Field applicable detection of hepatitis B virus using internal controlled duplex recombinase‐aided amplification assay and lateral flow dipstick assay

Abstract

Hepatitis B virus (HBV) is a widespread blood‐borne pathogen associated with the complication of liver cirrhosis and hepatocellular carcinoma, particularly in south‐east Asian and African countries where HBV is highly endemic and the budget and resources are limited. Therefore, simple, rapid, and portable field detection methods are crucial to efficiently control HBV infection. In this study, using heat‐treated DNA, we developed two‐field applicable detection assays for HBV based on recombinase‐aided amplification (RAA). One was an internal controlled duplex RAA assay using a portable real‐time fluorescence detection device, another was an instrument‐free visual observation assay using lateral flow dipsticks. The entire experimental time was greatly shortened to less than 40 minutes at 39.0°C. The sensitivities, specificities, and clinical performance of both assays were evaluated. Compared with quantitative polymerase chain reaction assay as a reference, our results demonstrated that the two RAA‐based assay obtained 97.18% and 95.77% of sensitivity, respectively, and the specificity was 100%, by testing a total of 157 serum samples with HBsAg positive. We conclude that the advantages of rapidity, simplicity, portability, and visualization of proposed two assays make them great potentials in point‐of‐care testing of HBV infection by untrained people in resource‐limited situations.

Reverse Transcription Recombinase-Aided Amplification Assay With Lateral Flow Dipstick Assay for Rapid Detection of 2019 Novel Coronavirus

“…The RAA requires only a simple thermostatic device (constant temperature 37–42°C) and a short reaction time (30 min). DNA amplification can be detected by gel electrophoresis, oligo chromatographic lateral flow dipstick (LFD) ( Bai et al., 2020 ; Xiong et al., 2020 ), or real-time fluorescence methods ( Wang J. et al., 2020 ; Wang W. et al., 2020 ), considered as a powerful detection means for Point of Care Test (POCT). Here, we developed a molecular POCT method for SARS-CoV-2 based on a reverse transcription RAA assay coupled with lateral flow dipstick (RT-RAA/LFD) conducted at 39.0°C for 30 min.…”

Rapid Detection of Pathogens in Wound Exudate via Nucleic Acid Lateral Flow Immunoassay

“…In addition, a competitive IAC was integrated that excludes false negative results and, in combination with the flow control, ensures the validity of the test result. Until now only a part of the published RPA approaches incorporated an IAC [ 46 , 81 , 82 , 83 , 84 , 85 , 86 , 87 ]. The main advantages over the non-competitive method are: (1) avoiding the risk of undesired interaction of multiple primers and (2) a more accurate reflection of the amplification, due to the identical primers and reaction conditions.…”

“…Among the isothermal amplification methods that can be interacted with LFA-based analysis, especially the recombinase polymerase amplification (RPA) has gained in importance as a cost-effective and reliable technology for POC diagnostics [ 37 ]. In the last years several articles were published that successfully combined RPA and LFA [ 36 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 ]. However, only a minority of them showed direct amplification of target nucleic acids from crude lysate [ 45 , 46 , 47 , 48 , 49 , 50 ].…”

“…In the last years several articles were published that successfully combined RPA and LFA [ 36 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 ]. However, only a minority of them showed direct amplification of target nucleic acids from crude lysate [ 45 , 46 , 47 , 48 , 49 , 50 ]. Most of these publications used purified nucleic acids [ 38 , 39 , 40 , 41 , 42 , 43 , 44 ] or commercial nucleic acid isolation kits [ 36 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 ] that require at least 6–13 hands-on steps and/or substantial sample preparation time (10–60 min), which adds to the total process time.…”

Development of a Recombinase-Aided Amplification Assay for Rapid Detection of Human Norovirus GII.4

“…The reaction is typically completed in approximately 30 min at 37-42 °C. RAA has been successfully applied in the detection of pathogens (Bai and Ma et al, 2020;Li and Yu et al, 2020;Wang and Cui et al, 2020; and single nucleotide polymorphisms (SNPs) (Duan and Li et al, 2018). Owing to its speed, low-cost, and high sensitivity, RAA is highly suitable for clinical applications, and is a potential assay for the point-of-care testing (POCT) for foodborne pathogens.…”