Culturing Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) for Diagnosis and Genome Sequencing

Author:

Zeng Zhiqi1,Guo Hua2,Chen Liping1,Lin Zhengshi1,Guan Wenda1,Wang Yutao1,Jiang Haiming1,Wu Xiao1,Yin Yong2,Gao Zelong2,Chen Canxiong1,Yang Zifeng13

Affiliation:

1. State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, the First Affiliated Hospital of Guangzhou Medical University , Guangzhou , China

2. Zhuhai Baso Diagnostics , Zhuhai , China

3. Guangzhou Key Laboratory for Clinical Rapid Diagnosis and Early Warning of Infectious Diseases , Guangzhou , China

Abstract

Abstract Objective The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid detection “re-positive” phenomenon is encountered clinically. The accuracy of a viral nucleic acid test is crucial to prevent reintroduction of the virus into the community. This study evaluated the effect of virus culturing on increasing the sensitivity and specificity of real-time polymerase chain reaction (RT-PCR) detection and viral genomic sequencing. Methods A series of tenfold dilutions of a SARS-CoV-2 viral stock were conducted and cultured for either 24 or 48 hours. The viral load of cultured samples was determined by RT-PCR. The cultured and non-cultured samples of 1x 50% tissue culture infectious dose (TCID50) were sequenced using metagenomic next-generation sequencing. The depth and coverage of SARS-CoV-2 genome were measured. Results The lowest viral load detectable in a sample with RT-PCR was 0.01 TCID50. After a 24-h culture, the viral ORF 1ab and N-gene cycle threshold (CT) values were reduced by 4.4 points and 1 point, respectively. One TCID50 viral load of post 24-h culture revealed the sequence depth reached an average of 752 reads, compared with 0.15 in the nonculture; furthermore, the coverage was 99.99% while 6.42% in the nonculture. Conclusion These results indicate that virus culturing can significantly increase the viral load, which can increase the certainty of true-positive detection of the viral nucleic acids, and improve the quality of virus genomic sequencing.

Funder

National Key Research and Development Program of China

Zhongnanshan Medical Foundation of Guangdong Province

Science and Technology Program of Foshan

Science and Technology Program of Guangzhou

China Evergrande

National Natural Science Foundation of Guangdong

National Natural Science Foundation of Guizhou

Science and Technology Bureau Project of Dongguan

Key Research and Development Program of Guangdong Province

Publisher

Oxford University Press (OUP)

Subject

Biochemistry (medical),Clinical Biochemistry

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