青岛市隐匿性乙型肝炎的分子流行病学调查研究

The molecular epidemic study on occult HBV infection in Qingdao

目的:由于国内目前尚无检测隐匿性乙型肝炎病毒(Occult Hepatitis B Virus;OHBV)感染的试剂盒且OHBV患者的特点是HBsAg阴性,血清中存在低水平的HBV DNA,用常规的检测方法不能检测出来;急需研制灵敏度高、特异性强的PCR方法,来探讨青岛市是否存在OHBV感染。方法根据OHBV血清图谱的特点随机收集了OHBV可疑样本100份,采用试剂盒和酚/氯仿两种方法提取DNA,PCR扩增及其引物设计按Kanneko等的方法进行并进行了改进。结果1.经改进的酚/氯仿法提取OHBV DNA优于试剂盒法。2.常规PCR引物组合109-588扩增,所有样品的109-588组合均无扩增产物;使用嵌套式PCR引物组合1763-1778-2017-2032进行扩增,33份样品出现了HBV特异性扩增带。3.在OHBV不同血清图谱样品中均检出OHBV DNA,总检出率为33%,其中anti-HBs+anti-HBc阳性组中OHBV DNA检出率最高(43.33%);anti-HBe+anti-HBc阳性组中OHBV DNA检出率最低(18.18%)。结论酚/氯仿法提取OHBV DNA经改良,优于试剂盒法,常规PCR方法不能检测出OHBV DNA,而嵌套式PCR方法可达到诊断OHBV的目的。本实验首次在青岛运用自行设计的OHBV检测体系,在OHBV可疑人群中检测出OHBV DNA。

Objective Because now there is no methods in the market to detect occult hepatitis B virus（OHBV） infection in our country and the specificity of OHBV is low level HBV DNA in the serum of HBsAg negative carriers which can not detected by routine methods in the market. Studying higher sensitivity and specificity method for the low level HBV DNA is eagerly needed. Method We collected 100 doubtful OHBV sample according to serum markers. Purification of HBV DNA was done by phenol-chloroform and method supplied by market. Oligonucleotide primers and PCR amplification of HBV DNA are according to Kaneko et al and also changed. Results 1 .The purification of HBV DNA by phenol-chloroform is better than the market methods.2.Routine PCR primer 109-588 amplified, there were no amplified product in all sample, but by 1763-177802017-2032 primer, there are 33 HBV DNA specificity amplified products. Total positive detecting rate is 33%.The highest positive rate is in anti-HBs plus anti- HBc group（43.33%）.The lowest positive rate is in anti-HBe plus anti-HBc group. Conclusion The changed phenelchloroform method for the purificating HBV DNA is better than the market methods. The detection of OHBV DNA can not done by routine PCR but Nested PCR can. Our study first detect OHBV DNA by changed Nested PCR in Qingdao OHBV doubtful samples and the positive rate higher than abroad. Our study have more significance in the control of OHBV spread in Qingdao.