一管法MLPA荧光探针溶解曲线分析技术检测15种高危型HPV方法的建立

Establishment of A Method to Detect 15 High Risk HPV Based on One Tube MLPA Melting Curve Analysis Technique

目的：结合多重连接依赖探针扩增（MLPA）和荧光探针溶解曲线分析技术，建立一种一管法同时检测并分辨15种高危型HPV的方法。方法：根据各HPV亚型的特异性序列设计MLPA探针，并在各亚型的ML—PA探针中加入溶解温度（Tm）不同的人工核酸序列作为识别标志；使用特殊的耐高温连接酶和热启动Taq酶，通过温度控制使探针杂交、连接、PCR在一管反应中顺序进行；以各HPV亚型10^2拷贝至10^6拷贝的标准品质粒作为模版进行敏感度实验；将10^2-10^6拷贝的HPV亚型标准品进行两两混合作为模版，检测本方法对混合样本的检测能力；使用本方法和HPV分型试剂盒（导流杂交法）对临床样本进行平行检测，比较2种方法的差异。结果：本方法可在一次反应内顺序完成MLPA探针杂交、连接、扩增3个步骤，扩增结果可通过不同荧光通道不同Tm值的溶解峰分辨不同的HPV亚型；本方法对标准品的检测灵敏度比经典的MLPA3步法反应下降约一个数量级；本方法可同时检测至少2种HPV亚型的混合感染；对临床阳性标本的检出率与导流杂交法结果基本一致。结论：本研究成功建立了一种利用一管法MLPA荧光探针溶解曲线技术检测15种高危型HPV的方法。

Objective： To establish an one-tube method to detect and distinguish 15 high risk HPV based on multiplex ligation dependent probe amplification and fluorescent probe melting curve analysis technique. Methods： Design MLPA probes targeting specific sequences of 15 high risk HPV subtypes and adding artificial nucleic sequences with different melting temperatures to each MLPA probes as i- dentification. Special high temperature tolerant ligase and hot-start PCR enzyme were used to hold hy- bridization, ligation and PCR process to run sequentially in one tube. Coping HPV subtype 10^2 to 10^6 copies standard plasmids as templates to detect the sensitivity of this method. Using 10^2 to 10^6 copies mixed standard plasmids to find the detection capability of mixed samples. Parallel detection of clinical samples was used to compare the difference of these two methods. Results： This method could detect at least 3 MLPA subtypes simultaneously. Different HPV subtypes can be distinguished by melting curves of fluorescent probes. The sensitivity of this method is lower an order of magnitude than classical 3 step method. This method had similar accuracy of flow-through hybridization method. Conclusions： Successfully established a 15-high risk HPV detecting method based on one tube MLPA fluorescent probe melting curve analysis technique.