Single-tube four-target lateral flow assay detects human papillomavirus types associated with majority of cervical cancers

Abstract

Isothermal nucleic acid amplification methods have many advantages for use at the point of care. However, there is a lack of multiplexed isothermal amplification tests to detect multiple targets in a single reaction, which would be valuable for many diseases, such as infection with high-risk human papillomavirus (hrHPV). In this study, we developed a multiplexed loop-mediated isothermal amplification (LAMP) reaction to detect the three most common hrHPV types that cause cervical cancer (HPV16, HPV18, and HPV45) and a cellular control for sample adequacy. First, we characterized the assay limit of detection (LOD) in a real-time reaction with fluorescence readout; after 30 min of amplification the LOD was 100, 10, and 10 copies/reaction of HPV16, HPV18, and HPV45, respectively, and 0.1 ng/reaction of human genomic DNA (gDNA). Next, we implemented the assay on lateral flow strips, and the LOD was maintained for HPV16 and HPV18, but increased to 100 copies/reaction for HPV45 and to 1 ng/reaction for gDNA. Lastly, we used the LAMP test to evaluate total nucleic acid extracted from 38 clinical samples; compared to qPCR, the LAMP test had 89% sensitivity and 95% specificity. When integrated with sample preparation, this multiplexed LAMP assay could be useful for point-of-care testing.

Keywords: HPV; Lateral flow assay; Loop-mediated isothermal amplification; Multiplex.

Figure 1.. Triplex real-time LAMP reaction with SiHa (HPV16), HeLa (HPV18) and MS751 (HPV45) DNA as target.

A-C) Amplification curves for a range of input copies of HPV DNA extracted from (A) SiHa cells, (B) HeLa cells and (C) MS751 cells. D-F) Time to detection for the triplex reaction with DNA extracted from (D) SiHa cells, (E) HeLa cells and (F) MS751 cells. G-I) Melt curves from the products produced in the triplex reaction with DNA extracted from (G) SiHa cells, (H) HeLa cells, and (I) MS751 cells.

Figure 2.. Four-plex real-time LAMP reaction with SiHa (HPV16), HeLa (HPV18), MS751 (HPV45) and human genomic DNA (gDNA) as target.

A-D) Amplification curves for a range of input copies of HPV DNA extracted from (A) SiHa cells, (B) HeLa cells and (C) MS75 cells and (D) human genomic DNA. E-H) Time to detection of DNA target in the four-plex reaction with DNA extracted from (E) SiHa cells, (F) HeLa cells, (G) MS751 cells and (H) gDNA. I-L) Melt curves from the products produced in the four-plex reaction with DNA extracted from (I) SiHa cells, (J) HeLa cells, (K) MS751 cells and (L) gDNA.

Figure 3.. Signal-to-background ratio of lines on lateral flow strips run with product from the four-plex reaction with different input copies.

A) SiHa DNA, B) HeLa DNA, C) MS751 DNA, and (D) human genomic DNA.

Figure 4.. HPV copies per LAMP reaction versus positivity on lateral flow strips.

For the sample that was positive for both HPV18 and HPV45, the higher of the two concentrations is shown and represented by HPV45.