CAJ | 학술논문

目的：探讨细胞培养／链特异性RT-PCR方法可否作为甲型肝炎（甲肝）灭活疫苗（ L-A-1减毒株）的病毒灭活验证试验方法。方法根据甲肝病毒（ HAV） L-A-1株基因组序列，设计5条基因特异性引物，提取HAV基因组RNA，应用设计的正向引物进行反转录，再进行两轮PCR扩增，通过检测HAV复制过程中的负链中间体，对甲肝灭活疫苗灭活验证试验方法进行探讨，并与《中华人民共和国药典》甲肝灭活疫苗HAV灭活验证试验方法进行比较。结果细胞培养／链特异性RT-PCR方法对HAV负链RNA特异、敏感。通过方法学验证试验表明，该方法的特异性、敏感性和重复性均良好。利用此方法对5批甲肝灭活疫苗（L-A-1减毒株）进行检测，结果全为阴性，与《中华人民共和国药典》甲肝灭活疫苗HAV灭活验证试验测定结果相同。结论细胞培养／链特异性RT-PCR方法快速、灵敏可靠，可作为检测甲肝灭活疫苗（ L-A-1减毒株） HAV灭活验证试验的方法。
목적：탐토세포배양／련특이성RT-PCR방법가부작위갑형간염（갑간）멸활역묘（ L-A-1감독주）적병독멸활험증시험방법。방법근거갑간병독（ HAV） L-A-1주기인조서렬，설계5조기인특이성인물，제취HAV기인조RNA，응용설계적정향인물진행반전록，재진행량륜PCR확증，통과검측HAV복제과정중적부련중간체，대갑간멸활역묘멸활험증시험방법진행탐토，병여《중화인민공화국약전》갑간멸활역묘HAV멸활험증시험방법진행비교。결과세포배양／련특이성RT-PCR방법대HAV부련RNA특이、민감。통과방법학험증시험표명，해방법적특이성、민감성화중복성균량호。이용차방법대5비갑간멸활역묘（L-A-1감독주）진행검측，결과전위음성，여《중화인민공화국약전》갑간멸활역묘HAV멸활험증시험측정결과상동。결론세포배양／련특이성RT-PCR방법쾌속、령민가고，가작위검측갑간멸활역묘（ L-A-1감독주） HAV멸활험증시험적방법。
Objective To investigate the integrated cell culture/strand specific reverse transcriptase-polymerase chain reac-tion( ICC/strand-specific RT-PCR ) method can be used as hepatitis A ( HAV ) inactivated vaccine ( L-A-1 attenuated strain) virus inactivation test method validation.Methods Five primers were designed according to genomic sequence of HAV L-A-1 attenuated strain, and the genomic RNA was extracted.Using designed positive primer to reverse transcription and two rounds of PCR amplification to detect the negative-strand intermediate.The effective inactivation tests of inactivated hepatitis A vaccine were carried out using ICC/strand-specific RT-PCR assay and compared with the method described in Pharmacopoeia of the People's Republic of China on inactivated hepatitis A vaccine inactivated HAV verification test.Re-sults The ICC/strand-specific RT-PCR had high specificity and sensitivity for HAV negative-strand RNA.The results showed that the specificity, sensitivity and repeatability of the assay were good.Five batch samples were all negative results detected by both ICC/strand-specific RT-PCR and the method described in Pharmacopoeia of the People's Republic of Chi-na.Conclusions The ICC/strand-specific RT-PCR is a novel, rapid and reliable method to test the effective inactivation test of inactivated hepatitis A vaccine.It could be used to evaluate the availability of routine inactivated hepatitis A test.