Molecular detection of human papillomavirus (HPV) in highly fragmented DNA from cervical cancer biopsies using double-nested PCR

Abstract

Archived Formalin-Fixed Paraffin-Embedded (FFPE) tissue specimens can be a valuable source of human papillomavirus (HPV) nucleic acids for molecular biological analyses in retrospective studies. Although successful amplification with polymerase chain reaction (PCR) is essential for analysis of HPV DNA extracted from cervical FFPE specimens, extensive DNA damage due to cross-linking and fragmentation results in poor yield. Therefore, techniques to improve the diagnostic rate and sensitivity from FFPE tissues through PCR is highly desired and of wider interest. To overcome this, a highly sensitive double-nested PCR methodology was designed and optimized for limited-resource laboratories coupled with an organic extraction of DNA. This method allows the detection of a broad range of HPV genotypes and also allowing the sequencing of the final amplicon. Validation of the new approach developed was done with an automated DNA extraction coupled with Real Time PCR. Results showed that the proposed method achieves 96.3% of HPV detection as compared to 100% Abbott m2000rt used as 'gold standard'. Moreover, the concordance rate between the two methods was equal for detecting HPV -16 or -18 genotypes. Nevertheless, the newly introduced assay has an advantage of: •Simultaneously identifying broad range of HPV genotypes besides HPV-16 and -18 from clinical samples.•It is an easy and cost-effective method that can be beneficial in resource-limited setting and can be employed for various molecular applications.•The method is indicated for highly degraded FFPE samples.

Keywords: DNA; DNA sequencing; Double-nested PCR; Double-nested PCR for detecting and genotyping HPV; FFPE; HPV; RFLP.

Graphical abstract

Fig. 1

Workflow diagram for HPV detection from FFPE samples.

Fig. 2

Comparative amplification of a DNA template from FFPE tissues using beta-globin and CYP2C8 primer pair. M: molecular marker (100 bp), NC: negative control, P: positive control. Agarose gel (2%) showing PCR amplified DNA fragments of different sizes within beta-globin (268 bp) on the left gel and CYP2C8 (107 bp) on the right gel gene locus using template from DNA from fragmented FFPE tissue. As seen, clearly PCRs was unsuccessful using beta-globin primer pair compared to CYP2C8 primers.

Fig. 3

Comparative amplification of HPV DNA template using one step PCR (MY09/11) protocol and nested PCR (MY09/11) from FFPE tissues. M: molecular marker (100 bp); P: positive control; NC: negative control. Numbers refer to samples. Both nested PCR carried out using 3 μl template DNA and PCR amplicons were run on 2.5% agarose gel stained with ethidium bromide. Nested PCR produce better yield of PCR product compared to one step PCR.

Fig. 4

RFLP digestion pattern of nested PCR. The digested PCR products from nested PCR (SB01/02 + MY09/11) (line 1–11) and DNA molecular weight (100 bp) marker (M) were electrophoretically visualised using MetaPhor^™ agarose gel (4%) (Lonza Rockland, ME, USA) stained with ethidium bromide showing digested PCR products from nested PCR using HpyCH4V restriction enzyme (Line 1–5, 8–10; HPV-16, line 7 and 11; HPV-33, line 6; HPV-45).

Fig. 5

Comparative amplification of HPV DNA template using PCR MY09/11 followed by GP5/6 primers and double-nested PCR protocol from FFPE tissues. M: molecular marker (100 bp), P: positive control; NC: negative control. Numbers refer to samples. NPCR: nested PCR; double-nPCR: double-nested PCR. Both PCR were carried out using 3 μl of the DNA template and amplicons (∼150 bp) were visualized on the 2% agarose gel stained with ethidium bromide.