Design and Evaluation of Innovative Loop-Mediated Isothermal Amplification-Lateral Flow Dipstick Kits for Advanced Detection of HPV16 and HPV18

Abstract

Background: Cervical cancer is a major cause of cancer-related mortality among women worldwide. Existing human papillomavirus (HPV) detection methods, such as PCR and immunohistochemistry (IHC), face barriers related to cost, infrastructure, and accessibility. Loop-mediated isothermal amplification (LAMP), a rapid, thermocycler-free nucleic acid amplification technique, combined with lateral flow dipstick (LFD) readout, enables simple, visual DNA detection. Objectives: This study developed and evaluated a novel LAMP-LFD assay to improve the detection of high-risk HPV16/18 in clinical specimens, with the aim of providing a rapid, sensitive, and user-friendly diagnostic tool for use in local healthcare settings. Methods: Cervical specimens were collected from women attending a gynecology clinic who were clinically suspected of having HPV16/18 infection, and DNA was extracted. HPV16/18 presence was initially confirmed by real-time PCR, which served as the reference standard for HPV16/18 detection. Subsequently, LAMP reactions using HPV16/18-specific primers were performed, and amplification products were detected using a molecular LFD system with gold-labeled antibodies. The same DNA samples were then analyzed using the LAMP-LFD assay. The sensitivity and specificity of the LAMP-LFD assay were compared with those of standard PCR methods. Diagnostic performance (sensitivity, specificity, predictive values, accuracy, and Cohen's kappa) was calculated by comparing LAMP-LFD results with real-time PCR. Included samples had adequate volume and acceptable DNA purity (A260/280 ratio: 1.8 - 2). All participants provided written informed consent. Exclusion criteria included sample contamination, insufficient volume, low-quality DNA, lack of signed consent, or failure to meet quality control standards to ensure sample integrity and reproducibility. Agreement between the LAMP-LFD assay and PCR was assessed using Cohen’s kappa coefficient, with 95% confidence intervals calculated. Results: Among the 50 clinical specimens, real-time PCR identified 20 HPV-positive and 30 HPV-negative specimens. Compared with PCR, the LAMP-LFD assay detected all PCR-positive samples but yielded two false-positive results for HPV16, resulting in high sensitivity (100%) and good specificity (93.3%). The specificity of the LAMP-LFD assay was also high, with minimal cross-reactivity observed between HPV16 and HPV18 targets. Compared with conventional PCR, the LAMP-LFD assay provided a faster turnaround time, required less specialized equipment, and was suitable for point-of-care applications. The sensitivity of LAMP is similar to or better than that of PCR (101 to 103 copies). LAMP specificity varies (67.5%-99.2%) and can be lower than that of PCR. Overall, the LAMP-LFD assay demonstrated 100% sensitivity and 93.3% specificity compared with real-time PCR. Conclusions: The developed LAMP-LFD assay is a rapid, sensitive, and user-friendly method for detecting high-risk HPV16 and HPV18 genotypes in clinical specimens. This approach has the potential to enhance HPV screening and cervical cancer prevention, particularly in resource-limited settings where access to conventional molecular diagnostics is restricted. The implementation of LAMP-LFD technology could facilitate timely diagnosis and management of HPV-associated diseases in local hospitals and field studies.