NEXTGENPCR: 30 cycli, 3 temperatures, less than 2 minutes

8-minute detection of SARS-CoV-2 plasmids with the 2019-nCoV CDC RUO Kit

8-minute detection of SARS-CoV-2 plasmids with the 2019-nCoV CDC RUO Kit

Introduction

“PCR is the most commonly used test for diagnosing coronavirus because it’s highly accurate. But other problems limit it. “It’s not getting the turnaround we need,” says Steven Wolinsky, an infectious diseases physician at Northwestern University.”1

Laboratories around the world are attempting to deploy widespread rapid screening for the novel coronavirus (2019-nCoV/SARS-CoV-2) identified in Wuhan, China. Countries with mass screening have been praised for managing local infections. “South Korea’s experience shows that “diagnostic capacity at scale is key to epidemic control,” says Raina MacIntyre, an emerging infectious disease scholar at the University of New South Wales, Sydney.”2 With many organizations supplying assays, test availability has risen, but few advances towards improving laboratory throughput. With current instrumentation, the US Centers for Disease Control (CDC) primers and probes that require over one hour and thirty five minutes to be completed, resulting in only a few hundred samples to be tested per day.

Molecular Biology Systems (MBS) introduced NEXTGENPCR, a machine capable of thermocycling at ultrafast speeds using standard reagents and reaction plate formats. In this brief, a method is described that allows for the qualitative detection of a SARS-CoV-2 control plasmid using the probe assays designed by the CDC to be completed in eight minutes.

NEXTGENPCR 8-minute protocol.

SARS-CoV-2 control plasmids (Integrated DNA Technologies (IDT), Inc., USA) were used for PCR protocol development. For dilution of control plasmids, nuclease-free H20 (Promega, USA) was used. PCR reactions (20ul) contained PerfeCTa® qPCR ToughMix, UNG (QuantaBio, USA). The primers and probes used were the 2019-nCoV RUO Kit (IDT) designed by the CDC. Reactions were loaded in 96-well × 20ul plates (MBS) and sealed with clear heat seals (MBS) in a heat sealer (MBS) for 1.5s. at 165°C. Reactions were performed using a NEXTGENPCR machine (MBS). The PCR protocol was incubation at 95°C for 30s. followed by 45 cycles at 95°C for 3s, 55°C for 5s. The sealed plates were placed directly on a Blue Bio Scan 1000F Imager (Intas Science Imaging, Germany). Fluorescence signal was captured with using the 4× setting. The images shown in this study, are the reactions still in the PCR plate wells. Gel electrophoresis was not required to image the probe hydrolysis reaction.

Primer/Probe Pos. Control
Ct +/- St. Dev
Neg. Control
Ct +/- St. Dev
RP 34.16 ± 0.2 Not detected
N2 33.69 ± 0.90 43.51
N1 33.4 ± 0.74 Not detected

 

Figure 1

Results

To establish comparative performance, a study was performed using the 2019-nCoV RUO Kit that tested for the presence of SARS-CoV-2 DNA using a control plasmid (1000 copies) as a template. A negative control without plasmid DNA was also tested. The PCR reactions were mixed as described in the Instructions for Use “CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel” (CDC-006-00019, Revision: 02). Thermocycling in the Bio-Rad CFX96 used the following program 95°C for 120s. followed by 45 cycles of 95°C for 3 s., 55°C for 3s. The threshold cycles are shown in Table 1 acquired by the Bio-Rad CFX96. The same 20ul reaction setup was loaded into MBS 96×20ul well plates and tested on the NEXTGENPCR machine with the same hold times. FAM fluorescence, from the hydrolyzed probe, was visualized with a blue light imager (Figure 1).

The transfer to NEXTGENPCR, which reduced ramp times to less than one second, resulted in a 25 minute protocol compared to 71 minutes on the Bio-Rad CFX96. To further decrease amplification time, the protocol was changed as described above in NEXTGENPCR 8-minute protocol. The results demonstrate that all three reactions produce fluorescent signal in the positive control reactions, but not in the negative control reactions (Figure 2).

Reactions were scaled down to a 5ul total volume, which resulted in testing for the presence of 250 copies of positive control plasmid (Figure 3). The standard CDC protocol and the accelerated MBS protocol both resulted in easily discernible qualitative results. These results suggest that scaling down the 2019-nCoV RUO Kit reaction size does not compromise reporting, yet it saves reagent and sample.

Figure 2
MBS 8’ Protocol in 20ul

Figure 3
MBS 25’ Protocol in 5ul

Figure 3
MBS 8’ Protocol in 5ul

Summary

Ultrafast PCR with NEXTGENPCR enables an 8 minute- 5ul 2019-nCoV CDC qPCR Probe-based amplification with direct observation on a blue light illuminator.

  1. > 91% decrease amplification time
  2. 22,860 samples could be amplified per day with a single NEXTGENPCR machine
  3. Reagent consumption reduced by 75% alleviates costs and availability of supply

References

  1. “The standard coronavirus test, if available, works well—but can new diagnostics help in this pandemic?” Science. Robert F. Service Mar. 22, 2020.
  2. “Coronavirus cases have dropped sharply in South Korea. What’s the secret to its success?” Science. Dennis Normile Mar. 17, 2020.

 


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