Comparative evaluation of LAMP and Nested-PCR for the diagnosis of bovine paratuberculosis

Safi, Shahabeddin; Heidarnejhad, Orkideh; Mosavari, Nader; Sakha, Mehdi; Afshar, Davoud; Moazami, Lale; Meshkat, Mohse; Keshavarz, Rouhollah; Taheri, Mohammad Mohammad; Pajoohi, Reza Aref; Tadayon, Keyvan; Reshadi, Samerand; Dashtipour, Shojaat

doi:10.1016/j.ijmyco.2014.09.015

Cited by 4 publications

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“…Recently, the use of LAMP in the detection of MAP was developed and similar LAMP results were reported [37]. In the present study, the LAMP-coupled LFD demonstrated a detection limit of 10 fg/μl of MAP DNA from the LAMP-amplified fecal samples and the developed assay successfully detected MAP not only in the bacterial cultures but also in the clinical fecal samples and the specificity was 100%.…”

Section: Discussionsupporting

confidence: 82%

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

et al. 2019

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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) .

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Section: Discussionsupporting

confidence: 82%

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

et al. 2019

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“…It is operated under a constant temperature (60 to 65 °C), and it eliminates the need for specialized thermal cycler equipment ( 32 ). This technique was first used to detect bacteria, and it is being used increasingly for the rapid detection of other bacteria, such as E. coli ( 33 – 36 ). An important advantage of LAMP is its ability to amplify specific sequences of DNA under isothermal conditions.…”

Section: Introductionmentioning

confidence: 99%

Visual Detection of Enterohemorrhagic Escherichia coli O157:H7 Using Loop-Mediated Isothermal Amplification

et al. 2016

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IntroductionEscherichia coli O157:H7, an important foodborne pathogen, can cause serious renal damage, which can also lead to mortality. Since a rapid and sensitive method is needed to identify this pathogenic agent, we evaluated Loop-Mediated Isothermal Amplification Assay (LAMP) to detect Escherichia coli O157:H7.MethodsWe used six primers that specifically identified the rfbE gene. To examine the sensitivity of the method, different dilutions were subjected to the LAMP reaction. Other bacterial strains also were investigated to determine the specificity of the test. The turbidity of the amplified products was assayed by visual detection. The amplified products were detected by addition of SYBR Green II to the reaction tubes.ResultsAmplification products were observed as a ladder-like pattern on the agarose gel. A white turbidity emerged in the positive tubes. Under UV light, the positive samples were green, whereas the negative samples were orange. The detection limit of the LAMP was 78 pg/tube, and this indicated that it was 100 times more sensitive than PCR for the detection of EHEC. No LAMP products were detected when template DNA of non-EHEC strains were used, suggesting high specificity of the LAMP assay.ConclusionThe results indicated that the LAMP assay is a valuable diagnostic assay to identify EHEC O157:H7. In addition, the simplicity, sensitivity, specificity, and rapidity of this assay make it a useful method to diagnose pathogens in primary labs without any need for expensive equipment or specialized techniques.

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“…LAMP test was carried out in tubes containing 25μl of final reaction mixture containing 10× Thermopol® Reaction Buffer (New England Biolabs, USA), MAP specific LAMP primers (F3, B3, FIP, BIP, FLP and BLP), MgSO 4 (New England Biolabs, USA), dNTP mix (New England Biolabs, USA), Betaine (Sigma Aldrich), 8 U Bst DNA polymerase 2 (New England Biolabs, USA), calcein dye (Sigma Aldrich), MnCl 2 , KCl, Triton X-100, nuclease free water and MAP DNA template. Based on the previous studies (Heidarnejhad et al, 2015;Safi et al, 2015), different combinations of a range of concentrations of each constituent (dNTPs (0.4 mmol/L ~1.6 mmol/L), betaine (0.8 mmol/L ~1.4 mmol/L), MgSO 4 (2 mmol/L ~9 mmol/ L) were examined for amplification efficiency. The amplification reaction was carried out in a thermal cycler between 59  C to 65  C within 30 to 80 min, to determine the optimal incubation temperature and time.…”

Section: Optimization Of Reaction Conditions For Lampmentioning

confidence: 99%

“…Lately, there was an interest in developing LAMP test for JD. Gene sequences like IS900 (Safi et al, 2015), HSPX (Enosava et al, 2003 and F57 (Enosava et al, 2003) have been used to develop LAMP primers. Firstly, Enosava et al (2003) optimized LAMP test on bacterial culture (20 different MAP strains) for MAP using IS900, HspX and F57.…”

Section: Development and Standardization Of Visual Loop Mediated Isotmentioning

confidence: 99%

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Development and Standardization of Visual Loop Mediated Isothermal Amplification (LAMP) Essay for Specific Diagnosis of Johne’s Disease

Singh

Gupta

Singh

et al. 2020

IJAR

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Mycobacterium avium subspecies paratuberculosis (MAP), causative agent of Johne’s disease (JD) is chronic granulomatous enteritis affecting domestic and wild ruminants. Since, MAP is not killed by pasteurization, it has been isolated from commercially pasteurized milk and milk products resulting exposure of human population to this pathogen through milk. Control and eradication of JD is considered difficult because of its insidious nature and lack of early, rapid and accurate diagnostic tests. Therefore in present study, a visual loop-mediated isothermal amplification (LAMP) assay method has been developed using a total of six primers including 2 outer (F3 and B3), 2 inner (FIP and BIP) and 2 loop (LF and LB) primers specific for MAP for the first time on ‘S 5’ strain of Mycobacterium avium subsp. paratuberculosis ‘Indian Bison type’ biotype. After laboratory standardization, final optimized reaction performed at 65°C for 45 min was achieved after titration of incubation time, temperature conditions and the reporter dye calcein. Sensitivity and specificity of the LAMP assay was optimized and compared with traditional IS900 PCR. The sensitivity of LAMP assay was found to detect 10fg (100%) of DNA and 95.7% specificity was recorded with respect to traditional IS900 PCR. Comparison showed that LAMP had 98.6% and 96.1% sensitivity and specificity of 96.1% and 92.3%, with respect to microscopy and culture exhibiting ‘Almost perfect’ strength of agreement. The study concluded that LAMP assay was a reliable and sensitive diagnostic test to detect MAP infection in feces and can also be used for the ‘mass screening’ of the milk samples with the help of less expertise.

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Comparative evaluation of LAMP and Nested-PCR for the diagnosis of bovine paratuberculosis

Cited by 4 publications

References 0 publications

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

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

Visual Detection of Enterohemorrhagic Escherichia coli O157:H7 Using Loop-Mediated Isothermal Amplification

Development and Standardization of Visual Loop Mediated Isothermal Amplification (LAMP) Essay for Specific Diagnosis of Johne’s Disease

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