Highly sensitive and rapid identification of coxsackievirus A16 based on reverse transcription multiple cross displacement amplification combined with nanoparticle-based lateral flow biosensor assay

Abstract

IntroductionOne of the main pathogens responsible for human hand, foot, and mouth disease (HFMD), coxsackievirus A16, has put young children’s health at danger, especially in countries in the Asia-Pacific region. Early quick identification is essential for the avoidance and control of the disorder since there are no vaccinations or antiviral medications available to prevent and manage CVA16 infection.MethodsHere, we describe the creation of an easy, speedy, and accurate CVA16 infection detection approach using lateral flow biosensors (LFB) and reverse transcriptionmultiple cross displacement amplification (RT-MCDA). A group of 10 primers was developed for the RT-MCDA system in order to amplify the genes in an isothermal amplification device while targeting the highly conserved region of the CVA16 VP1 gene. Then, without requiring any extra tools, RT-MCDA amplification reaction products might well be detected by visual detection reagent (VDR) and LFB.ResultsThe outcomes showed that 64°C within 40 min was the ideal reaction setting for the CVA16-MCDA test. Target sequences with <40 copies might be found using the CVA16-MCDA. There was no cross-reaction among CVA16 strains and other strains. The findings demonstrated that the CVA16-MCDA test could promptly and successfully identify all of the CVA16-positive (46/220) samples identified by the traditional real-time quantitative polymerase chain reaction (qRT-PCR) assays for 220 clinical anal swab samples. The whole process, such as the processing of the sample (15 min), the MCDA reaction (40 min), and the documenting of the results (2 min), could be finished in 1 h.ConclusionThe CVA16-MCDA-LFB assay, which targeted the VP1 gene, was an efficient, simple, and highly specific examination that might be used extensively in rural regions’ basic healthcare institutions and point-of-care settings.

Multiple cross displacement amplification combined with nanoparticle-based lateral flow biosensor for rapid and sensitive detection of Epstein-Barr virus

“…MCDA, loop-mediated isothermal amplification (LAMP)] as summarized in Table 4 , PCR methods pose the disadvantages of high cost (relying on complex thermal cycling equipment for nucleic acid amplification), intricate manipulation (requiring skilled laboratory technicians for operation), and time-consuming protocols (with the entire detection process lasting 3-4 hours) ( Vera-Sempere et al., 1998 ), limiting their widespread application in the PoC settings. ( Wang et al., 2015 ; Cheng et al., 2023 ). Consequently, there is an imperative need to develop a rapid, accurate, and sensitive new technique for EBV detection and diagnosis.…”

“…A distinct advantage of MCDA is the ability to rapidly and specifically amplify nucleic acids at constant temperatures (61-69°C), eliminating the need for expensive thermal cycling equipment. The amplification process can be completed within 40 minutes using simple equipment such as a metal bath, water bath pot, or a thermal cup ( Wang et al., 2015 ; Cheng et al., 2023 ). Due to its abovementioned advantages, MCDA has found extensive utility in testing various pathogens, such as chlamydia trachomatis, monkeypox virus, and SARS-COV-2, achieving the remarkable limit of detection below 15 copies per reaction ( Li et al., 2020 ; Chen et al., 2022 ; Zhou et al., 2023 ).…”

“…The whole process of MCDA-LFB method for EBV DNA detection, including sample preparation (15 minutes), MCDA (40 minutes), and result reading (2 minutes) can be completed within 60 minutes. This approach eliminates the reliance on complex thermal cycling equipment, simplifies the pathogen detection process, and fulfills the demand for PoC detection and rapid in-field testing ( Wang et al., 2017 ; Jiao et al., 2019 ; Cheng et al., 2023 ).…”

“…Our research suggests that employing only 0.5 µl of amplified products is sufficient for effective detection by the LFB assay. Additionally, applying 70% ethanol and sodium hypochlorite solution promptly to the workspace after analyzing the results can assist in mitigating DNA contamination ( Cheng et al., 2023 ). Addressing the challenge of minimizing such contamination remains a critical aspect for future studies in this field.…”

“…While the MCDA assay may not quantity the in vivo replication of EBV copies as efficiently as the qPCR technique, it remains extensively adopted for detecting various pathogens in clinical, biological, and environmental samples. This popularity arises from its distinctive advantages, including rapidity, cost-effectiveness, repeatability, as well as high specificity and sensitivity, as demonstrated in various studies ( Wang et al., 2016a ; Cao et al., 2021 ; Cheng et al., 2023 ; Zhou et al., 2023 ). In the MCDA system, only a single DNA polymerase with strand displacement activity is employed to amplify the target templates at a constant temperature.…”