TY - JOUR
T1 - Single-tube four-target lateral flow assay detects human papillomavirus types associated with majority of cervical cancers
AU - Barra, Maria
AU - Chang, Megan
AU - Salcedo, Mila P.
AU - Schmeler, Kathleen
AU - Scheurer, Michael
AU - Maza, Mauricio
AU - Lopez, Leticia
AU - Alfaro, Karla
AU - Richards-Kortum, Rebecca
N1 - Publisher Copyright:
© 2024 Elsevier Inc.
PY - 2024/5
Y1 - 2024/5
N2 - 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.
AB - 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.
KW - HPV
KW - Lateral flow assay
KW - Loop-mediated isothermal amplification
KW - Multiplex
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U2 - 10.1016/j.ab.2024.115480
DO - 10.1016/j.ab.2024.115480
M3 - Article
C2 - 38331373
AN - SCOPUS:85184842986
SN - 0003-2697
VL - 688
JO - Analytical Biochemistry
JF - Analytical Biochemistry
M1 - 115480
ER -