WHO HPV LabNet Regional Reference Laboratory for Europe, Institute of Microbiology, Centre Hospitalier Universitaire Vaudois, and University of Lausanne, 1011 Lausanne, Switzerland
J Clin Microbiol. 2011 Oct;49(10):3474-81. doi: 10.1128/JCM.05039-11. Epub 2011 Aug 10.
The genotyping of human papillomaviruses (HPV) is essential for the surveillance of HPV vaccines. We describe and validate a low-cost PGMY-based PCR assay (PGMY-CHUV) for the genotyping of 31 HPV by reverse blotting hybridization (RBH). Genotype-specific detection limits were 50 to 500 genome equivalents per reaction. RBH was 100% specific and 98.61% sensitive using DNA sequencing as the gold standard (n = 1,024 samples). PGMY-CHUV was compared to the validated and commercially available linear array (Roche) on 200 samples. Both assays identified the same positive (n = 182) and negative samples (n = 18). Seventy-six percent of the positives were fully concordant after restricting the comparison to the 28 genotypes shared by both assays. At the genotypic level, agreement was 83% (285/344 genotype-sample combinations; κ of 0.987 for single infections and 0.853 for multiple infections). Fifty-seven of the 59 discordant cases were associated with multiple infections and with the weakest genotypes within each sample (P < 0.0001). PGMY-CHUV was significantly more sensitive for HPV56 (P = 0.0026) and could unambiguously identify HPV52 in mixed infections. PGMY-CHUV was reproducible on repeat testing (n = 275 samples; 392 genotype-sample combinations; κ of 0.933) involving different reagents lots and different technicians. Discordant results (n = 47) were significantly associated with the weakest genotypes in samples with multiple infections (P < 0.0001). Successful participation in proficiency testing also supported the robustness of this assay. The PGMY-CHUV reagent costs were estimated at $2.40 per sample using the least expensive yet proficient genotyping algorithm that also included quality control. This assay may be used in low-resource laboratories that have sufficient manpower and PCR expertise.
HPV 基因分型对于 HPV 疫苗的监测至关重要。我们描述并验证了一种基于 PGMY 的低成本 PCR 检测方法（PGMY-CHUV），用于通过反向斑点杂交（RBH）对 31 种 HPV 进行基因分型。每种 HPV 基因型的检测限为 50 到 500 个基因组当量/反应。使用 DNA 测序作为金标准（n = 1,024 个样本），RBH 的特异性为 100%，敏感性为 98.61%。PGMY-CHUV 在 200 个样本上与经过验证的商业线性阵列（罗氏）进行了比较。两种检测方法都在 182 个阳性样本和 18 个阴性样本中得到了相同的结果。在将比较限制在两种检测方法共有的 28 种基因型后，76%的阳性结果完全一致。在基因水平上，两种检测方法的一致性为 83%（285/344 个基因型-样本组合；单一感染的 κ 值为 0.987，多重感染的 κ 值为 0.853）。59 个不一致的病例均与多重感染和每个样本中最弱的基因型有关（P < 0.0001）。PGMY-CHUV 对 HPV56 的检测灵敏度明显更高（P = 0.0026），并且可以在混合感染中明确识别 HPV52。在涉及不同试剂批次和不同技术人员的重复检测（n = 275 个样本；392 个基因型-样本组合）中，PGMY-CHUV 具有可重复性（κ 值为 0.933）。不一致的结果（n = 47）与多重感染样本中最弱的基因型显著相关（P < 0.0001）。成功参与能力验证也支持了该检测方法的稳健性。使用最便宜但仍具有足够性能的基因分型算法，每个样本的 PGMY-CHUV 试剂成本估计为 2.40 美元，该算法还包括质量控制。该检测方法可用于人力资源和 PCR 专业知识充足的资源有限的实验室。
