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

• Published in: Brazilian journal of microbiology Vol. 50; no. 4; pp. 1105 - 1114
• Main Authors: Punati, Rudrama Devi, Mallepaddi, Prudhvi Chand, Poonati, Revathi, Maity, Soumendra Nath, Sohal, Jagdip Singh, Polavarapu, Kavi Kishor B., Polavarapu, Rathnagiri
• Format: Journal Article
• Language: English
• Published: Cham Springer International Publishing 01.10.2019; Springer Nature B.V
• Subjects: Animal species; Animals; Assaying; Biomedical and Life Sciences; Biotin; Cattle; Cattle Diseases - diagnosis; Cattle Diseases - microbiology; Deoxyribonucleic acid; Detection limits; DNA; DNA Primers - genetics; Economic importance; Food Microbiology; Goat Diseases - diagnosis; Goat Diseases - microbiology; Goats; Intestine; IS900 gene; Life Sciences; Listeria; Livestock; Medical Microbiology; Microbial Ecology; Microbial Genetics and Genomics; Microbiology; Mycobacterium avium; Mycobacterium avium subsp. paratuberculosis - classification; Mycobacterium avium subsp. paratuberculosis - genetics; Mycobacterium avium subsp. paratuberculosis - isolation & purification; Mycology; Nucleic Acid Amplification Techniques - instrumentation; Nucleic Acid Amplification Techniques - methods; Paratuberculosis; Paratuberculosis - diagnosis; Paratuberculosis - microbiology; Pathogens; Point-of-Care Systems; Polymerase chain reaction; Salmonella; Sensitivity; Sensitivity and Specificity; Sheep; Sheep Diseases - diagnosis; Sheep Diseases - microbiology; Tuberculosis; Veterinary Microbiology - Research Paper; Yersinia enterocolitica; Lateral flow device (LFD); Loop-mediated isothermal amplification (LAMP); gene; Fluorescein isothiocyanate (FITC); Polymerase chain reaction (PCR); subsp; Point of care (POC); (; MAP; Insertion sequence 900 (IS900) gene; Mycobacterium avium subsp. paratuberculosis (MAP)
• ISSN: 1517-8382; 1678-4405
• DOI: 10.1007/s42770-019-00116-z

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.