膜反向点杂交技术在HIV-1耐药性点突变检测中的应用

目的以HIV基因组pol区序列为靶基因设计可鉴别HIV耐药性突变的寡核甘酸探针对，应用膜反向点杂交技术检测HIV-1耐药性点突变。方法以临床提取HIV-1患者血清为模板，扩增产物与点在尼龙膜上的5对特异的寡核苷酸探针杂交。通过标记在PCR上游引物5’端的荧光素显色得到结果，并将其与测序结果进行对比。结果采用反向点杂交共检测了9例野生型和21例突变耐药型样本，检测结果与测序结果符合率为74％。5对探针对于野生型和突变型的扩增产物均有较好的区分性，敏感性高且信号强弱与相应产物在样本所占比例相关。结论应用膜反向点杂交技术检测HIV耐药性点突变敏感、简便、快速，具有较高应用价值。

Application of reverse dot blot in detection of drug resistance mutations in HIV-1

Objective Oligonucleotide probes targeting pol region in HIV-1 were designed for reverse dot blot which was used to detect drug resistance mutations in HIV-1. Methods Amplicon containing drug resistance mutations were obtained by polymerase chain reaction with FITC-1abeled upstream primer. Then PCR products were hybridized with 5 pairs of probes that immobilised on Biodyne C membrane. Signals were detected by color development or chemilluminance and further contrasted with sequencing results. Results 9 wild-type samples and 21 mutant samples were tested with reverse dot blot, with 74% of the them showing concordant with sequencing results. These probes gave significant discrimination between wild-type and mutants. Their signal intensity was relative to proportion of certain PCR products. Conclusion Reverse dot blot may be widely used in detecting drug resistance mutations in HIV-1 for its sensitivity, simphcity, low cost and efficiency.