopsis’ PCRopsis™ reagents serve as an alternative to RNA and DNA extraction for PCR amplification of desired gene targets, particularly for COVID-19 testing. Below is a list of next-generation direct PCR technologies geared for amplification of SARS-CoV-2 and other viruses.

1.       Reagent RVD with RVD Enhancer: mediates direct PCR from diverse transport mediums, saliva and urine. Can also be used to amplify numerous bacterial species.

2.       Reagent SRVD: mediates direct PCR from saliva samples, even pure saliva samples.

3.       Reagent RVD-E: mediates direct PCR from dry swabs, without the need for transport mediums

4.       Reagent RVD-RT: mediates direct PCR at room temperature from samples in diverse transport mediums, without any heating step.

 

George Huang was speaking to Anna MacDonald, Science Writer for Technology Networks.