论文部分内容阅读
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 SARS CoV (SARS CoV) nucleic acid in clinical samples by double-site reverse transcription polymerase chain reaction (RT-PCR) and modified nested real-time RT-PCR to improve the reliability and sensitivity of the detection. Methods Thirty-three throat swab specimens of patients with suspected SARS and 27 medical observers were enrolled in this study. Real-time RT-PCR was used to detect SARS-CoV nucleic acid in 3 cases of severe acute respiratory syndrome (SARS) in Hangzhou. The amplified product was used to detect the nucleic acid sequence. At the same time, the semi-nested RT-PCR was used to detect the SARS-CoV nucleic acid fragment of the other three samples, and the real-time RT-PCR and modified real-time RT- Results The results of nested RT-PCR in 3 patients were positive in 2 cases, negative in 4 cases and 27 in medical observers. The nucleic acid sequence of the positive amplification product was completely consistent with the corresponding part of SARS-CoV genome. The nested RT-PCR and real-time RT PCR results of the other sites of the three patients were the same. Routine real-time RT-PCR showed a weak positive signal after about 35 cycles when detecting low-copy samples, but the modified nested real-time RT-PCR technique allowed strong positive signals to appear after 10 amplification cycles. Conclusion The double-site RT-PCR method is an effective method to improve the detection accuracy. The improved nested RT-PCR technique has higher sensitivity than the conventional real-time RT-PCR technique.