An Isothermal, Multiplex Amplification Assay for Detection and Genotyping of Human Papillomaviruses in Formalin-Fixed, Paraffin-Embedded Tissues
An Isothermal, Multiplex Amplification Assay for Detection and Genotyping of Human Papillomaviruses in Formalin-Fixed, Paraffin-Embedded Tissues
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DOI：
10.1016/j.jmoldx.2019.12.004
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发表时间：
2020-03-01
影响因子：
4.1
通讯作者：
Park, Kay J.
中科院分区：
文献类型：
--
作者：
Tang, Yi-Wei;Lozano, Lorena;Park, Kay J.
快速准确地识别人乳头瘤病毒（HPV）对临床管理和人群筛查都很重要。对经甲醛固定、石蜡包埋（FFPE）的宫颈/外阴和口咽诊断组织样本进行了Atila AmpFire多重HPV检测的分析验证。AmpFire检测结合了一种新型的等温多重扩增与实时荧光检测相结合，以检测15种高危（HR） HPV基因型并进行基因分型。用HPV基因型特异性序列克隆的质粒测定HPV16、HPV18和部分HR HPV基因型的检出限为2拷贝/反应，其余HR HPV基因型的检出限为20拷贝/反应。使用214个FFPE标本评估AmpFire检测在临床样品中的性能。AmpFire试验在一个临床标本中失败，无效率为0.5%。AmpFire检测在临床样本中检测HPV， HPV16的阳性率为100.0%，HPV18的阳性率为100.0%，非16/18 HR的阳性率为94.7%，HPV16、HPV18和非16/18 HR的阳性率为100%阴性。在重复性研究中获得定性检测一致。综上所述，Atila AmpFire HPV检测对15种HR HPV基因型的检测和基因分型具有出色的分析敏感性和特异性。样品处理简单，样本量要求小，周转时间快，几乎不需要仪器，使其非常适合于FFPE标本的HPV检测和基因分型。
Rapid and accurate identification of human papillomavirus (HPV) is important for both clinical management and population screening. Analytic validation of Atila AmpFire Multiplex HPV assays on formalinfixed, paraffin-embedded (FFPE) cervix/vulva and oropharynx diagnostic tissue samples was performed. The AmpFire assay incorporates a novel isothermal multiplex amplification coupled with real-time fluorescent detection to detect and genotype 15 high-risk (HR) HPV genotypes. Limits of detection determined by plasmids cloned with HPV genotype-specific sequences were 2 copies/reaction for HPV16, HPV18, and some HR HPV genotypes, and 20 copies/reaction for the remaining HR HPV genotypes. The performance of the AmpFire assays in clinical samples was evaluated using 214 FFPE specimens. The AmpFire assay failed in one clinical specimen for an invalid rate of 0.5%. The AmpFire assay detected HPV in clinical samples with positive percent agreements of 100.00/0 for HPV16, 100.0% for HPV18, and 94.7% for non-16/18 HR HPV, and 100% negative percent agreements for HPV16, HPV18, and non-16/18 HR HPV. Qualitative detection agreement was obtained in the reproducibility study. In summary, the Atila AmpFire HPV assay demonstrated excellent analytic sensitivity and specificity for detection and genotyping of 15 HR HPV genotypes. Assay parameters of simple specimen processing, small sample size requirement, rapid turnaround time, and being near instrument-free render it well suited for HPV detection and genotyping in FFPE specimens.