南方医科大学学报

南方医科大学学报 ›› 2013, Vol. 33 ›› Issue (06): 853-.

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肽核酸钳制PCR早期检测乙型肝炎病毒YMDD耐药变异

张迎迎，何海棠，杨洁，侯金林   

  • 出版日期:2013-06-20 发布日期:2013-06-20

Identification of hepatitis B virus YMDD point mutation using peptide nucleic acid clamping PCR

  • Online:2013-06-20 Published:2013-06-20

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摘要： 目的建立乙型肝炎病毒肽核酸钳制PCR（PNA-PCR）耐药监测方法，以提前监测乙型肝炎病毒YMDD耐药株的产生。方
法选取rtM204I（ATT）、rtM204V（GTG）突变型质粒与野生型质粒rtM204（ATG）按照不同比例混合后，用PNA-PCR法及直接测
序法对耐药位点进行检测，评价两种方法的灵敏度及特异性；选取临床耐药检测患者血清标本85例，采用PNA-PCR法及直接
测序法进行耐药检测，比较两种方法的耐药检出率。结果PNA-PCR法可在105倍野生型YMDD基因背景下检测出突变，灵敏
度为0.001%，可检测到最低限为101拷贝。而直接测序法可检测的基因突变的最低极限为104拷贝，检测灵敏度为10%。85例
临床耐药检测患者中，PNA-PCR法YMDD检测阳性率为85.9%（73/85∶YIDD∶40，YVDD∶23，YIDD+YVDD∶10），而PCR产物
直接测序法检阳性率仅为40%（34/85∶YIDD∶21，YVDD∶11，YIDD+YVDD∶2）。阴性对照，两种方法均未测出HBV病毒，两种
方法的特异性均较好。结论PNA-PCR方法在灵敏度及特异性上均优于直接测序法，而且操作简便，快捷，在科研和临床上都有
很好的应用前景。

Abstract: Objective To establish a peptide nucleic acid clamping PCR assay for detecting hepatitis B virus (HBV) drug
resistance mutation. Methods RtM204I (ATT) mutant, rtM204V (GTG) mutant and rtM204 (ATG) wild-type plasmids mixed at
different ratios were detected for mutations by PNA clamping PCR assay and direct sequencing, and the sensitivity and
specificity of the two methods were compared. Serum samples from 85 patients with chronic HBV infection were detected for
drug resistance using the two methods. Results The sensitivity of PNA-PCR assay was 0.001% in a 105-fold excess of wild-type
HBV DNA with a detection limit of 101 copies. The sensitivity of direct sequencing was 10% with a detection limit of 104 copies.
Mutants were detected in 73 of the 85 serum samples (85.9%), including YIDD in 40 samples, YVDD in 23 samples, and YIDD+
YVDD in 10 samples. The agreement of PNA-PCR assay with direct sequencing was only 40% (34/85, YIDD in 21 samples,
YVDD in 11 samples, and YIDD+YVDD in 2 samples). Neither of the two methods yielded positive results for the negative
control samples, suggesting their good specificity. Conclusion PNA-PCR assay appears to be a more sensitive and rapid assay
for detection of HBV genotypic resistance.