Establishment of the ultra-sensitive detection method for HIV integrase by rolling circle amplification combined with DNA nanosensor

doi:10.19871/j.cnki.xfcrbzz.2023.06.001

Abstract

Abstract: Objective A novel isothermal amplification assay (REIAD) for human immunodeficiency virus (HIV) integrase (IN) was developed using RCA and DNA nanosensors. Method Purified recombinant HIV IN or packaged HIV particles were extracted, and the LTR terminal sequence of att site and double-stranded DNA ring identified by IN were designed with the activity of HIV IN as the target, and then combined with DNA nanosensor detection technology. The LTR sequence was fixed on the surface of the slide and catalyzed to insert the end of the double-stranded DNA ring to produce a DNA ring with incision for interface RCA amplification and fluorescent labeling probe detection, and the HIV integrase isothermal amplification assay (REIAD) was established. The sensitivity of the detection method was evaluated and the detection limit was determined by detecting the gradient dilution of HIV IN and packaging HIV particles. The specificity of the assay was evaluated by detecting the effects of gradient dilution mouse leukemia virus (MLV) and HIV IN inhibitor raltegravir. By adding HEK293 whole cell extract and 10% fetal bovine serum cell culture medium to gradient diluted HIV IN, the feasibility of detecting plasma samples from patients was evaluated. Result The detection of graded diluted HIV IN and packaged HIV particles was completed by REIAD method at 37℃, integration for 50 minutes, and rolling ring amplification for 30 minutes. The minimum detection limits of recombinant HIV IN and packaged HIV particles were up to 15 fmol and 2 TU/μl, and there was no cross-reaction with MLV. It has good specificity. Conclusion The REIAD detection method established in this study has high sensitivity, good specificity, simple operation and low platform requirements, and is expected to provide a new detection method for the early detection of HIV virus.

Key words: Nanosensor, HIV testing, Integrase, Roll circle amplification

CLC Number:

R447

Chen Fuming, Wang Jing, Li Furong. Establishment of the ultra-sensitive detection method for HIV integrase by rolling circle amplification combined with DNA nanosensor[J]. Electronic Journal of Emerging Infectious Diseases, 2023, 8(6): 1-8.

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