Detection of HPV16 in Cervical Carcinoma Using Fully Automated In Situ PCR System.
Suresh Thakur, Swapna Mysore, Sheeba Guddu, AR Poongothai, Hongwei Wang, Krishan Kalra. BioGenex, Hyderabad, AP, India; BioGenex, San Ramon, CA
Background: Molecular detection of Human Papilloma Virus (HPV) DNA is currently the gold standard for detection of HPV. Various methods that are in use include, dot blotting, southern blotting, ISH, CISH, hybrid capture and PCR. Unlike conventional methods like PCR, in situ PCR (ISP) can help in detection of low copy nucleic acid targets (DNA/ mRNA) while preserving the histological context. In this study, we have reported the use of fully automated detection of HPV16 in cervical carcinoma tissue using ISP and established concordance between ISP and CISH.
Design: Cohort of 20 cervical carcinoma samples is used for the study. SiHa cell lines (1-2 copies of HPV16) have served as experimental control. Tissues from human breast, colon carcinoma and normal cervix are also tested. PCR primers are designed to amplify the E6 region of HPV16. Fluorescein labeled dUTP's are incorporated during PCR amplification and subsequently, amplicons are detected with sequential addition of anti-fluorescein antibody, and a poly-HRP labeled secondary antibody followed by final color development with DAB chromogen. HPV16 CISH is performed using single oligo-probe labeled with penta-fluorescein at 5' end. We have used Xmatrx®- a fully automated walk-away staining system to perform the complete assay.
Results: In this study, we are reporting for the first time successful detection of HPV16 using fully automated in situ PCR followed by chromogenic detection. The sensitivity of this detection is found to be as low as 1-2 copies of HPV16 using SiHa cell lines. All the 20 cervical carcinoma samples in this cohort detected positive for HPV16 by ISP and 15/20 are found to be positive by CISH. The results showed 75% of concordance between HPV16 ISP and HPV16 CISH using automated detection system. Further, there is no HPV16 signal detected from breast, colon carcinoma and normal cervix tissues.
Conclusions: Fully automated ISP detection is observed to be 25% more sensitive when compared to CISH with an ability to detect even 1-2 copies of HPV16 using SiHa cell lines. Fully automated ISP offers rapid and sensitive method for histological localization of HPV. Further, this is useful for the early screening of HPV infection in cervical scrapings and cervical intraepithelial neoplasia, where early detection of very small quantities of HPV is critical for prediction of clinical outcome.
Monday, February 28, 2011 1:00 PM
Poster Session II # 259, Monday Afternoon