Schmitz Martina, Scheungraber Cornelia, Herrmann Jörg, Teller Karin, Gajda Mieczyslaw, Runnebaum Ingo B, Dürst Matthias
Klinik für Geburtshilfe und Frauenheilkunde, Universitätsklinikum Jena, Germany.
J Clin Virol. 2009 Apr;44(4):302-7. doi: 10.1016/j.jcv.2009.01.006. Epub 2009 Feb 14.
High-risk HPV DNA detection has become a valuable tool for the triage of borderline, questionable and abnormal cytologic findings in cervical carcinoma screening programs. This knowledge is largely based on studies which could only discriminate between low-risk (LR-) and high-risk (HR-) HPV groups. However, it is becoming increasingly clear that HPV genotyping may allow further risk stratification and may offer different treatment options in the future.
To establish a fast and cost-effective system not only for genotyping but also for quantification of viral DNA.
Development and validation of a 5' exonuclease fluorescent probe multiplex real-time PCR assay (TaqMan format) for the detection and quantification of the 7 most frequent HR-HPV types (16, 18, 31, 33, 45, 52 and 58) which account for over 87% of cervical carcinomas world-wide. Two PCR reactions are required to detect the designated HPV types.
Experiments with plasmid constructs of all 18 HR-HPV DNA showed that the multiplex real-time PCR assay was highly sensitive and specific. Evaluation of DNA extracted from archived cell pellets of cervical scrapes by the multiplex assay and the GP5+/6+-EIA showed identical genotyping for 234 of 261 (89.6%) samples and an almost perfect agreement when considering all typing results (kappa 0.901). Viral load did not correlate with disease progression within the CIN spectrum but significant differences were evident when comparing all CIN with the group lacking CIN (p=0.0028) or with the cancer group (p=0.0001).
Our multiplex assay will be useful to address questions related to viral persistence at the genotype level, the kinetics of viral load and disease recurrence.
高危型人乳头瘤病毒（HPV）DNA检测已成为宫颈癌筛查项目中对意义不明确的、可疑的及异常细胞学检查结果进行分流的重要工具。这一认知主要基于那些仅能区分低危（LR-）和高危（HR-）HPV组别的研究。然而，越来越清楚的是，HPV基因分型可能会实现进一步的风险分层，并可能在未来提供不同的治疗选择。
建立一种不仅用于基因分型，还能对病毒DNA进行定量分析的快速且经济高效的系统。
开发并验证一种5'核酸外切酶荧光探针多重实时PCR检测法（TaqMan法），用于检测和定量7种最常见的高危型HPV（16、18、31、33、45、52和58型），这些类型在全球范围内导致了超过87%的宫颈癌。检测指定的HPV类型需要进行两次PCR反应。
对所有18种高危型HPV DNA的质粒构建体进行的实验表明，该多重实时PCR检测法具有高度的敏感性和特异性。通过多重检测法和GP5+/6+-酶免疫测定法对宫颈刮片存档细胞沉淀中提取的DNA进行评估，结果显示，261个样本中的234个（89.6%）样本的基因分型相同，考虑所有分型结果时一致性几乎完美（kappa值为0.901）。病毒载量与宫颈上皮内瘤变（CIN）范围内的疾病进展无关，但将所有CIN与无CIN组（p = 0.0028）或癌症组（p = 0.0001）进行比较时，差异显著。
我们的多重检测法将有助于解决与基因型水平的病毒持续存在、病毒载量动力学及疾病复发相关的问题。
