Detection of severe acute respiratory syndrome probable patients' virus RNA in Hangzhou by using a two loci and a modified nested real-time reverse transcription polymerase chain reaction
Ye, R.; Pan, J-cao.; Huang, Z-cheng.; Wang, H.; Wang, H-qiu.; Wei, D-fang.; Xu, K.; Wen, H-gen.; Chen, K-kai.
Zhonghua Yu Fang Yi Xue Za Zhi 39(2): 129-132
2005
ISSN/ISBN: 0253-9624 PMID: 15842838 Document Number: 585168
Objective To detect the RNA of severe acute respiratory syndrome virus (SARS-CoV) by using reverse transcription polymerase chain reaction (RT-PCR) targeted for a two loci and. a modified nested real-time RT-PCR as to improving the reliability and sensitivity of tests. Methods A nested RT-PCR was used for detecting one fragment of SARS-CoV RNA in oropharyngeal swabs from 3 SARS probable patients 4 SARS suspect patients and other 27 patients with fever in Hangzhou, and the nested RT-PCR product from one SARS probable patient was sequenced. Meanwhile in these 3 SARS probable patients, other three RT-PCR methods, including a hemi-nested RT-PCR targeted for another fragment of SARS-CoV RNA, a real-time RT-PCR and a modified nested real-time RT-PCR, were employed to detect SARS-CoV RNA. Results Two positives were found in the 3 SARS probable patients, and none positive in 4 SARS suspect patients and other 27 patients with fever, using the nested RT-PCR. The sequence of the nested RTPCR product from one SARS probable patient was identified with the counterpart of SARS-CoV genomes published in public database. The results of the hemi-nested RT-PCR, the real-time RT-PCR and the modified nested real-time RT-PCR in the 3 SARS patients were consistent with the one of the nested RT-PCR. During detecting specimen with low copies of RNA, a weak positive signal was produced after about 35 cycles in the real-time RT-PCR, but a strong positive signal was found only after 10 cycles in the modified nested real-time RT-PCR. Conclusion It might improve the reliability of test by employing RT-PCR targeted for two or more fragments in SARS-CoV genome. The modified nested real-time RT-PCR might have higher sensitivity than the routine real-time RT-PCR.