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[Establishment and preliminary application of quantitative real-time PCR assay for the detection of SARS-CoV-2 subgenomic nucleocapsid RNA].
Zhu, X J; Chen, Y; Wu, B; Ge, Y Y; Wu, T; Qiao, Q; Zhao, K C; Cui, L B.
  • Zhu XJ; NHC Key laboratory of Enteric Pathogenic Microbiology/Institute of Pathogenic Microbiology, Jiangsu Provincial Center for Disease Control and Prevention/Jiangsu Province Engineering Research Center of Health Emergency, Nanjing 210009, China.
  • Chen Y; NHC Key laboratory of Enteric Pathogenic Microbiology/Institute of Pathogenic Microbiology, Jiangsu Provincial Center for Disease Control and Prevention/Jiangsu Province Engineering Research Center of Health Emergency, Nanjing 210009, China.
  • Wu B; NHC Key laboratory of Enteric Pathogenic Microbiology/Institute of Pathogenic Microbiology, Jiangsu Provincial Center for Disease Control and Prevention/Jiangsu Province Engineering Research Center of Health Emergency, Nanjing 210009, China.
  • Ge YY; NHC Key laboratory of Enteric Pathogenic Microbiology/Institute of Pathogenic Microbiology, Jiangsu Provincial Center for Disease Control and Prevention/Jiangsu Province Engineering Research Center of Health Emergency, Nanjing 210009, China.
  • Wu T; NHC Key laboratory of Enteric Pathogenic Microbiology/Institute of Pathogenic Microbiology, Jiangsu Provincial Center for Disease Control and Prevention/Jiangsu Province Engineering Research Center of Health Emergency, Nanjing 210009, China.
  • Qiao Q; NHC Key laboratory of Enteric Pathogenic Microbiology/Institute of Pathogenic Microbiology, Jiangsu Provincial Center for Disease Control and Prevention/Jiangsu Province Engineering Research Center of Health Emergency, Nanjing 210009, China.
  • Zhao KC; NHC Key laboratory of Enteric Pathogenic Microbiology/Institute of Pathogenic Microbiology, Jiangsu Provincial Center for Disease Control and Prevention/Jiangsu Province Engineering Research Center of Health Emergency, Nanjing 210009, China.
  • Cui LB; NHC Key laboratory of Enteric Pathogenic Microbiology/Institute of Pathogenic Microbiology, Jiangsu Provincial Center for Disease Control and Prevention/Jiangsu Province Engineering Research Center of Health Emergency, Nanjing 210009, China.
Zhonghua Yu Fang Yi Xue Za Zhi ; 57(2): 268-272, 2023 Feb 06.
Article in Chinese | MEDLINE | ID: covidwho-2289052
ABSTRACT

Objective:

To establish a rapid and specific quantitative real-time PCR (qPCR) method for the detection of SARS-CoV-2 subgenomic nucleocapsid RNA (SgN) in patients with COVID-19 or environmental samples.

Methods:

The qPCR assay was established by designing specific primers and TaqMan probe based on the SARS-CoV-2 genomic sequence in Global Initiative of Sharing All Influenza Data (GISAID) database. The reaction conditions were optimized by using different annealing temperature, different primers and probe concentrations and the standard curve was established. Further, the specificity, sensitivity and repeatability were also assessed. The established SgN and genomic RNA (gRNA) qPCR assays were both applied to detect 21 environmental samples and 351 clinical samples containing 48 recovered patients. In the specimens with both positive gRNA and positive SgN, 25 specimens were inoculated on cells.

Results:

The primers and probes of SgN had good specificity for SARS-CoV-2. The minimum detection limit of the preliminarily established qPCR detection method for SgN was 1.5×102 copies/ml, with a coefficient of variation less than 1%. The positive rate of gRNA in 372 samples was 97.04% (361/372). The positive rates of SgN in positive environmental samples and positive clinical samples were 36.84% (7/19) and 49.42% (169/342), respectively. The positive rate and copy number of SgN in Wild strain were lower than those of SARS-CoV-2 Delta strain. Among the 25 SgN positive samples, 12 samples within 5 days of sampling time were all isolated with virus; 13 samples sampled for more than 12 days had no cytopathic effect.

Conclusion:

A qPCR method for the detection of SARS-CoV-2 SgN has been successfully established. The sensitivity, specificity and repeatability of this method are good.
Subject(s)

Full text: Available Collection: International databases Database: MEDLINE Main subject: SARS-CoV-2 / COVID-19 Type of study: Diagnostic study / Prognostic study Limits: Humans Language: Chinese Journal: Zhonghua Yu Fang Yi Xue Za Zhi Year: 2023 Document Type: Article Affiliation country: Cma.j.cn112150-20220407-00331

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Full text: Available Collection: International databases Database: MEDLINE Main subject: SARS-CoV-2 / COVID-19 Type of study: Diagnostic study / Prognostic study Limits: Humans Language: Chinese Journal: Zhonghua Yu Fang Yi Xue Za Zhi Year: 2023 Document Type: Article Affiliation country: Cma.j.cn112150-20220407-00331