Optimization of PCR based detection of human papillomavirus DNA from urine specimens

Abstract

Background: Human papillomavirus (HPV) causes cervical cancer. Current screening requires a yearly pelvic exam and Pap smear. However, these procedures are impractical for screening all women at risk for disease. Urine sampling has been successfully utilized to screen for Chlamydia trachomatis (CT) and Neisseria gonorrhoreae (NG) infections and has been considered for HPV DNA detection by several investigators. However, no study to date has been performed to specifically optimize HPV detection in urine.

Objectives: To compare handling and extraction techniques in order to optimize the HPV specific PCR system in urine specimens.

Study design: Examination of 10 characteristics that may contribute to PCR inhibition in urine was performed utilizing 10SG mulitstixs. Five different DNA extraction methods were compared in spiked specimens and in 10 clinical specimens. After the optimal extraction technique was identified, concentration of the sample with and without prior dilution was compared to the original protocol. Lastly, specimen handling was compared between immediate processing, refrigerating overnight, or freezing overnight.

Results and conclusions: the presence of protein in urine enhanced amplification while nitrites decreased amplification. Of the extraction methods tested, the QIAamp DNA Mini Kit demonstrated the best amplification from urine samples spiked with HPV DNA and clinical specimens. The addition of a dilution step and a concentration step before applying the Qiagen protocol further increased amplification of beta-globin (from 50 to 63%) and the HPV L1 gene (from 13 to 33%). Lastly, refrigerating the specimens at 4 degrees C overnight appears to produce better amplification (62% beta-globin and 17% HPV positive) than either immediate processing (46% beta-globin and 13% HPV+) or freezing the specimen for 24h prior to processing (46% beta-globin and 10% HPV+). In these studies, amplification was low despite optimization. Additional improvements are required prior to clinical application of a urine-based HPV DNA detection system.