摘要
目的探讨建立一种巢式PCR结合Sanger测序检测乙型肝炎病毒耐药基因位点的方法,为临床抗病毒药物的选择提供依据。方法采用特异性引物对120例HBV-DNA阳性样本(病毒载量>5×10^(2)copies/mL)进行HBV-DNA基因扩增,然后利用基因测序仪对扩增产物进行基因分型及耐药位点的检测。结果本实验室建立的巢氏PCR结合Sanger测序的最低检测下限为5×10^(2)copies/mL;86例使用了核苷类似物治疗的患者(用药组)中55例为B型HBV,31例为C型HBV。86例用药患者中有37例检测出耐药突变(突变率为43%),耐药位点检测阳性率明显高于未用药对照(P<0.05),其中以L180M+M204I/V、rtM204V/I、N236T和rtA181V/T突变为主,分别占37.8%、29.7%、13.5%和10.8%;34例未用药患者未检测出耐药突变基因;耐药组的HBV-DNA、AFU、IL-6水平高于非耐药组,差异有统计学意义(P<0.05)。结论本方法能够对低载量HBV进行基因分型和耐药基因位点的检测,方法便捷高效,对于低水平HBV病毒载量的标本(HBV-DNA<1×10^(3))也能检测出准确突变位点,为乙肝药物使用提供了技术支持,可推广使用。
Objective To establish a nested PCR combined with Sanger sequencing method to detect the drug resistance gene sites of hepatitis B virus,to provide a basis for the selection of clinical antiviral drugs.Methods 120 HBV-DNA positive samples(viral load>5%)were tested with specific primers×10^(2) copies/mL,and then the amplified products were genotyped and the drug resistance sites were detected by gene sequencer.Results The lowest detection limit of Nest PCR combined with Sanger sequencing established in the laboratory was 5×10^(2) copies/mL;Among 86 patients treated with nucleoside analogues(medication group),55 cases were type B HBV and 31 cases were type C HBV.Drug resistance mutations were detected in 37 of 86 patients(mutation rate was 43%),and the positive rate of drug resistance sites was significantly higher than that of untreated controls(P<0.05).L180M+M204I/V,rtM204V/I,N236Tand rtA181V/T mutations were the main mutations,accounting for 40.5%,29.7%,13.5% and 10.8% respectively;No drug resistance mutation gene was detected in 34 patients without drug use;The levels of HBV-DNA,AFU and IL-6 in drug-resistant group were higher than those in non drug-resistant group,the differences were statistically significant(P<0.05).Conclusion This method can detect the genotyping and drug-resistant gene sites of low load HBV,the method is convenient and efficient.It is suitable for samples with low HBV viral load(HBV-DNA<1×10^(3))it can also detect accurate mutation sites,which provides technical support for the use of hepatitis B drugs and can be popularized.
作者
艾文彬
王定保
AI Wenbin;WANG Dingbao(Clinical Center trial,the First People's Hospital of Xiaogan,Xiaogan,Hubei 432100,China)
出处
《检验医学与临床》
CAS
2022年第S02期74-77,共4页
Laboratory Medicine and Clinic
