Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification

Summary

This work summarizes steps on developing different assays for SARS-CoV-2 detection using a two color ddPCR system. The steps are elaborate and notes have been included on how to improve the assays and experiment performance. These assays may be used for multiple SARS-CoV-2 RT-ddPCR applications.

Abstract

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) is the gold standard for SARS-CoV-2 diagnosis as no permanent solution is available. However effective this technique may be, research has emerged showing its limitations in detection and diagnosis especially when it comes to low abundant targets. In contrast, droplet digital PCR (ddPCR), a recent emerging technology with superior advantages over qPCR, has been shown to overcome the challenges of RT-qPCR in diagnosis of SARS-CoV-2 from low abundant target samples. Prospectively, in this article, the capabilities of RT-ddPCR are further expanded by showing steps on how to develop simplex, duplex, triplex probe mix, and quadruplex assays using a two-color detection system. Using primers and probes targeting specific sites of the SARS-CoV-2 genome (N, ORF1ab, RPP30, and RBD2), the development of these assays is shown to be possible. Additionally, step by step detailed protocols, notes, and suggestions on how to improve the assays workflow and analyze data are provided. Adapting this workflow in future works will ensure that the maximum number of targets can be sensitively detected in a small sample significantly improving on cost and sample throughput.

Introduction

Polymerase chain reaction (PCR), a well-recognized technique, has undergone several transformations since its advent to become a powerful technique capable of providing answers to nucleic acid research. These transformations have been a constant improvement of the old technique. These transformations can be summarized into three generations¹. The first generation is conventional PCR that relies on gel electrophoresis to quantify and detect amplified targets. The second generation is quantitative real time PCR (qPCR) that can detect samples in real time and rely on a standard curve to directly quantify targets in a sample. The third generation, ....

Protocol

Ethical statement
Wuhan Institute of Virology (WHIOV) is among the labs and institutes approved by China CDC of Wuhan city to conduct research on SARS-CoV-2 and detect COVID-19 from clinical samples. Research on developing new diagnostic techniques for COVID-19 using clinical samples has also been approved by the ethical committee of Wuhan Institute of Virology (2020FCA001).

1. Sample processing workflow (Figure 1A)

NOTE: Throughout the protocol, it is important to use separate rooms with dedicated pipettes for sample handling (extraction and storage), reagent/ma....

Results

In a proof-of-concept study, the multiplex assays analytical performance was tested on clinical and research samples¹⁹. The performance of the multiplex assays was superior to that of an RT-PCR¹⁹. Since low numbers of droplets may indicate a problem during droplet generation, in this article a cutoff of 10,000 droplets per well was set based on empirical data.

A good separation between positive and negative droplets with minimal rain interference.......

Discussion

Few resources are available on how to develop RT-ddPCR assays for SARS-CoV-2 detection. Though not used in this article, standard samples with known copies may be used to develop and optimize assays. In this work however, SARS-CoV-2 samples grown in Vero-E6 cells were spiked in a background of human genomic RNA and used as standard samples to develop the assays. Proper primer and probe sequences are essential when developing assays. Since most preliminary work on SARS-CoV-2 RT-ddPCR used the China CDC primer and probes t.......

Materials

Name	Company	Catalog Number	Comments
32-channel fully automatic nucleic acid extractor Purifier 32	Genfine Biotech	FHT101-32	Automated extractor for RNA
AutoDG Oil for Probes	BioRad	12003017	QX200 AutoDG consumable
ddPCR 96-Well Plates	BioRad	12003185
ddPCR Supermix for Probes (No dUTP)	BioRad	1863024	Making ddPCR assay mastermix
DG32 AutoDG Cartridges	BioRad	1864108	QX200 AutoDG consumable
Electronic thermostatic water bath pot	Beijing Changfeng Instrument and Meter Company	XMTD-8000	Heat inactivation of samples
FineMag Rapid Bead Virus DNA/RNA Extraction Kit	Genfine Biotech	FMY502T5	Magnetic bead extraction of inactivated RNA samples
Pierceable Foil Heat Seals	BioRad	1814040
Pipet Tips for the AutoDG	BioRad	1864120	QX200 AutoDG consumable
Pipet Tip Waste Bins for the AutoDG	BioRad	1864125	QX200 AutoDG consumable
PrimeScript RT Master Mix (Perfect Real Time)	TaKaRa	RR036A	cDNA generation
PX1 PCR Plate Sealer	BioRad	1814000	Seal the droplet plate from AutoDG
QuantaSoft 1.7 Software	BioRad	10026368	Data acquisition and analysis
QuantaSoft Analysis Pro 1.0	BioRad	N/A	Data analysis
QX200 Automated Droplet Gererator (AutoDG)	BioRad	1864101	QX200 AutoDG consumable
QX200 Droplet Reader	BioRad	1864003	Droplet reading and data acquisition
T100 Thermal Cycler	BioRad	1861096	Droplet target amplification (PCR) and cDNA generation