Rapid In Situ Hydrogel LAMP for On-Site Large-Scale Parallel Single-Cell HPV Detection

Abstract

Rapid human papillomavirus (HPV) screening is urgently needed for preventing and early diagnosis of cervical cancer in rural areas. To date, no HPV nucleic acid test (NAT) can be implemented within a single patient visit starting from clinical samples. Here, we develop a hydrogel loop-mediated isothermal amplification (LAMP) method in a fashion of large-scale parallel (about 1000 cells) in situ HPV DNA detection in clinical cervical exfoliated cells at the single-cell level. It can be used with a hotplate and smartphone to obtain HPV NAT results in less than 30 min, which is especially suitable for the on-site scenario. We apply this rapid HPV NAT on 40 clinical cervical exfoliated cell samples and compare the results to a clinical gold standard quantitative polymerase chain reaction (qPCR) method [area under curve (AUC), 1.00]. Meanwhile, our assay can provide HPV infection information for large-scale parallel single clinical cervical exfoliated cells, which cannot be received from traditional NAT methods. Our findings suggest the potential of in situ hydrogel LAMP as a powerful tool for clinical HPV screening and fundamental research.

StratoLAMP: Label-free, multiplex digital loop-mediated isothermal amplification based on visual stratification of precipitate

“…The primer and target sequences are listed in SI Appendix , Tables S3 and S4 . The detection of HPV 16 and HPV 18 was used to demonstrate the generalizability of the method, and the primers were synthesized by a company (Sangon Biotech Co., Shanghai) using the sequences in SI Appendix , Tables S5 and S6 ( 28 ). The concentrations of the primers were measured using a plate reader (Infinite M Nano, Tecan) at 280 nm and diluted using nuclease-free water according to need.…”

Portable, and ultrasensitive HR-HPV tests based on nucleic acid biosensors

“…Compared with the traditional PCR, LAMP-LFA can complete detection in a shorter time with no relying on large equipment, but its sensitivity and specificity are slightly lower than the standard, and it is prone to contamination during the reaction ( Landaverde et al., 2020 ). The FIP primer in the LAMP system was changed to a thiol group ( Wang et al., 2022 ) so that the product was covalently linked to the hydrogel matrix ( Figure 2C ). After the hydrogel becomes solid, LAMP reaction is carried out in-situ , and the combination of the amplification product and DNA dye is limited in the microstructure of the gel, forming bright spots on HPV infected cervical cells.…”