MultNAT HPV16/18 Assay: A Simple, Rapid, and Low‐Cost Polymerase Chain Reaction Method for the Detection of HPV16 and HPV18 Suitable for Point‐of‐Care Testing

ABSTRACT Aim Persistent high‐risk HPV infection can lead to the development of precancerous lesions, potentially progressing to invasive cancer. HPV status serves not only as a prognostic marker for squamous cell carcinoma (HNSCC) like oropharyngeal squamous cell carcinomas (OPSCC) but also significantly influences clinical management and treatment decisions. Detecting high‐risk HPV infection is crucial for effective intervention and cancer prevention, especially in resource‐limited settings. This study aimed at investigating a point‐of‐care testing method based on the TaqMan qPCR technique for the rapid detection of HPV16 and HPV18 in clinical specimens. Methods The sensitivity and specificity of the MultNAT HPV16/18 assay were determined with simulated cervical swab and saliva matrix samples. Clinical cervical swab specimens were collected from 368 inpatients including 268 cervical cancer‐associated cases and 100 benign/noncancer cases of Xiaoshan Hospital from January 2023 to December 2025. To demonstrate that this detection method was also applicable for detecting HPV in saliva samples from patients with OPSCC, we tested 235 salivary samples self‐collected by internal company employees. This study evaluated the presence of HPV in the clinical specimens including cervical swab and salivary specimens using the MultNAT method and the conventional fluorescence quantitative PCR technique. Results The limit of detection (LOD) of the MultNAT HPV16/18 assay was 100 copies/mL, whereas the test results of the other 32 common pathogens causing OPC or colonizing in the upper respiratory tract showed no cross‐reactivity. Of 368 cervical swab specimens, MultNAT HPV16/18 assay gave a positive indication in 224 specimens, including 218 HPV16‐positive and 6 HPV18‐positive cases. Compared with conventional fluorescence quantitative PCR, the positive coincidence rate and the negative coincidence rate of MultNAT HPV16/18 assay was found to be 100.00% and 98.60%, respectively. Of the 368 cervical swab specimens, two specimens were determined to be negative by the conventional fluorescence quantitative PCR but were positive for the MultNAT HPV16/18 assay. The two samples were re‐extracted and confirmed to be positive by conventional fluorescence quantitative PCR. Of the 235 salivary samples, one sample was HPV16‐positive by the MultNAT HPV16/18 assay and conventional fluorescence quantitative PCR. Sanger sequencing confirmed this result. Conclusions MultNAT HPV16/18 assay is suitable as an initial test for HR‐HPV diagnosis due to its simplicity, low turnaround time, and high sensitivity and specificity.