改良分子信标-双重实时荧光PCR快速检测SARS病毒

Modified molecular beacon-based dual real-time PCR for detection of SARS virus and its application

目的建立改良分子信标-双重实时荧光PCR检测SARS病毒的方法,用于SARS的早期诊断和动物溯源。方法利用改良分子信标技术、装甲RNA和双片段双色荧光技术,根据GenBank公布的SARS病毒聚合酶基因1b的阅读开放框架结构的保守序列,自行设计一对引物和探针,以部分临床标本的酶联吸附实验结果和传统细胞培养方法作为对照,建立分子信标检测SARS病毒的方法。对368份临床标本(咽漱液、血液、粪便、尿液)、52份细胞培养液和50份动物标本进行荧光PCR扩增。结果分子信标检测SARS病毒的方法灵敏度为10～100个拷贝ml,与流感病毒等呼吸道病毒无交叉反应。分子信标检测368份临床标本,20份阳性。其中确诊病例阳性率为21.27%(1047),确诊病例的咽漱液阳性率为43.48%,还分别从粪便和血清中检测到SARS病毒。52份细胞培养液,29份阳性,阳性率为55.77%。50份动物标本,23份阳性,阳性率为46%。结论改良分子信标-双重实时荧光PCR检测SARS病毒方法灵敏度高、特异性强,可用于SARS的临床早期诊断和动物溯源。

Objective\ To develop the modified molecular beacon-based dual fluorescent PCR assays for detection of SARS virus. The assay was applied to the early clinic diagnosis & animal tracking. Methods On the basis of the obtained core sequence of open reading frame 1b of the coronavirus polymerase gene sequences, which was published in GenBank, using modified molecular beacon probe, artifical virus techinique and two different fragments amplification with different fluoresce, one set of primers and probe were designed. Then fluorescent PCR assays for specific and sensitive detection of the SARS virus was established, while the ELISA & the traditional method were used as control. 368 clinical specimens such as the throat swab, serum, feces, and urine from different cases, 52 cell cultures and 50 animal specimens were detected by the molecular beacon-based PCR.Results The sensitivity of real -time PCR was 10-100 copies/ml, there was no cross reaction to other respiratory viruses such as influenza virus etc. Of 368 specimens, 20 were positive by using molecular beacon-based fluorescence PCR. The positive rate of SARS case ~10/47) were 21.27%, the positive rate of the throat swab of SARS cases ~10/23) were 43.87%. Among 52 SARS cell cultures, 29 were positive. The positive rate of SARS cell cultures was 55.77%. Of 50 animal specimens, 23 were positive. The positive rate was 46%. Furthermore, SARS virus RNA was detected in feces and in serum during the acute phase. Conclusion The molecular beacon-based PCR is sensitive and specific, it could be applied to the early diagnosis and animal tracking. This molecular beacon-based PCR kit is useful for the different units.