Defining detection threshold and improving analytical proficiency of HPV testing in clinical specimens

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• 作者：Fang-Yu Chao ; Angel Chao ; Chu-Chuen Huang ; Swei Hsueh ; Jung-Erh Yang ; Huei-Jean Huang ; Lei-Chiung Wang ; Cheng-Tao Lin ; Hung-Hsueh Chou ; Chyong-Huey Lai
• 关键词：Human papillomavirus ; Viral load ; Detection threshold ; Cervical cancer
• 刊名：Gynecologic Oncology
• 出版年：2010
• 出版时间：May 2010
• 年：2010
• 卷：117
• 期：2
• 页码：302-307
• 全文大小：219 K

文摘

Objective

Previous studies have shown that SPF1/GP6+ PCR followed by HPV Blot has high sensitivity and high reproducibility for the detection of human papillomavirus (HPV). However, there have been reports of false-negative results. In the present study we endeavoured to improve the diagnostic performance of HPV DNA testing in cervical swab specimens. We also aimed to identify viral load thresholds.

Methods

We examined DNA from cervical swabs of 7 women with a cervical intraepithelial neoplasia grade 2 or worse (CIN2+) who were negative by HPV DNA testing. For control purposes, 106 cytology negative/HPV-negative samples were included. PCR was performed with either 1 μL of DNA solution or different amounts of input DNA (25, 50, and 100 ng). We tested two different commercial alkaline phosphatases (ALPs) at two distinct reaction times. The overall conclusions were based on HPV Blot, type-specific PCR, direct sequencing, and real-time quantitative PCR (qPCR).

Results

All seven patients with CIN2+ disease turned HPV-positive when 100 ng of input DNA and E6 type-specific PCR were used. Discrepant and false-negative results were obtained using different amounts of input DNA. Different commercial ALPs showed a significant impact on detection performance. Analysis of viral load indicated that the detection threshold for HPV infection using SPF1/GP6+ PCR plus HPV Blot was approximately 10²–10³ copies.

Conclusions

Reliable determination of HPV status is crucially dependent on the amount of input genomic DNA.