Simple and field amenable loop-mediated isothermal amplification-lateral flow dipstick assay for detection of west Nile virus in human clinical samples

Abstract

Aim: West Nile encephalitis caused by infection with the West Nile virus (WNV) is endemic in many regions of the world and is a global public health threat. The aim of this report was to develop a method using colorimetry-based reverse-transcription loop-mediated isothermal amplification (cRT-LAMP) and RT-LAMP combined with lateral-flow dipstick (LFD) for rapidly detecting WNV in low-infrastructure settings. Methods and Results:The primers for the cRT-LAMP and RT-LAMP-LFD assays were designed based on env gene of the WNV. Primers concentration, temperature and time were optimized for cRT-LAMP and RT-LAMP-LFD. The diagnostic performance of the cRT-LAMP and RT-LAMP-LFD assays was evaluated using human serum samples from 110 patients who were clinically suspected to be infected with WNV. The RT-LAMP was performed in a heating block at 63°C for 40 min. The LAMP amplicons were visible in the lateral-flow dipstick within 5 min. The detection limit of the developed cRT-LAMP and RT-LAMP-LFD assays was 10 copies and this assay showed a high degree of specificity for WNV. Compared with quantitative real-time RT-PCR assay, the kappa value of cRT-LAMP and RT-LAMP-LFD were 0.970. Conclusions:These results showed that the newly developed WNV-specific cRT-LAMP and RT-LAMP-LFD assays can be employed as an alternative method for screening of WN-suspected human samples. The results revealed that the assay could potentially identify the virus without interference from human serum samples. Collectively, all results revealed that cRT-LAMP and RT-LAMP-LFD assays offer a suitable field-based diagnosis of WNV. Significance and Impact of the Study:The cRT-LAMP and LAMP-LFD platform for the detection of WNV is rapid, accurate and simple-to-perform. Our present method has not only a short turnaround time but also avoided cross-contamination problem. Moreover, the use of simple lateral flow dipsticks broadens its application potential for the point-of-care use in resource-limited settings during outbreak situations. To the best of our knowledge, this is the first report for the development of cRT-LAMP and LAMP-LFD assays for rapid, simple, specific and sensitive detection of WNV using human clinical samples and EvaGreen dye.

Establishment and application of a loop‐mediated isothermal amplification−lateral flow dipstick (LAMP–LFD) method for detecting Clostridium piliforme

“…Because LAMP involves the use of four or six primers, the likelihood of primer dimer formation is high, resulting in false‐positive results owing to template‐free amplification (Rolando et al., 2020 ; Schneider et al., 2019 ). LFD is used to detect LAMP products (Mescolini et al., 2022 ; Tomar et al., 2022 ). LAMP–LFD is more specific to the biotin and fluorescein labelling of LAMP products obtained by molecular probe hybridization and combined with a double sandwich.…”

“…The high tolerance of LAMP to enzyme inhibitors allows for onsite assessment of clinical and biological samples without sample enrichment (Das et al., 2022 ). LAMP assays have been successfully used to detect the pathogens of many infectious diseases, including SARS‐CoV‐2 (Nuchnoi et al., 2023 ; Ooi et al., 2022 ), porcine epidemic diarrhoea virus and porcine circovirus type 2 (Areekit et al., 2022 ), West Nile virus (Tomar et al., 2022 ), herpesvirus of turkeys (Mescolini et al., 2022 ), Treponema pallidum (Becherer et al., 2020 ), Haemophilus ducreyi (Becherer et al., 2020 ), Ascaridia galli (Panich et al., 2023 ), Toxoplasma gondii (Xue et al., 2021 ), Ebola virus (Bonney et al., 2020 ) and Helicobacter pylori (Horiuchi et al., 2019 ; Panich et al., 2023 ). However, LAMP may result in non‐specific binding owing to the formation of primer dimers (Garg et al., 2022 ).…”

“…In a study, a novel technique based on LAMP and a lateral flow dipstick (LFD) was developed for early detection of C. piliforme . This technique reduced the risk of false‐positive results caused by primer dimer formation (Tomar et al., 2022 ). Labelling the primers enables the visualization of LAMP products in combination with LFD, and the results can be judged by observing the control and the test lines, reducing the likelihood of false‐positive results caused by the non‐specific binding of amplification products.…”