Development and Evaluation of a Novel One-Step RT-qPCR Targeting the Vero Gene for the Identification of False-Positive Results Caused by Inactivated Virus Vaccine Contamination

• Published in: Vaccines (Basel) Vol. 11; no. 2; p. 372
• Main Authors: Zheng, Xin-Qi, Yang, Wan-Bao, Xie, Lin, Wei, Zi-Han, Zhuang, Jiang-Xing, Yang, Tian-Ci
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 01.02.2023; MDPI
• Subjects: Acids; Amplification; Assaying; Coefficient of variation; Contamination; Coronaviruses; COVID-19; COVID-19 vaccines; Deactivation; Design; Disease transmission; false positive; False positive reactions; Genes; Hospitals; Identification and classification; Immunization; inactivated virus vaccine; Infections; Performance evaluation; RT-qPCR; SARS-CoV-2; Sensitivity; Severe acute respiratory syndrome coronavirus 2; Vaccines; Vero gene; Viral diseases; Viral infections; China; SARS-CoV-2; inactivated virus vaccine; RT-qPCR; false positive; Vero gene
• DOI: 10.3390/vaccines11020372
• ISSN: 2076-393X

To identify false-positive SARS-CoV-2 test results caused by novel coronavirus inactivated vaccine contamination, a novel RT-qPCR targeting the ORF1ab and N genes of SARS-CoV-2 and Vero gene was developed. The amplification efficiency, precision, and lower limit of detection (LLOD) of the RT-qPCR assay were determined. A total of 346 clinical samples and 132 environmental samples were assessed, and the diagnostic performance was evaluated. The results showed that the amplification efficiency of the ORF1ab, N, and Vero genes was 95%, 97%, and 93%, respectively. The coefficients of variation of Ct values at a concentration of 3 × 10 copies/mL were lower than 5%. The LLOD for the ORF1ab, N, and Vero genes reached 8.0, 3.3, and 8.2 copies/reaction, respectively. For the 346 clinical samples, our RT-qPCR assay identified SARS-CoV-2-positive and SARS-CoV-2-negative samples with a sensitivity of 100.00% and a specificity of 99.30% and novel coronavirus inactivated vaccine-contaminated samples with a sensitivity of 100% and a specificity of 100%. For the environmental samples, our RT-qPCR assay identified novel coronavirus inactivated vaccine-contaminated samples with a sensitivity of 88.06% and a specificity of 95.38%. In conclusion, the RT-qPCR assay we established can be used to diagnose COVID-19 and, to a certain extent, false-positive results due to vaccine contamination.