逆转录-半套式-聚合酶链反应在乙脑早期诊断中的应用  

Early diagnosis of Japanese encephalitis by RT seminested PCR

作　　者：黄庆生[1] 马文煜[1] 姜绍谆[1] 张富泉[1] 李别虎[1] 肖毅[1] 刘利兵[1] 

机构地区：[1]第四军医大学微生物学教研室

出　　处：《细胞与分子免疫学杂志》1998年第4期258-261,共4页Chinese Journal of Cellular and Molecular Immunology

摘　　要：目的：为了探讨ＲＴ－ＰＣＲ用于乙脑临床诊断的可行性。方法：根据乙脑病毒Ｅ蛋白基因区设计了３条引物，采用逆转录－半套式－聚合酶链反应（ＲＴ－ｓｅｍｉｎｅｓｔｅｄ－ＰＣＲ）方法，从实验室到临床应用上，对该方法的敏感性和特异性、临床检测的可靠性进行了探索，并与ＩｇＭＥＬＩＳＡ捕捉法进行了比较。结果：本方法所用引物的敏感性高，特异性强。在此基础上，检测了４７例临床上确诊为乙脑患者的标本（特异性ＩｇＭ阳性），４３例ＲＴ－ｓｅｍ－ｉｎｅｓｔｅｄ－ＰＣＲ结果为阳性，与ＩｇＭ检测的符合率为９１％。在２０例特异性ＩｇＭ阴性、临床上怀疑为乙脑的患者中，１２例ＲＴ－ｓｅｍｉｎｅｓｔｅｄ－ＰＣＲ结果为阳性，阳性率为６０％。结论：上述结果提示，ＲＴ－ｓｅｍｉｎｅｓｔｅｄ－ＰＣＲ用于乙脑患者的临床诊断是可行的，若与ＩｇＭＥＬＩＳＡ捕捉法相结合可进一步提高阳性率。另外，对１６例核酸检测阳性的患者进行了追踪检查，结果有１４例患者恢复期标本的ＲＮＡ检测仍为阳性，提示该病毒并未随着症状的消失而立即消失。Aim: To evaluate the possibility of RT PCR in clinical diagnosis of Japanese encephalitis (JE). Methods: A set of sense and antisense primers were synthesized (according to the sequence of JEV SA 14 RNA) to establish a RT seminested PCR for detecting Japanese encephalitis virus(JEV). We optimized various parameters of the RT PCR after several tries. Results: The results showed that the method appeared highly sensitive and specific. In further experiments with clinical specimens, 47 samples of IgM(+) JE patients, 20 samples of suspected JE patients with IgM(-) were tested by the RT PCR followed by the seminested PCR. Specific amplification was occurred in 43/47 and 12/20 of the patients . Conclusion: It suggested that RT PCR combined with seminested PCR could provide a sensitive and specific method for laboratory diagnosis of JE.

关 键 词：乙型脑炎 诊断 RT-PCR ELISA 早期诊断 

分 类 号：R512.320.4[医药卫生—内科学]