Detection of SARS-CoV-2 RNA by direct RT-qPCR on nasopharyngeal specimens without extraction of viral RNA

被引:54
作者
Hasan, Mohammad Rubayet [1 ,2 ]
Mirza, Faheem [1 ]
Al-Hail, Hamad [1 ]
Sundararaju, Sathyavathi [1 ]
Xaba, Thabisile [1 ]
Iqbal, Muhammad [1 ]
Alhussain, Hashim [3 ]
Yassine, Hadi Mohamad [3 ]
Perez-Lopez, Andres [1 ,2 ]
Tang, Patrick [1 ,2 ]
机构
[1] Sidra Med, Dept Pathol, Doha, Qatar
[2] Weill Cornell Med Coll Qatar, Doha, Qatar
[3] Qatar Univ, Doha, Qatar
来源
PLOS ONE | 2020年 / 15卷 / 07期
关键词
PCR;
D O I
10.1371/journal.pone.0236564
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
To circumvent the limited availability of RNA extraction reagents, we aimed to develop a protocol for direct RT-qPCR to detect SARS-CoV-2 in nasopharyngeal swabs without RNA extraction. Nasopharyngeal specimens positive for SARS-CoV-2 and other coronaviruses collected in universal viral transport (UVT) medium were pre-processed by several commercial and laboratory-developed methods and tested by RT-qPCR assays without RNA extraction using different RT-qPCR master mixes. The results were compared to that of standard approach that involves RNA extraction. Incubation of specimens at 65 degrees C for 10 minutes along with the use of TaqPathT 1-Step RT-qPCR Master Mix provides higher analytical sensitivity for detection of SARS-CoV-2 RNA than many other conditions tested. The optimized direct RT-qPCR approach demonstrated a limit of detection of 6.6x10(3) copy/ml and high reproducibility (co-efficient of variation = 1.2%). In 132 nasopharyngeal specimens submitted for SARS-CoV-2 testing, the sensitivity, specificity and accuracy of our optimized approach were 95%, 99% and 98.5%, respectively, with reference to the standard approach. Also, the RT-qPCR C-T values obtained by the two methods were positively correlated (Pearson correlation coefficient r = 0.6971, p = 0.0013). The rate of PCR inhibition by the direct approach was 8% compared to 9% by the standard approach. Our simple approach to detect SARS-CoV-2 RNA by direct RT-qPCR may help laboratories continue testing for the virus despite reagent shortages or expand their testing capacity in resource limited settings.
引用
收藏
页数:9
相关论文
共 50 条
[41]   Comparison of RT-dPCR and RT-qPCR and the effects of freeze-thaw cycle and glycine release buffer for wastewater SARS-CoV-2 analysis [J].
Huge, Bonnie Jaskowski ;
North, Devin ;
Mousseau, C. Bruce ;
Bibby, Kyle ;
Dovichi, Norman J. ;
Champion, Matthew M. .
SCIENTIFIC REPORTS, 2022, 12 (01)
[42]   Development of a robust TaqMan probe-based one-step multiplex RT-qPCR for simultaneous detection of SARS-CoV-2 and Influenza A/B viruses [J].
Abbasi, Hamidreza ;
Nikoo, Hadi Razavi ;
Fotouhi, Fatemeh ;
Khosravi, Ayyoob .
BMC MICROBIOLOGY, 2023, 23 (01)
[43]   A SARS-CoV-2 Negative Antigen Rapid Diagnostic in RT-qPCR Positive Samples Correlates With a Low Likelihood of Infectious Viruses in the Nasopharynx [J].
Correa, Isadora Alonso ;
Faffe, Debora Souza ;
Galliez, Rafael Mello ;
Goncalves, Cassia Cristina Alves ;
Maia, Richard Araujo ;
da Silva, Gustavo Peixoto ;
Moreira, Filipe Romero Rebello ;
Mariani, Diana ;
Campos, Mariana Freire ;
Leitao, Isabela de Carvalho ;
de Souza, Marcos Romario ;
Cunha, Marcela Sabino ;
Nascimento, erica Ramos dos Santos ;
Ribeiro, Liane de Jesus ;
da Cruz, Thais Felix Cordeiro ;
Policarpo, Cintia ;
Gonzales, Luis ;
Rodgers, Mary A. ;
Berg, Michael ;
Vijesurier, Roy ;
Cloherty, Gavin A. ;
Hackett, John, Jr. ;
Ferreira, Orlando da Costa ;
Castineiras, Terezinha Marta Pereira Pinto ;
Tanuri, Amilcar ;
da Costa, Luciana Jesus .
FRONTIERS IN MICROBIOLOGY, 2022, 13
[44]   SARS-CoV-2 RT-qPCR testing of pooled saliva samples: A case study of 824 asymptomatic individuals and a questionnaire survey in Japan [J].
Oba, Junna ;
Taniguchi, Hiroaki ;
Sato, Masae ;
Takanashi, Masaki ;
Yokemura, Moe ;
Sato, Yasunori ;
Nishihara, Hiroshi .
PLOS ONE, 2022, 17 (05)
[45]   Comparison between RT-qPCR for SARS-CoV-2 and expanded triage in sputum of symptomatic and asymptomatic COVID-19 subjects in Ecuador [J].
Torres, Ariel ;
Fors, Martha ;
Rivero, Tamaris ;
Pantoja, Karina ;
Ballaz, Santiago .
BMC INFECTIOUS DISEASES, 2021, 21 (01)
[46]   Factors associated with weak positive SARS-CoV-2 diagnosis by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) [J].
Rawat, Priyank ;
Zerbato, Jennifer M. ;
Rhodes, Ajantha ;
Chiu, Chris ;
Tran, Thomas ;
Rasmussen, Thomas A. ;
Druce, Julian ;
Lewin, Sharon R. ;
Roche, Michael .
PATHOLOGY, 2022, 54 (05) :623-628
[47]   RT-LAMP assay combining multi-fluorescent probes for SARS-CoV-2 RNA detection and variant differentiation [J].
Talap, Jadera ;
Shen, Minzhe ;
Yu, Lushan ;
Zeng, Su ;
Cai, Sheng .
TALANTA, 2022, 248
[48]   Post-lockdown detection of SARS-CoV-2 RNA in the wastewater of Montpellier, France [J].
Trottier, Julie ;
Darques, Regis ;
Mouheb, Nassim Ait ;
Partiot, Emma ;
Bakhache, William ;
Deffieu, Maika S. ;
Gaudin, Raphael .
ONE HEALTH, 2020, 10
[49]   Alternative detection of SARS-CoV-2 RNA by a new assay based on mass spectrometry [J].
Stelzl, Evelyn ;
Kessler, Harald H. ;
Mustafa, Hans G. ;
Mustafa, Maria E. ;
Santner, Brigitte, I ;
Seier, Josef ;
La Torre, Marco ;
Haushofer, Alexander C. .
CLINICAL CHEMISTRY AND LABORATORY MEDICINE, 2021, 59 (12) :1998-2002
[50]   Comparison of analytical sensitivity and efficiency for SARS-CoV-2 primer sets by TaqMan-based and SYBR Green-based RT-qPCR [J].
Tao, Yile ;
Yue, Yang ;
Qiu, Guangyu ;
Ji, Zheng ;
Spillman, Martin ;
Gai, Zhibo ;
Chen, Qingfa ;
Bielecki, Michel ;
Huber, Michael ;
Trkola, Alexandra ;
Wang, Qiyuan ;
Cao, Junji ;
Wang, Jing .
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 2022, 106 (5-6) :2207-2218