CAJ | 학술논문

目的建立一种新的基于焦磷酸测序技术的HBV前C区/基本核心启动子(pre-C/BCP)突变的检测方法,并验证该方法的准确性、重复性、可靠性及进行初步临床检测.方法通过对基因库中100个HBV基因序列的比对分析,设计出专用的pre-C/BCP焦磷酸测序的PCR扩增引物及测序引物,并以标准质粒作为标准品建立其相应的检测方法.通过对标准质粒及PCR扩增产物、经Sanger测序的HBV血清及基因芯片检测结果分析,以验证本检测方法检测的标本的特异度及本方法的检测可靠性.最后对60例HBV临床血清标本进行了初步的检测.结果本研究成功地建立了pre-C/BCP突变热点的焦磷酸测序方法.其检测结果与pre-C/BCP质粒,Sanger测序法的检测结果符合率达100%(Kappa=1.0),基因芯片检测结果的诊断符合率为91.7%.同一批血清标本的重复率达97.8%,阳性PCR产物测序的重复率100%.充分证明了本方法的准确性、重复性、可靠性.初步批量化检测60份临床标本结果表明,本方法能一次批量检出包括pre-C/BCP的单一、多位点的突变,并发现了一例少见的BCP T1758C突变.结论本实验所建立的pre-C/BCP突变热点的焦磷酸测序方法是一种可一次性检测HBV pre-C/BCP多位点突变的高通量的检测方法.
목적 건립일충신적기우초린산측서기술적HBV전C구/기본핵심계동자(pre-C/BCP)돌변적검측방법,병험증해방법적준학성、중복성、가고성급진행초보림상검측.방법 통과대기인고중100개HBV기인서렬적비대분석,설계출전용적pre-C/BCP초린산측서적PCR확증인물급측서인물,병이표준질립작위표준품건립기상응적검측방법.통과대표준질립급PCR확증산물、경Sanger측서적HBV혈청급기인심편검측결과분석,이험증본검측방법검측적표본적특이도급본방법적검측가고성.최후대60례HBV림상혈청표본진행료초보적검측.결과 본연구성공지건립료pre-C/BCP돌변열점적초린산측서방법.기검측결과여pre-C/BCP질립,Sanger측서법적검측결과부합솔체100%(Kappa=1.0),기인심편검측결과적진단부합솔위91.7%.동일비혈청표본적중복솔체97.8%,양성PCR산물측서적중복솔100%.충분증명료본방법적준학성、중복성、가고성.초보비양화검측60빈림상표본결과표명,본방법능일차비량검출포괄pre-C/BCP적단일、다위점적돌변,병발현료일례소견적BCP T1758C돌변.결론 본실험소건립적pre-C/BCP돌변열점적초린산측서방법시일충가일차성검측HBV pre-C/BCP다위점돌변적고통량적검측방법.
Objective To develop a clinically useful assay for detecting the mutations of HBV pre-C/BCP based on the pyrosequencing and accuracy, reproducibility and reliability of this assay was evaluated. Methods The pyrosequencing primers for HBV pre-C/BCP mutation were designed through the cluster analysis among one hundred HBV gene sequences. After the amplification of the fragment of pre-C/BCP with the template of pre-C/BCP mutation plasmids, the pyrosequencing method for pre-C/BCP detection was initially set up with this standard sample. The accuracy, reliability and reproducibility of the pre-C/BCP pyrosequencing were confirmed through the pre-C/BCP plasmids as a standard sample when compared with Sanger/genechip sequencing method pre-C/BCP pyrosequencing assay was applied for detecting pre-C/BCP mutation types of 60 chnical serum samples in HBV patients. Results The pre-C/BCP mutation detection assay based on pyrosequencing has been established in our study. The coincidence rate between pyrosequencing and Sanger squencing was 100%. The coincidence rate between the result of pyrosequencing and of genechip method was 91.7%. The reproducibility of this assay was 97. 8%. It indicates the pre-C/BCP pyrosequencing is a high-accurate method with, good-reproducibility and high-reliability. And multi-site detection can be achieved by pyrosequencing one time. A rare mutation T1758C was also detected. Conclusion Pyrosequencing for pre-C/BCP mutations assay is high-throughout method for simultaneous detection of multi-site mutation.