Tags

Type your tag names separated by a space and hit enter

Rapid determination of infectious SARS-CoV-2 in PCR-positive samples by SDS-PMA assisted RT-qPCR.
Sci Total Environ. 2021 Jul 17; 797:149085.ST

Abstract

The ongoing COVID-19 pandemic has generated a global health crisis that needs well management of not only patients but also environments to reduce SARS-CoV-2 transmission. The gold standard RT-qPCR method is sensitive and rapid to detect SARS-CoV-2 nucleic acid, but does not answer if PCR-positive samples contain infectious virions. To circumvent this problem, we report an SDS-propidium monoazide (PMA) assisted RT-qPCR method that enables rapid discrimination of live and dead SARS-CoV-2 within 3 h. PMA, a photo-reactive dye, can react with viral RNA released or inside inactivated SARS-CoV-2 virions under assistance of 0.005% SDS, but not viral RNA inside live virions. Formation of PMA-RNA conjugates prevents PCR amplification, leaving only infectious virions to be detected. Under optimum conditions, RT-qPCR detection of heat-inactivated SARS-CoV-2 resulted in larger than 9 Ct value differences between PMA-treated and PMA-free groups, while less than 0.5 Ct differences were observed in the detection of infectious SARS-CoV-2 ranging from 20 to 5148 viral particles. Using a cutoff Ct difference of 8.6, this method could differentiate as low as 8 PFU live viruses in the mixtures of live and heat-inactivated virions. Further experiments showed that this method could successfully monitor the natural inactivation process of SARS-CoV-2 on plastic surfaces during storage with comparable results to the gold standard plaque assay. We believe that the culture-free method established here could be used for rapid and convenient determination of infectious SARS-CoV-2 virions in PCR-positive samples, which will facilitate better control of SARS-CoV-2 transmission.

Authors+Show Affiliations

CAS Key Laboratory of Emerging Pathogens and Biosafety, Centre for Biosafety Mega-Sciences, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China; University of Chinese Academy of Sciences, Beijing 100049, China.CAS Key Laboratory of Emerging Pathogens and Biosafety, Centre for Biosafety Mega-Sciences, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.CAS Key Laboratory of Emerging Pathogens and Biosafety, Centre for Biosafety Mega-Sciences, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China; University of Chinese Academy of Sciences, Beijing 100049, China.CAS Key Laboratory of Emerging Pathogens and Biosafety, Centre for Biosafety Mega-Sciences, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.CAS Key Laboratory of Emerging Pathogens and Biosafety, Centre for Biosafety Mega-Sciences, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China; University of Chinese Academy of Sciences, Beijing 100049, China.CAS Key Laboratory of Emerging Pathogens and Biosafety, Centre for Biosafety Mega-Sciences, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China; University of Chinese Academy of Sciences, Beijing 100049, China.CAS Key Laboratory of Emerging Pathogens and Biosafety, Centre for Biosafety Mega-Sciences, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China; University of Chinese Academy of Sciences, Beijing 100049, China.CAS Key Laboratory of Emerging Pathogens and Biosafety, Centre for Biosafety Mega-Sciences, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China; University of Chinese Academy of Sciences, Beijing 100049, China. Electronic address: yangh@wh.iov.cn.CAS Key Laboratory of Emerging Pathogens and Biosafety, Centre for Biosafety Mega-Sciences, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China; University of Chinese Academy of Sciences, Beijing 100049, China. Electronic address: hpwei@wh.iov.cn.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

34293609

Citation

Hong, Wei, et al. "Rapid Determination of Infectious SARS-CoV-2 in PCR-positive Samples By SDS-PMA Assisted RT-qPCR." The Science of the Total Environment, vol. 797, 2021, p. 149085.
Hong W, Xiong J, Nyaruaba R, et al. Rapid determination of infectious SARS-CoV-2 in PCR-positive samples by SDS-PMA assisted RT-qPCR. Sci Total Environ. 2021;797:149085.
Hong, W., Xiong, J., Nyaruaba, R., Li, J., Muturi, E., Liu, H., Yu, J., Yang, H., & Wei, H. (2021). Rapid determination of infectious SARS-CoV-2 in PCR-positive samples by SDS-PMA assisted RT-qPCR. The Science of the Total Environment, 797, 149085. https://doi.org/10.1016/j.scitotenv.2021.149085
Hong W, et al. Rapid Determination of Infectious SARS-CoV-2 in PCR-positive Samples By SDS-PMA Assisted RT-qPCR. Sci Total Environ. 2021 Jul 17;797:149085. PubMed PMID: 34293609.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Rapid determination of infectious SARS-CoV-2 in PCR-positive samples by SDS-PMA assisted RT-qPCR. AU - Hong,Wei, AU - Xiong,Jin, AU - Nyaruaba,Raphael, AU - Li,Junhua, AU - Muturi,Elishiba, AU - Liu,Huan, AU - Yu,Junping, AU - Yang,Hang, AU - Wei,Hongping, Y1 - 2021/07/17/ PY - 2021/05/11/received PY - 2021/07/05/revised PY - 2021/07/13/accepted PY - 2021/7/23/pubmed PY - 2021/7/23/medline PY - 2021/7/22/entrez KW - COVID-19 KW - Inactivation KW - Infectious particles KW - PMA KW - RT-qPCR KW - SARS-CoV-2 SP - 149085 EP - 149085 JF - The Science of the total environment JO - Sci Total Environ VL - 797 N2 - The ongoing COVID-19 pandemic has generated a global health crisis that needs well management of not only patients but also environments to reduce SARS-CoV-2 transmission. The gold standard RT-qPCR method is sensitive and rapid to detect SARS-CoV-2 nucleic acid, but does not answer if PCR-positive samples contain infectious virions. To circumvent this problem, we report an SDS-propidium monoazide (PMA) assisted RT-qPCR method that enables rapid discrimination of live and dead SARS-CoV-2 within 3 h. PMA, a photo-reactive dye, can react with viral RNA released or inside inactivated SARS-CoV-2 virions under assistance of 0.005% SDS, but not viral RNA inside live virions. Formation of PMA-RNA conjugates prevents PCR amplification, leaving only infectious virions to be detected. Under optimum conditions, RT-qPCR detection of heat-inactivated SARS-CoV-2 resulted in larger than 9 Ct value differences between PMA-treated and PMA-free groups, while less than 0.5 Ct differences were observed in the detection of infectious SARS-CoV-2 ranging from 20 to 5148 viral particles. Using a cutoff Ct difference of 8.6, this method could differentiate as low as 8 PFU live viruses in the mixtures of live and heat-inactivated virions. Further experiments showed that this method could successfully monitor the natural inactivation process of SARS-CoV-2 on plastic surfaces during storage with comparable results to the gold standard plaque assay. We believe that the culture-free method established here could be used for rapid and convenient determination of infectious SARS-CoV-2 virions in PCR-positive samples, which will facilitate better control of SARS-CoV-2 transmission. SN - 1879-1026 UR - https://www.unboundmedicine.com/medline/citation/34293609/Rapid_determination_of_infectious_SARS-CoV-2_in_PCR-positive_samples_by_SDS-PMA_assisted_RT-qPCR. L2 - https://linkinghub.elsevier.com/retrieve/pii/S0048-9697(21)04157-7 DB - PRIME DP - Unbound Medicine ER -
Try the Free App:
Prime PubMed app for iOS iPhone iPad
Prime PubMed app for Android
Prime PubMed is provided
free to individuals by:
Unbound Medicine.