套式逆转录PCR检测戊型肝炎病毒RNA的临床应用

Detection of HEV RNA by Nested RT-PCR and its clinical significance

目的评价套式逆转录PCR检测血清戊肝病毒(HEV)RNA的临床意义。方法对GenBank数据库中的戊型肝炎病毒全基因组序列进行分析比对,并针对国内流行的HEV株序列,选择位于ORF2的保守区域设计引物,建立套式逆转录聚合酶链式反应(nRT-PCR)方法,并检测126例急性戊型肝炎患者,并与检测抗-HEV IgM的方法进行比较。结果 126例急性戊型肝炎患者血清中有67例HEV-RNA阳性,对照组血清HEV-RNA检测均为阴性;与血清抗-HEV IgM检测结果比较,HEV-RNA检测的总符合率为80.91%,两种方法的检测结果具有良好的一致性;nRT-PCR方法检测HEV-RNA与血清抗-HEV IgM检测方法存在明显的差异,不能相互替代,而有一定的互补性;3例患者的首份血清检测为HEV-RNA阳性,但抗-HEV IgM为阴性,系列追踪检测,均相继出现抗-HEV IgM,急性戊型肝炎患者血清HEV-RNA的检出多在发病的1～12d。结论应用nRT-PCR方法能对急性散发戊型肝炎患者血清中的HEV RNA进行定性检测,且有较高的特异性和敏感性,临床使用可以提高对HEV早期诊断的水平,具有一定的临床应用价值。

OBJECTIVE To investigate the clinical significance of detection of hepatitis E virus （HEV） RNA in serum from patients with acute hepatitis E using nRT-PCR. METHODS The conserved region at ORF2 was used for the design of primers based on the alignment result of HEV registered sequences and other related sequences in GenBank. Two pairs of primers were designed by bioinformatics software. Then nested RT-PCR （nRT-PCR） technology was wsed to amplify Hepatitis E virus RNA of 126 patients. The nested RT-PCR technology was performed to detect HEV RNA in all these sera in comparison to anti-HEV IgM detected by ELISA assay. RESULTS A total of 67 of sera from 126 cases were positive for HEV RNA measured by nRT-PCR. All of the controls were negative. The results of nRT-PCR and anti-HEV IgM （ELISA） were concordant in 80. 91~ samples. The two methods appeared a good concordance. Moreover, there was significant difference between the two methods, so they had complementarity and could not be substituted with each other. The first serum samples from three patients were positive for HEV RNA and negative for anti-HEV IgM. Following studies showed all the three sera samples were positive for anti-HEV IgM. HEV RNA in serum of the patients with acute HEV infection could be detected mostly from the first to the twelfth day. CONCLUSION The nRT-PCR method had high specificity and can be applied to the qualitative detection of the serum with hepatitis E virus, nRT-PCR is more sensitive than the assay of ELISA （detected anti-HEV IgM） in this study, and it may improve the early diagnosis of HEV for clinical diagnosis.