Development of Multiplex Real-Time RT-qPCR Assays for the Detection of SARS-CoV-2, Influenza A/B, and MERS-CoV

Summary

We present two probe-based in-house one-step RT-qPCR kits for common respiratory viruses. The first assay is for SARS-CoV-2 (N), Influenza A (H1N1 and H3N2), and Influenza B. The second is for SARS-Cov-2 (N) and MERS (UpE and ORF1a). These assays can be successfully implemented in any specialized laboratory.

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) is a serious threat to the general public's health. During influenza seasons, the spread of SARS-CoV-2 and other respiratory viruses may cause a population-wide burden of respiratory disease that is difficult to manage. For that, the respiratory viruses SARS-CoV-2, Influenza A, Influenza B, and Middle East respiratory syndrome (MERS-CoV) will need to be carefully watched over in the upcoming fall and winter seasons, particularly in the case of SARS-CoV-2, Influenza A, and Influenza B, which share similar epidemiological factors like susceptible populations, mode of transmission, and clinical syndromes. Without target-specific assays, it can be challenging to differentiate among cases of these viruses owing to their similarities. Accordingly, a sensitive and targeted multiplex assay that can easily differentiate between these viral targets will be useful for healthcare practitioners. In this study, we developed a real-time reverse transcriptase-PCR-based assay utilizing an in-house developed R3T one-step RT-qPCR kit for simultaneous detection of SARS-CoV-2, Influenza A, Influenza B, and SARS-CoV-2, MERS-CoV. With as few as 10 copies of their synthetic RNAs, we can successfully identify SARS-CoV-2, Influenza A, Influenza B, and MERS-CoV targets simultaneously with 100% specificity. This assay is found to be accurate, reliable, simple, sensitive, and specific. The developed method can be used as an optimized SARS-CoV-2, Influenza A, Influenza B, and SARS-CoV-2, MERS-CoV diagnostic assay in hospitals, medical centers, and diagnostic laboratories as well as for research purposes.

Introduction

The pandemic of the ongoing coronavirus disease 2019 (COVID-19) is caused by the novel coronavirus known as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)¹. Due to SAR-CoV-2's strong contagiousness and capacity for rapid transmission, the COVID-19 pandemic emerged in Wuhan City, China, and spread quickly throughout the world. This eventually led to the start of respiratory distress signs and even death^2,3,4. COVID-19 has been declared a pandemic in more than 213 countries, expecting a steep increase in the number of confirmed case....

Protocol

1. Taq polymerase expression and purification

1. Construct a plasmid with a cleavable hexa-histidine tag at the C-terminus of the enzyme.
2. Transform 50 ng of the expression vector into E. coli BL21-(DE3) strain following the standard protocol³⁸.
3. Inoculate the transformed cells in four 6 L flasks each containing 2 L of 2YT media broth at 37 °C with shaking at 170 rpm until the OD 600 of 0.8 or cell number 6.4 x 10⁸ is reached.

Discussion

There is a heavy economic burden on the healthcare system worldwide resulting from the high infection and mortality rates due to the spread of common respiratory viruses such as SARS-CoV-2, Influenza A/B and MERS-CoV variants^12,19,20. Motivated by the sense of responsibility towards alleviating this burden, we realized the need for a quick, precise and accessible diagnostic assay such as RT-qPCR to distinguish between these comm.......

Materials

Name	Company	Catalog Number	Comments
0.45 μm filter cups	Thermo Scientific	291-4545
10X Tris-Glycine SDS running buffer	Novex	LC2675
6-well tissue culturing plates	Corning	353046
Ammonium sulfate	Fisher Scientific	A701-3
Ampicillin	Corning	61-238-RH
Cation exchange (HiTrap SP HP) 5 mL	Cytiva	17-1152-01
D-(+)-Biotin, 98+%	Thermo Scientific	A14207.60
DH10Bac competent cells	Fisher Scientific	10361012
Dialysis bag (Snakeskin 10,000 MWC)	Thermo Scientific	68100
Dithiothreitol (DTT)	Thermo Scientific	R0862
Dnase/Rnase Free Distilled Water	Ambion	AM9930
dNTPs	Thermo Scientific	R0192
E. coli BL21(DE3) competent cells	Invitrogen	C600003
EDTA	Fisher Scientific	BP120-1
Elution Buffer	Qiagen	19086
ESF 921 insect cell culture medium (Insect cells media)	Expression Systems	96-001-01
FBS Solution	Gibco	A38400-01
Fugene (transfection reagent)	Promega	E2311
Gentamicin	Fisher Scientific	15750060
Glycerol	Sigma Aldrich	G5516-500
IGEPAL CA-630	Sigma Aldrich	I8896-100ml
Imidazole	Sigma Aldrich	56750-1Kg
Influenza A (H1N1) synthetic RNA	Twist Bioscience	103001
Influenza A (H3N2)  synthetic RNA	Twist Bioscience	103002
Influenza B synthetic RNA	Twist Bioscience	103003
IPTG	Gold Biotechnology	I3481C100
Kanamycin	Gibco	11815-032
LB Agar	Fisher Scientific	BP1425-500
LB Broth media	Fisher Scientific	BP1426-500
Lysozyme	Sigma Aldrich	L6876-10G
Magnesium Chloride	Sigma Aldrich	13152-1Kg
MERS-CoV synthetic RNA	Twist Bioscience	103015
MicroAmp Fast Optical 96-well Reaction plates with Barcode (0.1 mL)	Applied Biosystems	10310855
Mini- PROTEAN TGX Precast Gel	Bio-Rad	456-1093
Miniprep kit	Qiagen	27106
Ni-NTA Excel (HisTrap Excel) 5 mL	Cytiva	17-3712-06
Ni-NTA HP (HisTrap HP) 5 mL	Cytiva	17-5248-02
Optical Adhesice Covers (PCR Compatible,DNA/Rnase/PCR Inhibitors Free	Applied Biosystems	4311971
Potassium Chloride	Fisher Bioreagents	BP366-1
Primers and Probes	Integrated DNA Technologies, Inc.
Protease Inhibitor Mini tablets EDTA-Free	Thermo Scientific	A32955
Protein marker	Fermentas	26616
RT-qPCR machine (QuantStudio 7 Flex)	Applied Biosystems
S.O.C medium	Fisher Scientific	15544034
SARS-CoV-+A2:C442 synthetic RNA	Twist Bioscience	102024
Sf9 insect cells	Gibco	A35243
Sodium Chloride	Sigma Aldrich	S3014-1Kg
StrepTrap XT 5 mL	Cytiva	29401323
Tetracycline	IBI Scientific	IB02200
Tris Base Molecular Biology Grade	Promega	H5135
Tris-HCl	Affymetrix	22676
Tween 20	Sigma Aldrich	P1379-100ml
X-Gal	Invitrogen	B1690