Application of a Multiplex PCR to cervical cells collected by a paper smear for the simultaneous detection of all mucosal human papillomaviruses and typing of HR HPV types 16 and 18

Abstract

A simple Paper Smear (PS) method for dry collection and storage of cervical specimens has been employed to develop an easy multiplex PCR for simultaneous detection of generic HPVs as well as typing of high risk HPV16 and 18, the two clinically most important HPV genotypes that are responsible for more than 80% of cervical cancer. Multiplexing was performed with a very small amount of DNA eluted by boiling from a single paper smear punch in a single tube and using a mixture of four pairs of primers MY09/11, HPV16, HPV18 and beta-globin gene. Sixty HPV positive biopsies and corresponding paper smear specimens from cervical cancer patients and cervical smears from 100 healthy women with or without abnormal cytology were collected both as paper smear as well as in PBS. Detection of HPV DNA from cervical biopsies collected in PBS and corresponding cervical scrapes on paper smear or in PBS by conventional and multiplex PCR showed a concordance of 100% and adequacy of 93%. Similar comparative study in cervical scrapes from normal women also revealed 100% concordance. The technique was validated in a multicentric study at four different national laboratories. Paper smears collected by different centres showed variable adequacy (73% to 82%) but, use of multiple PS discs for DNA extraction reduced inadequacy significantly. Integration of paper smear with multiplex PCR for detection and typing of HPV is highly convenient, efficient, simple and cost-effective method for large scale clinico-epidemiological study and also suitable for HPV vaccine monitoring programs in resource poor settings.