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目的建立辛德毕斯病毒核酸的SYBRGREENΙ荧光PCR检测方法。方法根据辛德毕斯病毒基因组核苷酸序列特性设计引物。病毒在BHK21细胞上繁殖扩增后提取RNA和逆转录。以病毒cDNA为模板分别进行SYBRGREENΙ荧光PCR和常规RTPCR扩增,并对SYBRGREENΙPCR法的灵敏性、特异性、重复性等进行分析。结果最适退火温度为55℃,最适引物浓度为0.5μmol/L。用该方法检测2株SIN病毒株YN87448和XJ160结果均为阳性,而对其他虫媒病毒如甲病毒属Geta病毒、乙脑病毒、Batai病毒、Banna病毒、环状病毒及西方马脑炎病毒合成模板检测时结果均为阴性。根据病毒空斑形成实验结果,将YN87448病毒悬液进行连续10倍稀释后分别用常规PCR和SYBRGREENΙ荧光PCR方法进行检测。结果SYBRGREENΙ荧光PCR检测敏感性比常规PCR方法要高近100倍,检出下限可达0.1PFU/ml。对模拟感染的人血清标本检测结果表明人血清中成分对检测体系无明显影响。对151份不明原因发热和病毒性脑炎患者的血清或脑脊液标本进行检测,结果检测到6份标本阳性。结论本实验建立了辛德毕斯病毒特异性核酸的SYBRGREENΙ荧光PCR检测方法,实验结果显示了较好的特异性、广谱性,初步证实可应用于临床标本的检测,为将来用于临床和调查辛德毕斯病毒在我国的流行情况提供了新的技术手段。
Objective To establish a SYBRGREEN 1 fluorescent PCR assay for Sindbis virus nucleic acid. Methods Primers were designed according to the nucleotide sequence of Sindbis virus. The virus is propagated on BHK21 cells and amplified after RNA extraction and reverse transcription. SYBRGREEN I fluorescence PCR and routine RTPCR amplification were performed using the viral cDNA as a template, respectively, and the sensitivity, specificity, repeatability and the like of the SYBRGREEN I PCR method were analyzed. The optimum annealing temperature was 55 ℃, and the optimal primer concentration was 0.5 μmol / L. The two strains of SIN virus YN87448 and XJ160 were positive by this method, while others were positive for other Arbovirus such as alphavirus Geta virus, Japanese encephalitis virus, Batai virus, Banna virus, circular virus and western equine encephalitis virus The results of the template test were negative. According to the experimental results of virus plaque formation, the suspension of YN87448 virus was serially diluted by 10 times and tested by conventional PCR and SYBRGREEN 1 fluorescent PCR respectively. Results The sensitivity of SYBRGREEN 1 fluorescence PCR was 100 times higher than that of conventional PCR, and the detection limit was up to 0.1 PFU / ml. The results of the human serum samples mock-infected showed that the composition of human serum had no significant effect on the test system. Serum or cerebrospinal fluid samples from 151 patients with unexplained fever and viral encephalitis were tested, and 6 samples were positive. Conclusion The SYBRGREENI fluorescent PCR detection method of Sindbis virus-specific nucleic acid was established in this experiment. The experimental results showed good specificity and broad spectrum. It was initially confirmed that it could be used in the detection of clinical specimens for future clinical and investigational studies. The virus in our country has provided a new epidemic of technical means.