Development and evaluation of LAMP-coupled lateral flow device for the detection of MAP in livestock at point of care resource-limited areas

Abstract

The Mycobacterium avium subspecies paratuberculosis (MAP) causes paratuberculosis (Johne's disease), a systemic and chronic inflammation of intestine that affects bovine, small ruminants like goat and sheep. The disease has a greater economic importance in cattle and in small ruminants. But its effective control is impeded due to lack of rapid and accurate diagnostics. The present study is aimed at developing a LAMP-coupled lateral flow device (LFD) for rapid detection of paratuberculosis in livestock animal species such as cattle and in small ruminants at resource-limited areas. LAMP primers with biotin and FITC end tags were designed for IS900 gene specific for MAP. To determine sensitivity of LAMP assay, 10-fold serial dilutions were made from 10 ng/μl MAP stock DNA and were compared with PCR. The detection limits of LAMP-coupled LFD were defined and reactions were repeated for reproducibility. The specificity was evaluated using other infectious bacteria such as M. bovis, M. tuberculosis, Brucella abortus, Leptospira interrogan, Yersinia enterocolitica, Salmonella typhimurium, Listeria monocytogens, and Staphylococcus aureus. A total of 95 samples turned positive for LAMP-coupled LFD out of 389 fecal samples. All the cultural-positive and PCR-positive samples showed positive in LAMP-coupled LFD. Nine samples with negative cultures turned positive in LAMP assay. The overall sensitivity and specificity of the LAMP-coupled LFD assays were 100% and 97.02% respectively in comparison with the culture as the gold standard method. The sensitivity detection limit of developed assay was 10 fg/μl and specificity was 100%. This assay successfully detected MAP not only by using bacterial DNA but also in clinical fecal samples. The clear band formation at control and test positions was observed on LAMP-coupled LFD. The developed assay is a simple, rapid, easy to perform, and is very useful in early diagnosis of Mycobacterium avium subsp. paratuberculosis at point of care resource-limited areas. Keywords Loop-mediated isothermal amplification (LAMP). Lateral flow device (LFD). Insertion sequence 900 (IS900) gene. Mycobacterium avium subsp. paratuberculosis (MAP). Fluorescein isothiocyanate (FITC). Polymerase chain reaction (PCR) .

The development and use of Actiphage® to detect viable mycobacteria from bovine tuberculosis and Johne’s disease‐infected animals

“…These difficulties mean that alternative, more rapid detection methods for mycobacteria are often used. PCR has proven to be a powerful tool for the specific detection of mycobacterial signature DNA sequences from a variety of sample matrices (Dinnes et al, 2007;Priyadarshini et al, 2017), and most recently loop-mediated isothermal amplification (LAMP) methods have been successfully developed for the specific detection of MAP (Punati et al, 2019). However, when used as a diagnostic test, efficient release of DNA is crucial for the success of any PCR or nucleic acid-based method, especially when this is being used to quantify the number of cells present in a sample.…”

Efficacy of LAMP assay for Mycobacterial spp. detection to prevent treatment delays and onset of drug resistance: a systematic review and meta-analysis

“…In 53.33% (n = 16) of studies, more than one detection method was used. In 16.66% (n = 5) of studies, combination of three methods was used while in only two studies (6.66%), combination of four different methods was reported ( 17 , 18 ).…”

A rapid detection method on-site for Zygosaccharomyces based on loop-mediated isothermal amplification (LAMP) combined with a lateral flow dipstick (LFD)

“…Furthermore, it is simpler to perform, and merely use constant-temperature equipment, such as a water bath or Mini Dry Bath within 60 min. This can be potentially used for tests in the field (Punati et al, 2019;Joon et al, 2019;Tumino et al, 2020). In addition, LAMP has been extensively applied in food-borne pathogens, allergens, and genetically modified organisms in food analysis (Hara-Kudo et al, 2010;Rostamkhani et al, 2011;Saharan et al, 2015;Shi et al, 2017;Yuan et al, 2018;Li et al, 2019;Zhuo et al, 2019;Tumino et al, 2020).…”

“…In addition, LAMP has been extensively applied in food-borne pathogens, allergens, and genetically modified organisms in food analysis (Hara-Kudo et al, 2010;Rostamkhani et al, 2011;Saharan et al, 2015;Shi et al, 2017;Yuan et al, 2018;Li et al, 2019;Zhuo et al, 2019;Tumino et al, 2020). Furthermore, LAMP products traditionally observed by gel electrophoresis and hydroxynaphthol blue (HNB) or SYBR are either difficult to distinguish by fluorescent dyes, which need special equipment and the process is time-consuming, or easily exposed to aerosol pollution due to the opening of the lid (Shi et al, 2017;Punati et al, 2019;Zhang et al, 2019). Moreover, the amplified products are visualized directly on an LFD with one or two bands, which is more suitable for on-site detection (Shi et al, 2017).…”