基因Ⅰ、Ⅳ型戊型肝炎病毒高灵敏度通用引物的设计和初步应用

Design and Preliminary Application of a Set of Highly Sensitive Universal RT-PCR Primers for Detecting Genotype I/IV Hepatitis E Virus

设计针对国内流行的戊型肝炎病毒(HEV)基因Ⅰ、Ⅳ型,在这两型序列的保守区域设计了一套RT-PCR引物--E引物,并将E引物与目前较常用的3对通用引物(Meng、ConORF1和ConORF2引物)比较了检测基因Ⅰ、Ⅳ型HEV的灵敏度.对基因Ⅰ型HEV,E引物能检出的稀释度为105,参考引物能检出的稀释度为102～104;对基因Ⅳ型HEV,E引物能检出的稀释度为102,参考引物能检出的稀释度为101～102.在17份HEV-IgM阳性血清中,E引物检出5份,检出率为29.4%;参考引物只能检出1份或2份,检出率最高为11.8%.E引物在33份HEV-IgM阳性的隐性感染血清中检出6份,阳性率18.2%;在79份HEV-IgM阳性的临床肝炎血清中检出36份,阳性率45.6%.以上结果初步表明,对于在国内流行的基因Ⅰ、Ⅳ型HEV,E引物的检测灵敏度要高于目前常用的通用引物.

Genomic sequences of genotypes I, II, III and IV of hepatitis E virus were aligned and the conserved region was selected for designing a set of reverse-transcription polymerase chain reaction (RT-PCR) primers, which were denominated as E primers. The sensitivity of E primers and three sets of reported primers (Meng, ConORF1 and ConORF2) were compared by using HEV genotypes I and IV challenged monkeys stools and sera of hepatitis E patients. The detection limit of E primers for genotype I HEV in monkey stools was 10 to 1000 times lower than that of reference primers, and the same or 10 times lower than that of reference primers for genotype IV HEV. When amplified by RT-PCR using E primers, five out of seventeen (29.4%) anti-HEV IgM positive patients sera were positive, and just 1 or 2 samples were positive when RT-PCR were performed using reference primers. Thus, the E primers would be a better choice for detection of HEV RNA in samples from China, where only genotype IV and genotype I HEV were reported.