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PCR and Genotyping for HPV in Cervical Cancer Patients - PubMed

PCR and Genotyping for HPV in Cervical Cancer Patients

Pradyot Prakash et al. J Glob Infect Dis. 2016 Jul-Sep.

Abstract

Aims: 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).

Settings and design: Cross-sectional observational study.

Materials and 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.

Statistical analysis used: 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.

Keywords: Cervical intraepithelial neoplasia; Pap smear; molecular diagnosis; polymerase chain reaction; squamous cell carcinoma.

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Figures

Figure 1
Figure 1

Gel picture of amplicons obtained after nested multiplex polymerase chain reaction for the detection of human papilloma virus. Lane 1 and 16: 100bp DNA ladder, Lane 2: Positive control (SiHa cell line) 457 bp E6 and 134 bp L1 amplicons, Lane 3: Negative control (Milli-Q water), Lane 4, 6, 7, 9-15: Carcinoma cervix positive for human papilloma virus (134 bp), Lane 7: Carcinoma cervix positive for human papilloma virus-16 (457 bp)

Figure 2
Figure 2

Gel picture of MY/GP nested polymerase chain reaction products of human papilloma virus positive carcinoma cervix. Lane 1: 100 bp DNA ladder, Lane 2, 3: Positive control (SiHa cell line and HeLa cell line DNA), Lane 16: Negative control (Milli-Q water), Lane 4, 6, 8, 9, 10, 11, 15: Carcinoma cervix positive for human papilloma virus (142 bp),

Figure 3
Figure 3

Sequence analyses for L1 gene sequence for GP+ primers compatibility

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