GP5+/6+PCR followed by reverse line blot analysis enables rapid and high-throughput identification of human papillomavirus genotypes
GP5+/6+PCR followed by reverse line blot analysis enables rapid and high-throughput identification of human papillomavirus genotypes
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DOI：
10.1128/jcm.40.3.779-787.2002
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发表时间：
2002-03-01
影响因子：
9.4
通讯作者：
Snijders, PJF
中科院分区：
文献类型：
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作者：
van den Brule, AJC;Pol, R;Snijders, PJF
本研究应用通用引物（GP 5 +/6+）PCR产物的非放射性反向线印迹（RLS）技术，建立了一种简便、快速的37种嗜粘液型人乳头瘤病毒（HPV）分型方法。该系统的优点不仅在于以简单的形式，每天每个膜可以同时分型多达42个PCR产物，而且在于在剥离后，膜可以容易地再杂交至少15次而没有信号损失。RLB表现出高度的特异性，其灵敏度与酶免疫分析（EIA）中使用类型特异性寡核苷酸探针进行的常规分型相同。用HPV阳性宫颈刮片（n = 196）和宫颈癌前病变活检（n = 100）评价RLB分型的性能。这些样本中检测到的HPV基因型分布与基于文献数据的预期分布一致。此外，RLB和EIA分型程序进行了比较，在GP 5 +/6+ PCR产物的210个高危HPV阳性的妇女参加了以人群为基础的筛查计划的宫颈刮片的高危HPV类型的分型。分型程序的总体一致率为96.5%（kappa值，0.77）。RLB在检测多个RPV感染以及单个感染方面是成功的。总之，GP 5 +/6+ PCR-RLB程序似乎是一种可靠且简单的方法，对于需要高通量HPV分型的大型流行病学研究、基于人群的宫颈癌筛查计划和疫苗接种试验可能具有重要价值。
In this study, we developed a simple and fast typing procedure for 37 mucosotropic human papillomavirus (HPV) types using a nonradioactive reverse line blotting (RLS) procedure for general primer (GP5+/6+) PCR products. This system has the advantages not only that in a simple format, up to 42 PCR products can be simultaneously typed per membrane per day, but also that after stripping, the membranes can be easily rehybridized at least 15 times without a loss of signal. RLB appeared highly specific, and its sensitivity was identical to that of conventional typing performed with type-specific oligonucleotide probes in an enzyme immunoassay (EIA). The performance of RLB typing was evaluated with samples of HPV-positive cervical scrapings (n = 196) and biopsies of cervical premalignant lesions (n = 100). The distribution of HPV genotypes detected in these samples was in line with the distribution expected on the basis of literature data. In addition, RLB and EIA typing procedures were compared for the typing of high-risk HPV types in GP5+/6+ PCR products of 210 cervical scrapings from high-risk HPV-positive women who participated in a population-based screening program. The typing procedures had an excellent overall agreement rate of 96.5% (kappa value, 0.77). RLB was successful in detecting multiple RPV infections as well as single infections. In conclusion, the GP5+/6+ PCR-RLB procedure appeared to be a reliable and simple approach that may be of great value for large epidemiological studies, population-based cervical cancer screening programs, and vaccination trials that require high-throughput HPV typing.