简便HBV基因型S基因PCR—RFLP分型方法的建立

To establish a new and simple method of genotyping of hepatitis B virus by PCR-RFLP to analysis on S region

目的建立简便HBV基因型s基因PCR-限制性片段长度多态性（RFLP）分型方法（以下简称“简便PCR—RFLP法”），并评价其临床应用价值。方法临床诊断研究。查阅并比较GenBank中128株HBV（基因型A～D型）S基因片段（s区nt253-687），设计限制性内切酶HinfI、EarI、ApoI鉴别A～D基因型分型方法，并评价简便PCR—RFLP法检测HBVDNA的最低检测下限、重复性；然后，用该方法检测50例慢性乙型重型肝炎患者的HBV基因型，同时用直接测序法验证简便PCR—RFLP法检测HBV基因型的一致性。结果建立了简便PCR．RFLP法HBV基因分型的方案，通过限制性内切酶HinfI一次酶切就可分出我国流行的B型、C型、D型等毒株及B／C混合型。随机抽取3份标本，不同稀释浓度下，用简便PCR—RFLP法重复检测HBV基因型，分型结果均与原血清完全一致，且最低检测下限约为7～9IU／ml。经HinfI酶一步酶切后，用简便PCR—RFLP法从50例患者标本中，检出B型23份、c型10份；经直接测序法验证，从PCR产物中检出B型23份、c型10份，2种方法检测B、C型基因结果完全一致（Kappa=1．00，P=0．001），简便PCR—RFLP法检出B／C混合型9份，优于PCR产物直接测序法（0份，X2=18．00，P=0．001）。HinfI酶一步酶切后分出的ABD型8份，进一步酶切及应用PCR产物直接测序法检测均为B型变异株。结论建立的简便PCR—RFLP法对HBV基因分型有较高的灵敏度和重复性，且在检出混合基因型方面具有优势，更为简便。

Objective A method was established for genotyping of hepatitis B virus （HBV A-D genotype） , based on the PCR-restriction fragment length polymorphism （RFLP） created by Hinf I , Ear I , Apo I action on an amplified segment of the S region. Methods Clinical diagnosis research. One hundred and twenty-eight HBV S sequences obtained from GeneBank were analyzed for restriction enzyme sites that would be genotype-speeifie. Restriction patterns following digestion with restriction enzymes Hif I , Ear I , Apo I were determined to identify A-D HBV genotypes. The method was used to detect the HBV genotype of fifty severe hepatitis patients due to chronic hepatitis B in China. Then the detection results were confirmed by direct sequencing. Results The new genotyping method was established, named simple PCR-RFLP, which could identify HBV genotypes A to D. Genotypes B, C, B/C and A or D could be determined by a single step digestion with Hif I. Eight patients of genotype A/B/C classified by single step digestion with Hif I were conformed as genotype B variant by further digestion and direct sequencing. Extracted randomly and diluted into different concentration, three specimens were tested for genotype of HBV repeatedly and respectively. The results were all in accord with the originals, and the lowest detection limit of HBV DNA was 7 -9 IU/ml. This was particularly useful in China where genotypes B and C were predominant. Twenty-three of genotype B and ten genotype C patients were classified from these fifty severe hepatitis B patients by a single step digestion with Hif I through the simple PCR-RFLP method. The same results were also obtained by direct sequencing of PCR products （ Kappa = 1.00, P = 0. 001 ） . The simple PCR-RFLP method was superior to direct sequencing in detecting HBV B/C polyinfection （9 cases and 0 case; X2= 18.00, P =0. 001 ）. Conclusions Both the sensitivity and repetitiveness of Simple PCR-RFLP method are satisfaetory. It is superior to direct sequencing in detecting HBV B/C polyinfection, and simple, convenient.