用逆转录聚合酶链反应方法检测SARS冠状病毒

Detection of severe acute respiratory syndrome probable patients’ virus RNA in Hangzhou by using a two loci and a modified nested rea l-time reverse transcription polymerase chain reaction

目的 应用双位点逆转录聚合酶链反应 (RT-PCR)和改良巢式实时RT PCR方法检测临床标本中SARS冠状病毒(SARS CoV)核酸,以提高检测的可靠性和灵敏度。方法 对 2003年杭州市 3例严重急性呼吸综合征(SARS)临床确诊患者、4例疑似和 27名医学观察者的咽拭子标本用巢式实时RT-PCR检测SARS- CoV核酸,对阳性扩增产物进行核酸序列测定,同时对 3例患者的标本应用半巢式RT -PCR检测另一位点SARS- CoV核酸片段,并应用常规实时RT- PCR技术及改良实时RT -PCR技术进行检测。结果 3例患者的巢式RT -PCR检测结果 2例阳性, 4例疑似和 27名医学观察者均为阴性;阳性扩增产物的核酸序列与SARS- CoV基因组相应部分序列完全一致。3例患者标本的另一位点的半巢式RT -PCR及实时RT PCR结果均相同。在检测低拷贝数标本时,常规实时RT- PCR技术在约 35个循环后出现弱阳性信号,但改良的巢式实时RT -PCR技术可使强阳性信号在 10个扩增循环后出现。结论 双位点RT- PCR方法是提高检测准确性的有效方法,改良巢式实时RT- PCR技术较常规实时RT- PCR技术具有更高的灵敏度。

Objective To detect the RN A of severe acute respiratory syndrome virus (SARS-CoV) by using reverse transcri ption 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 t ests.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-Co V RNA.Results Two positives were found in the 3 SARS pro bable patients, and none positive in 4 SARS suspect patients and other 27 patien ts with fever, using the nested RT-PCR.The sequence of the nested RT-PCR pro duct 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 dete cting 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 fou nd only after 10 cycles in the modified nested real-time RT-PCR.Co nclusion It might improve the reliability of test by employing RT-P CR 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.