Detection and Typing of Human Papilloma Virus by Multiplex PCR with Type-Specific Primers

The primary underlying cause of cervical cancer is infection with one or more high-risk (HR) types of the human papilloma virus (HPV). Detection and typing of HPV have been commonly carried out by PCR-based assays, where HPV detection and typing are two separate procedures. Here, we present a multiplex PCR-based HPV typing assay that detects 20 HPV types (15 HR, 3 probably HR and 2 low risk) using type-specific primers and agarose gel electrophoresis. 46 cervical, urethral, and biopsy samples were analyzed by both Multiplex PCR and PGMY09/11 consensus PCR, and results were compared. 611 samples were further analyzed by Multiplex PCR, 282 were positive for HR HPV, and 101 showed multiple HR HPV infections. The relatively ease and economic accessibility of the method and its improved ability to detect high-risk HPV types in multiple HPV-infected samples make it an attractive option for HPV testing.

Detection and typing of HPV have been commonly carried out by PCR-based assays, where HPV DNA is amplified by consensus primers and then typed by restriction enzyme analysis (RFLP), hybridization with type-specific probes, or direct sequencing of the amplicons, among the most common methods [13]. Recently, methods that use multiplex PCR amplification with type-specific primers have been reported, where detection and typing are deducted from the amplification pattern of capillary electrophoresis [14].
Here, we present a multiplex PCR-based HPV typing assay that detect 20 HPV types (15 HR), 3 probably HR and 2 low risk (LR) using type-specific primers and agarose gel electrophoresis.  Primer selection for each reaction tube mix was carried out in silico [15] and experimentally to ensure primer compatibility, and a primer pair specific for β-globin was included as positive control [16]. Sequences of primers included in each reaction mix, with predicted amplification product size and digestion product sizes with indicated restriction enzymes are shown in Table 1 HPV type was assigned based on the amplification pattern. In cases where band interpretation was not clear, an additional PCR amplification with specific primers was performed to confirm. Selected PCR amplified fragments were cloned into pGem-T vector (Promega, USA), each cloned product was sequenced with universal forward and reverse primers to confirm fragment identity. Additionally, selected amplified fragments were digested with restriction enzymes AluI, HaeIII, RsaI, or MspI (New England Biolabs, USA), and digestion patterns were observed in a 2% agarose gel to also confirm fragment identity.

PGMY09/11
Consensus PCR. HPV consensus PCR was performed using primers PGMY09/PGMY11 designed to amplify a fragment of the HPV L1 gene of approximately 450 bp as previously described [17]. HPV genotype was assigned by sequencing of amplified fragments using primers PGMY11.
In this report, we present an assay based in HPV DNA amplification with type-specific primers in a Multiplex PCR format to detect and type single or multiple HR HPV infections in samples of different sources. Primers specific to each of 15 high-risk, 3 probably high-risk, and 2 low-risk HPV types were included in seven independent Multiplex PCR reactions. Typing was assigned based on the amplification pattern. As a result of having specific primers, stringent PCR conditions can be set to increase the clarity of results by reducing the presence of amplification artifacts, and all HPV types in a sample are amplified by their specific primer pair. Also, detection and HPV typing are accomplished at the same time, without the need of an additional protocol for typing after HPV detection (see examples of HPV detection in Figure 1).
In order to evaluate the newly developed HPV detection assay, 83 samples (46 cervical and 16 urethral scrapes and 21 tissue biopsies) were analyzed with Multiplex PCR and PGMY09/11 consensus PCR. Positive high risk is determined when a HR or probable HR HPV type is detected, and negative high risk is determined when a LR HPV type or no HPV infection is detected (Table 2). 47 samples reported negative results by both Multiplex PCR (Multiplex) and by PCR with PGMY09/11 primers (PGMY), while no samples were reported positive by PGMY and negative by Multiplex. 19 samples were reported positive by both Multiplex and PGMY, but 17 samples were reported positive by Multiplex and negative by PGMY. Of those, 12 samples did not produce any Particularly important is the capacity of detection of HPV multiple infections. A total of 611 samples (232 cervical and 164 urethral scrapes and 215 tissue biopsies) were analyzed by Multiplex PCR, including 83 samples mentioned above. 324 (53.03%) samples were negative for HR HPV, and 282 (46.15%) samples were positive for HR HPV. Only 5 (0.82%) samples (1 cervical and 4 paraffin-embedded tissue biopsies) failed to amplify the β-globin control gene and were reported as being not informative. 35% (101) of HR HPV positive samples had infections with two or more HR HPV types, representing 16% of the total number of samples analyzed. Detecting all HR HPV present in a sample is important in patient treatment to asses prevalence of infection and response to treatment.
Multiplex PCR HPV detection and typing are simple and potentially affordable. After DNA extraction and Multiplex PCR amplification, detection and typing of HPV are deduced from the amplification pattern observed in an agarose gel electrophoresis. This is particularly important in low-income countries. According to a study in Peru [31], simple, effective, and cost-efficient HPV testing is the best option for primary cervical screening. The entire cost in Mexico of the equipment and reagents for DNA extraction, amplification, agarose gel electrophoresis, and documentation is approximately $22,000 USD, and many research laboratories in Mexico already have all the necessary equipments.

Conclusions
Multiplex PCR HPV can detect single or multiple HR HPV infections in cervical and urethral scrapes and paraffinembedded tissue biopsies. The relatively ease and economic accessibility of the method can potentially have an impact in HPV screening in low-income countries like Mexico, and its improved ability to detect high-risk HPV types in multiple 4 ISRN Microbiology  HPV-infected samples makes it an attractive option for HPV testing.