PCR and Genotyping for HPV in Cervical Cancer Patients.
OBJECTIVE
To devise nested multiplex polymerase chain reaction (NMPCR) protocol for detection of mucosal human papilloma viruses (HPVs) and typing of HPV-16 and -18 in formalin-fixed, paraffin-embedded (FFPE) tissues of carcinoma cervix (CaCx).


METHODS
Cross-sectional observational study.


METHODS
NMPCR was done for simultaneous detection of HPV, targeting 134 bp L1 capsid gene employing GP+/mGP+ primers and typing of genotypes-16 and -18, targeting E6/E7 gene from 34 FFPE tissue blocks of CaCx and cervical intraepithelial neoplasia (CIN). Detection of 142 bp consensus sequence of L1 capsid gene was performed by nested PCR employing MY/GP+ primers. Sequencing of selected PCR amplicons of the later protocol obtained from control cell line DNA and 5 select samples were done for validation of the NMPCR protocol.


METHODS
Calculation of percentage from the Microsoft Excel Software.


RESULTS
Of 26 FFPE samples of CaCx, 17 (65.3%) samples were found positive for HPV by NMPCR. Amplicons of 142 bp L1 capsid gene employing MY/GP+ primers were observed in 11 (42.3%) samples of CaCx. Nearly 25% samples of CIN were positive for HPV. On sequence analysis, it was observed that the sample typed as HPV-16 by NMPCR was found to be the same on sequencing of amplicons obtained after MY/GP+ nested PCR.


CONCLUSIONS
This study indicates the usefulness of our NMPCR protocol for detection of mucosal HPVs and typing of HPV-16 and -18 from FFPE tissue samples of CaCx. The NMPCR protocol may be used to detect HPV and type common genotypes-16 and -18 in fresh tissue of cervical biopsy or scrape samples for screening of CaCx.
Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India.
Pradyot
P
Prakash
Department of Pathology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India.
Shashikant C U
SC
Patne
Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India.
Ashish Kumar
AK
Singh
Department of Pathology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India.
Mohan
M
Kumar
Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India.
Mukti Nath
MN
Mishra
Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India.
Anil Kumar
AK
Gulati
Cervical intraepithelial neoplasia; Pap smear; molecular diagnosis; polymerase chain reaction; squamous cell carcinoma