实时荧光定量PCR方法检测乙型肝炎病毒基因型

A real-time PCR method for determination of hepatitis B virus genotype

目的建立一种快速准确的实时荧光PCR方法检测乙型肝炎病毒基因型。用此方法对安徽地区乙肝患者进行检测,了解该地区HBV基因型分布情况。方法首先用实时荧光定量PCR方法对150例安徽地区乙肝患者进行HBVDNA定量检测和分型检测,然后对其中载毒量大于5000IU/mL的113例标本和1例1000IU/mL≤载毒量<5000IU/mL进行测序分型检测,验证所建立方法的准确性,了解安徽地区HBV基因型分布情况。结果150例HBV样本中HBVDNA含量<1000IU/mL30例,分型检测结果均为阴性,对载毒量≥5000IU/mL的样本检出率为97.35%(110/113);对1000IU/mL≤载毒量<5000IU/mL的样本检出率为14.29%(1/7);对全部DNA阳性样本的检出率为92.5%(111/120)。HBV分型检测与HBVDNA测序检测的114例样本中,只要是HBVB基因型、C基因型或B/C混合型,乙型肝炎病毒基因分型检测都能检出来(共111例),其中部分样本经分型检测属于B/C混合基因型,测序结果都是混合基因型中占优势的基因型结果,总体符合率为100%。未能被本法检出的样本共3例,3例样本经测序分型均为D型。120例病毒载毒量≥1000IU/mL乙肝患者中B型病例占51.67%(62/120);C型占29.17%(35/120);B/C混合型占11.67%(14/120)。PCR测序检出为D型3例,占2.5%(3/120)。结论应用TaqMan探针的实时荧光定量PCR方法能快速对我国HBV主要基因型B基因型、C基因型进行检测,结果准确可靠,特异性高。安徽地区的HBV基因型以B型为主,C型次之,亦存在着D型的少数感染病例和B/C的混合感染。

Objective A rapid method for the determination the genotype of hepatitis B virus was developed by real-time PCR.The distribution of HBV genotype in Anhui district was investigated using this method.Methods The copies and genotypes of HBV DNA of 150 patients from Anhui district were determined by the real-time florescence quantitative PCR.HBV DNA sequence was carried out for the 113 samples which HBV DNA copy was more than 5 000 IU/mL,and for one sample which HBV DNA copy was from 1 000 IU/mL to 5 000 IU/mL.The validation of PCR method was performed by the DNA sequence.Results The copies of HBV DNA in 30 patients were less than 1 000 IU/mL among 150 patients and genotype test was negative.The positive ratio of genotype was 97.35 % for the copies of more than 5 000 IU/mL,14.29 % for the copies from 1 000 IU/mL to 5 000 IU/mL and 92.5 % for the all DNA positive samples.Conclusion The real-time florescence quantitative PCR was a rapid,accuracy and specific method which could be used for the characteristic of HBV genotype.HBV genotype B was main genotype,genotype C took second place,and genotype D and B/C mix infectious was seldom in Anhui district.