Detection assay for HPV16 and HPV18 by loop-mediated isothermal amplification with lateral flow dipstick tests

Abstract

Cervical cancer is the third highest cause of death in developing countries and most commonly results from high‑risk human papillomavirus (HR‑HPV) infection. Among HR‑HPV genotypes, HPV16 and HPV18 are the most prevalent in cervical cancers. Therefore, the present study aimed to develop a detection assay for HPV16 and HPV18 infection using loop‑mediated isothermal amplification (LAMP) with lateral flow dipstick (LFD) tests. This assay is a simplified, user‑friendly method for the visual detection of HPV genotypes. DNA was extracted from clinical tissue samples, and HPV genotyping was performed using nested polymerase chain reaction (PCR). The clinical samples were demonstrated to include 44 HPV16‑positive, 18 HPV18‑positive and 80 HPV‑negative samples. All DNA samples were also used as templates for a LAMP reaction (30 min at 65˚C), and subsequently, a fluorescein isothiocyanate‑labelled probe was hybridized with the reaction product. Finally, the LFD test was performed. The sensitivity of the LAMP‑LFD test was higher than LAMP‑turbidity, exhibiting up to 100‑fold higher sensitivity for HPV16 and 10‑fold higher sensitivity for HPV18. All HPV16 and HPV18‑positive samples generated positive results in both assays; however, 22 samples detected as HPV‑negative by LAMP‑turbidity exhibited positive results by LAMP‑LFD test (22 of 80 samples). Therefore, these samples were further examined using quantitative (q)PCR. The results demonstrated that 20 out of the 22 samples designated positive by LAMP‑LFD, but negative by LAMP turbidity, gave a positive result with qPCR, while the remaining 2 samples were negative by qPCR. The present results suggested that LAMP‑LFD provided higher sensitivity than LAMP‑turbidity and nested PCR. Thus, the LAMP‑LFD test developed in the present study might be useful for the detection of HPV16 and HPV18 in local hospitals.

Development of a Novel Multiple Cross-Linking Spiral Amplification for Rapid and Sensitive Detection of HPV16 DNA

“…In the case of sensitivity, the MCLSA proposed here could detect as little as 5.4 × 10 1 copies per reaction of HPV-16 DNA, which was 10-fold more sensitive than the LAMP and qPCR assay. Interestingly, we obtained a lower LoD values when amplified using the previously reported LAMP primers [7], which sensitivity was similar to that of the LAMP combined with lateral flow dipstick tests reported by Kumvongpin et al [13]. Factors such as professional operators, changes in chemical reagents, and primer purity will affect the slight changes of amplification results to some extent.…”

“…based techniques are complicated, time consuming, and involve a thermal cycling for heating and cooling [13]. Thus, there is still a need for a rapid, cost-effective, and precise method to determine HR-HPV infection status in HNSCC.…”

Human Papillomavirus – Role in Cervical Carcinogenesis and Methods of Detection

“…Další dipstick test s fluorescenční detekcí využil namísto PCR tzv. izotermální amplifikaci [22]. Na rozdíl od PCR reakce, která vyžaduje termocykler (z důvodu cyklování teplot) a delší čas, dokáže speciální polymeráza, při izotermální amplifikaci amplifikovat DNA při konstantní teplotě (obvykle kolem 60 °C) během kratší doby (za < 1 hod).…”

Molecular Diagnostics of Dengue by Reverse Transcription-Loop Mediated Isothermal Amplification (RT-LAMP) in Disposable Polyester-Toner Microdevices

“…Other advantages, such as sensitivity, specificity, cost-effective instrumentation, and less time consumption, contribute to the use of LAMP in molecular diagnostics. [24][25][26][27] Furthermore, LAMP products can be detected quickly by visual detection through turbidity, 27,28 lateral flow, 29,30 metal precipitation, 27,31 and fluorescence using a deoxyribonucleic acid (DNA) intercalator dye. 12,32 The RT-LAMP assay has been successfully used for molecular dengue diagnostics.…”