GROUP-SPECIFIC DIFFERENTIATION BETWEEN HIGH- AND LOW-RISK HUMAN PAPILLOMAVIRUS GENOTYPES BY GENERAL PRIMER-MEDIATED PCR AND 2 COCKTAILS OF OLIGONUCLEOTIDE PROBES
GROUP-SPECIFIC DIFFERENTIATION BETWEEN HIGH- AND LOW-RISK HUMAN PAPILLOMAVIRUS GENOTYPES BY GENERAL PRIMER-MEDIATED PCR AND 2 COCKTAILS OF OLIGONUCLEOTIDE PROBES
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DOI：
10.1128/jcm.33.4.901-905.1995
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发表时间：
1995-04-01
影响因子：
9.4
通讯作者：
WALBOOMERS, JMM
中科院分区：
文献类型：
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作者：
JACOBS, MV;HUSMAN, AMD;WALBOOMERS, JMM
近年来，通用引物介导的PCR检测已被开发用于检测广谱的人乳头瘤病毒（HPV）基因型。在这项研究中，一种能够对高风险进行简单的群体特异性区分的程序（HPV-16、-18、-31、-33、-35、-39、-45、-51、-52、-54、-56和-58）和低风险（HPV-6、-11、-34、40、-42、-43和44）HPV的通用引物介导的（GP 5 +/GP 6+）PCR。通过计算机辅助序列分析，从150-bp的GP 5 +/GP 6+扩增区域的内部部分中选择19种不同HPV基因型的特异性寡核苷酸（30-mer）。这些寡核苷酸探针在来自同一组HPV类型的PCR产物的Southern印迹分析中测试特异性。未发现交叉杂交。寡聚探针的灵敏度从HPV-16和-18等扩增良好的HPV类型的毫微微克水平到HPV-39和-51等扩增较差的HPV类型的皮克水平不等。当单独和混合应用寡核苷酸探针时，达到了这些灵敏度。在这些结果的基础上，组成了两种鸡尾酒寡核苷酸探针，能够特异性和灵敏地区分低风险和高风险HPV类型。
In recent years, general primer-mediated PCR assays have been developed to detect a broad spectrum of human papillomavirus (HPV) genotypes. In this study, a procedure enabling a simple group-specific differentiation of high-risk (HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -54, -56, and -58) and low-risk (HPV-6, -11, -34, 40, -42, -43, and 44) HPVs following an HPV general primer-mediated (GP5+/GP6+) PCR is presented. By computer-assisted sequence analysis, oligonucleotides (30-mers) specific for 19 different HPV genotypes were selected from the internal part of the 150-bp GP5+/GP6+-amplified region. These oligo probes were tested for specificity in a Southern blot analysis of PCR products derived from the same panel of HPV types. No cross-hybridizations were found. The sensitivities of the oligo probes varied from the femtogram level for the well-amplified HPV types like HPV-16 and -18 to the picogram level for the less-well-amplified HPV types like HPV-39 and -51. These sensitivities were reached when the oligo probes were applied both individually and in a cocktail. On the basis of these results, two cocktail oligo probes that enabled a specific and sensitive differentiation between low- and high-risk HPV types were composed.