RT-PCR (Real time -PCR) in Coronavirus Disease 2019 (COVID-19) test

View Knowledge Base - Landscape Coronavirus Disease 2019 test (COVID-19 test) in vitro -- A comparison of PCR vs Immunoassay vs Crispr-Based test>>

Author:
Huajun Bai¹ Xiaolong Cai^{1, 2*} Xiaoyan Zhang¹
1. R&D Center, GeneMedi Co.Ltd., Shanghai, P.R. China (www.genemedi.net)
2. Hanbio Research Center, Hanbio Tech Co. Ltd., Shanghai, P.R. China (www.hanbio.net)

Abstract:
The outbreak of COVID-19, caused by 2019 novel coronavirus (2019-nCoV), has been a global public health threat and caught the worldwide concern. Scientists throughout the world are sparing all efforts to explore strategies for the determination of the 2019-nCoV virus and diagnosis of COVID-19 rapidly. Several assays are developed for COVID-19 test , including RT-PCR, coronavirus antigens-based immunoassays, and CRISPR-based strategies (Cas13a or Cas12a), etc. Different assays have their advantages and drawbacks, and people should choose the most suitable assay according to their demands. Here, we make a brief introduction about these assays and give a simple overview of them, hoping to help doctors and researchers to select the most suitable assay for the Coronavirus Disease 2019 test (COVID-19 test) .

RT-PCR (Real time -PCR) in Coronavirus Disease 2019 (COVID-19) test

1) Principles for diagnostics

Figure 2. Principle of qRT-PCR.

2) Advantages and disadvantages

Advantages:
① High sensitivity.
② Easy to be operated on a large scale.

Disadvantages:
① Professional technician and special apparatus are required to perform RT-PCR experiments and analyze the data, which costs so much.
② The standard positive control affects the experimental accuracy, and a false negative result may occur due to improper handling.

3) Optional Targets, primers, and probes from different departments

Different departments have their own systems for the determination of COVID-19 by targeting various open reading frames of SARS-CoV-2, which are shown as follows. Primers and probes of Orf1ab and N (nucleocapsid gene) are commonly used for COVID-19 test in China. Three pairs of primers and probes of N (nucleocapsid gene, N1, N2, N3) are applied in the US CDC for COVID-19. And primers and probes of E (envelope gene) are utilized for COVID-19 test in Europe.

Organisation	Target	Forward primer (5’-3’)	Reverse primer (5’-3’)	Probe (5’-3’)
University of Hong Kong, Beijing Center for Disease Prevention and Control, Capital Medical University Beijing Research Center for Preventive Medicine	orf1b	TGGGGYTTTACRGGTAACCT (Forward; Y=C/T, R=A/G)	AACRCGCTTAACAAAGCACTC (Reverse; R=A/G)	TAGTTGTGATGCWATCATGACTAG (Probe in 5’-FAM/ZEN/3’-IBFQ format; W=A/T)
	N	TAATCAGACAAGGAACTGATTA	CGAAGGTGTGACTTCCATG	FAM-GCAAATTGTGCAATTTGCGG-IBFQ
Charité Virology, Berlin, Germany; Olfert Landt, Tib-Molbiol, Berlin, Germany; Erasmus MC, Rotterdam, The Netherlands; Public Health England, London	RdRP	GTGARATGGTCATGT GTGGCGG	CARATGTTAAASACA CTATTAGCATA	FAM-CAGGTGGA ACCTCATCAGGA GATGCBBQ
	E gene	ACAGGTACGTTAATA GTTAATAGCGT	ATATTGCAGCAGTAC GCACACA	FAM-ACACTAGC CATCCTTACTGC GCTTCGBBQ
	N	CACATTGGCACCCGC AATC	GAGGAACGAGAAGA GGCTTG	FAM-ACTTCCTCA AGGAACAACATT GCCABBQ
The US Centers for Disease Control and Prevention (CDC), Integrated DNA Technologies	N1	GACCCCAAAATCAGC GAAAT	TCTGGTTACTGCCAG TTG AATCTG	FAM-ACCCCGCA T TACGTTTGGTGG ACC-BHQ1
	N2	TTACAAACATTGGCCGCAAA	GCGCGACATTCCGAAGAA	FAM-ACAATTTGCCCCCAGCGCTTCAG-BHQ1
	N3	GGGAGCCTTGAATACACCAAAA	TGTAGCACGATTGCAGCATTG	FAM-AYCACATTGGCACCCGCAATCCTG-BHQ1
	RNAse P	AGATTTGGACCTGCGAGCG	GAGCGGCTGTCTCCACAAGT	FAM–TTCTGACCTGAAGGCTCTGCGCG–BHQ-1

Table 1. Optional Targets, primers, and probes from different departments

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RT-PCR (Real time -PCR) in Coronavirus Disease 2019 (COVID-19) test

RT-PCR (Real time -PCR) in Coronavirus Disease 2019 (COVID-19) test

Reference

Collection of COVID-19 landscape knowledge base

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