CAJ | 학술논문

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Hinf Ⅰ、Ear Ⅰ、Apo Ⅰ鉴别A～D基因型分型方法,并评价简便PCR-RFLP法检测HBV DNA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Hinf Ⅰ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 ～9 IU/ml.经Hinf Ⅰ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).Hinf Ⅰ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.
목적 건립간편HBV기인형S기인PCR-한제성편단장도다태성(RFLP)분형방법(이하간칭“간편PCR-RFLP법”),병평개기림상응용개치.방법 림상진단연구.사열병비교GenBank중128주HBV(기인형A～D형)S기인편단(S구nt253-687),설계한제성내절매Hinf Ⅰ、Ear Ⅰ、Apo Ⅰ감별A～D기인형분형방법,병평개간편PCR-RFLP법검측HBV DNA적최저검측하한、중복성；연후,용해방법검측50례만성을형중형간염환자적HBV기인형,동시용직접측서법험증간편PCR-RFLP법검측HBV기인형적일치성.결과 건립료간편PCR-RFLP법HBV기인분형적방안,통과한제성내절매Hinf Ⅰ일차매절취가분출아국류행적B형、C형、D형등독주급B/C혼합형.수궤추취3빈표본,불동희석농도하,용간편PCR-RFLP법중복검측HBV기인형,분형결과균여원혈청완전일치,차최저검측하한약위7 ～9 IU/ml.경Hinf Ⅰ매일보매절후,용간편PCR-RFLP법종50례환자표본중,검출B형23빈、C형10빈；경직접측서법험증,종PCR산물중검출B형23빈、C형10빈,2충방법검측B、C형기인결과완전일치(Kappa=1.00,P=0.001),간편PCR-RFLP법검출B/C혼합형9빈,우우PCR산물직접측서법(0빈,x2=18.00,P=0.001).Hinf Ⅰ매일보매절후분출적ABD형8빈,진일보매절급응용PCR산물직접측서법검측균위B형변이주.결론 건립적간편PCR-RFLP법대HBV기인분형유교고적령민도화중복성,차재검출혼합기인형방면구유우세,경위간편.
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-specific.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 satisfactory.It is superior to direct sequencing in detecting HBV B/C polyinfection,and simple,convenient.