A New Method of Multiplex Polymerase Chain Reaction Coupled with Invader Reaction for Multiplex Human Papillomavirus Visual Typing

A visualized and cost-effective method was established for multiple human papillomavirus (HPV) typing detection with high sensitivity and good specificity. Specific primers and probes were designed based on sequence differences of HPV-DNA Ll gene region of different subtypes. After single-tube multiplex polymerase chain reaction (PCR) amplification, visualized typing detection was then realized by cascade invader reactions coupled with gold nanoparticle (AuNPs) probes hybridization under an intensive multi-tube typing scheme. The analytical specificity and sensitivity of the method were evaluated. The established assay was further applied to detection of clinical cervical swab samples. The results indicated that the visualized multiplex typing method of 15 kinds of HPV subtypes through 6 detection tubes was successfully established. The assay demonstrated high specificity with no cross-reaction among different subtypes under several artificial sample concentrations (from 10 & DEG; to 103 copy/reaction) and enabled highly sensitive typing detection of as low as 10 & DEG; copy/reaction. The analytical sensitivity of three subtypes (HPV56, HPV66, HPV45) could reach 10 & DEG; copy/reaction; seven subtypes (HPV73, HPV59, HPV18, HPV39, HPV6, HPV58, HPV31) reached 101 copy/reaction and the detection sensitivity of the other five subtypes (HPV68, HPV35, HPV51, HPV16, of HPV11) were 102 copy/reaction. Successful typing detection of 27 samples which were consistent with a PCR-reverse dot blot HPV typing kit verified the clinical applicability of the method. The proposed visualized assay for multiple HPV typing detection had high sensitivity and specificity. The final typing results could be distinguished with naked eyes without special equipment. The method had the potential to be used as a tool for HPV typing detection especially in resource-limited regions.