摘要
目的建立PCR—MALDI—TOFMS检测乙型肝炎病毒针对核苷类药物耐药突变位点的检测方法，探讨PCR-MALDI-TOFMS技术的影响因素，为正确应用该技术提供参考。方法采用PCR-MALDI-TOFMS检测10份HBV质粒标准品及100份HBV阳性临床标本（均单独或者联合使用过拉米夫定、阿德福韦酯、恩替卡韦和替比夫定等核苷类药物），并用PCR产物测序进行验证。结果100份HBV阳性的临床标本，PCR-MALDI-TOFMS检测耐药阳性结果31份，阴性结果69份。与测序结果相比完全一致的有94份（94％），6份检测结果不一致，其中3份质谱与测序结果均为耐药阳性，但质谱检测结果耐药位点多于测序。PCR-MALDI—TOFMS检测灵敏度为100copies／μl，突变型的检出阈值为5％。结论PCR—MALDI—TOFMS技术检测HBV耐药位点变异具有高灵敏度，高准确率，高通量和自动化的特点，适合用于临床对HBV耐药位点进行基因检测。
Objective To establish a rapid method for detection of drug-resistance mutation in HBV, based on PCR-MALDI-TOF MS, and to explore the influential factors on this method. Methods One hundred blood serum samples, which were collected from chronic HBV patients with single drug-resistance or multiple drug-resistance of Lamivudin, Adefovi, Entecavir and Telbivudine, and 10 kinds of mutant HBV plasmids were analyzed using PCR-MALDI-TOF MS and confirmed by PCR-based sequencing. Results Of 100 samples detected, thirty-one samples were positive for drug-resistance and 69 samples were negative. The PCR-MALDI-TOF MS results of 94 samples were completely consistent with PCR-based sequencing. Six samples were inconsistent , of which three samples were positive by the two methods, but more mutation loci were detected by PCR-MALDI-TOF MS than sequencing. The consistent rate of two methods was 94%, detection sensitivity was up to 100 copies/μl, and the cut off value of detectable mutation level was 5%. Conclusion PCR-MALDI-TOF MS could be used for rapid and simple analysis of the drug resistance for the clinical application with features of high sensitivity and accuracy, high throughput and automation.
出处
《中华检验医学杂志》
CAS
CSCD
北大核心
2011年第3期213-217,共5页
Chinese Journal of Laboratory Medicine
关键词
肝炎病毒
乙型
抗药性
病毒
变异(遗传学)
光谱法
质量
电喷雾电离
Hepatitis B virus
Drug resistance, viral
Variation （ genetics ）
Spectrometry, mass, electrosprayionization
