LAMP technology: Rapid identification of Brucella and Mycobacterium avium subsp. paratuberculosis

Trangoni, Marcos David; Gioffré, Andrea; Cerón‐Cucchi, María Esperanza; Caimi, Karina; Ruybal, Paula; Zumárraga, Martín José; Cravero, Silvio

doi:10.1590/s1517-838246220131206

Cited by 16 publications

(12 citation statements)

References 27 publications

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“…; Trangoni et al . ). A problem of targeting the IS900 sequence with common PCR and LAMP is a cross‐reactivity with closely related mycobacteria such as M. scrofulaceum (Iwamoto et al .…”

Section: Discussionmentioning

confidence: 97%

“…Other studies on MAP detection with LAMP also targeted the IS900 gene. The detection limit of MAP was 0Á5 pg 25 ll À1 , 100 fg 25 ll À1 and 4 fg 25 ll À1 when targeting IS900 with LAMP (Enosawa et al 2003;Iwamoto et al 2003;Trangoni et al 2015). A problem of targeting the IS900 sequence with common PCR and LAMP is a cross-reactivity with closely related mycobacteria such as M. scrofulaceum (Iwamoto et al 2003).…”

Section: Discussionmentioning

confidence: 99%

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Development and validation of a loop‐mediated isothermal amplification assay—a rapid and sensitive detection tool forMycobacterium aviumsubsp.paratuberculosisin small ruminants

et al. 2019

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Aims The aim of this study was to design an assay for the identification of Mycobacterium avium subsp. paratuberculosis (MAP) to be used in faeces and milk samples of small ruminants with a loop‐mediated isothermal amplification (LAMP) system, as a time‐saving and user‐friendly method in contrast to real‐time PCR. Methods and Results For the detection of MAP in milk and faeces of small ruminants, we developed a set of primers, specific for the target gene ISMap02. The analytical sensitivity of LAMP, when targeting ISMap02, showed a DNA detection limit of 10 fg μl−1. After performing spiking experiments with two MAP reference strains, DSM 44133 and ATCC 19698T, the limit of detection, using the LAMP protocol described herein were 3·8 MAP CFU per ml milk and 12·5 MAP CFU per gram faeces. All LAMP results during the establishment of the assay were compared to those of the real‐time PCR results. An internal amplification control was incorporated into the assay to exclude false‐negative results produced and had no significant negative impact on the analytical sensitivity. Validation of the assay was confirmed by testing field samples of faeces and revising the results with real‐time PCR. Conclusion Our study conducted the first MAP detection system with a LAMP targeting ISMap02. Due to the positive results we encourage the use of LAMP in combination with ISMap02, when detecting MAP in faeces samples, as an alternative to targeting other genes as f57 or IS900. Further research on MAP detection in different matrices like raw milk, tissue or sperm with this system is recommended. Significance and Impact of the Study This study provides new achievements in MAP diagnostic. Especially small ruminants do not show signs of diarrhoea until the terminal stage of the illness. The greatest task in fighting MAP is to rule out animals, which shed MAP with faeces and milk before showing symptoms of Johne's disease. Worldwide there is a need to eradicate animals, which are low MAP shedders to stop the illness spreading in animal holdings. MAP detection with LAMP is time saving, easy to use, does not need expensive equipment, as, for example, PCR kits and can be used without access to laboratories. The target gene ISMap02 was shown to be a specific insertion element for MAP and is a reliable aim in future MAP detection studies.

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“…; Trangoni et al . ). A problem of targeting the IS900 sequence with common PCR and LAMP is a cross‐reactivity with closely related mycobacteria such as M. scrofulaceum (Iwamoto et al .…”

Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

Development and validation of a loop‐mediated isothermal amplification assay—a rapid and sensitive detection tool forMycobacterium aviumsubsp.paratuberculosisin small ruminants

et al. 2019

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“…Teknik deteksi molekuler melalui amplifikasi DNA seperti polymerase chain reaction (PCR) (OIE 2011), loop-mediated isothermal amplification (LAMP) (Trangoni et al 2015), finger printing, telah banyak digunakan untuk diagnosis Brucellosis pada hewan dan manusia. Melalui teknik molekuler PCR mampu mengidentifikasi biotipe B. abortus secara cepat, sensitif dan spesifik sehingga dapat digunakan untuk identifikasi kekerabatan genotipe isolat Brucella sp yang ada di dunia (Habtamu et al 2013).…”

Section: Pendahuluanunclassified

DNA Amplification Technique for Detection of Bovine Brucellosis

Noor¹

2018

WARTAZOA

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Brucellosis is one of cattle diseases which causes a very significant economic loss and categorized as zoonotic disease. Early detection of Brucellosis in livestock is very important to prevent the spread of disease to livestock and humans. The success of Brucellosis control depends on rapid, sensitive and specific detection methods. The aim of this paper is to review several methods of Brucellosis detection in cattle. Currently, the detection of Brucellosis in Indonesia is using serological and isolation methods. The latter method is the gold standard of Brucellosis diagnosis, however, its sensitivity is low. Therefore, molecular techniques with DNA amplification have been developed and applied in many countries both in livestock and humans because they are more sensitive, specific and rapid in detecting Brucella sp in blood, milk and semen samples. Various DNA amplification methods for detection of Brucellosis that have been developed including polymerase chain reaction (PCR), finger printing and loop-mediated isothermal amplificatiom (LAMP). Both PCR and LAMP are more sensitive and specific in detecting Brucella sp than conventional techniques. PCR technique has advantages in detecting Brucella sp species to serotype and biovar levels. In addition, PCR reagents are cheaper and easier to obtain than LAMP eventhough, LAMP procedure is simpler and faster.

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“…Two extra primers, LF (loop forward) and LB (loop backward), are also incorporated in order to accelerate the amplification of reaction as well as enhance the specificity [10]. LAMP has been shown to be a sensitive and specific method for the detection of veterinary pathogens [11][12][13][14]. The detection of M. agalactiae by LAMP was first reported by Rekha et al [15].…”

Section: Introductionmentioning

confidence: 99%

Validation of Loop-Mediated Isothermal Amplification (LAMP) Field Tool for Rapid and Sensitive Diagnosis of Contagious Agalactia in Small Ruminants

Tumino

Tolone

Parco

et al. 2020

Animals

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Contagious agalactia (CA), an infectious disease of small ruminants, caused by Mycoplasma agalactiae, is responsible for severe losses to dairy sheep production with substantial socioeconomic impacts on small-scale farmers. The diagnosis of CA is still problematic, time-consuming and requires well-equipped labs for confirmation of outbreaks. Therefore, rapid, accurate and cost-effective diagnostic tests are urgently needed. This work aims to validate a novel Loop-Mediated Isothermal Amplification (LAMP) test, based on the p40 target gene, for the detection of M. agalactiae in dairy sheep in order to confirm its potential practical use as a rapid and cheap field test. The LAMP system proposed in this study consists of a portable device composed of real-time fluorometer with the automatic interpretation of results displayed in a tablet. A total of 110 milk samples (90 positives and 20 negatives) were analysed to optimise the analysis procedure and to investigate the efficacy and robustness of the LAMP method. All samples were analysed using LAMP and conventional real-time PCR to compare the diagnostic sensitivity of the methods. The sensitivity of the LAMP was 10-fold higher than that of real-time PCR, with a detection limit up to 103 CFU/ml. The LAMP assay was able to detect M. agalactiae in 81 of 90 (90%, 95%CI 0.84–0.96) positive milk samples compared to 69 (77%, 95%CI 0.59–0.95) positive samples detected by real-time PCR; no positive signal occurred for any of the negative milk samples in either test. Therefore, the LAMP assay was found to be more sensitive than real-time PCR, low-cost, easy to perform, fast and not affected by contamination, indicating its potential as an effective diagnostic tool in the field level for the diagnosis of CA.

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LAMP technology: Rapid identification of Brucella and Mycobacterium avium subsp. paratuberculosis

Cited by 16 publications

References 27 publications

Development and validation of a loop‐mediated isothermal amplification assay—a rapid and sensitive detection tool forMycobacterium aviumsubsp.paratuberculosisin small ruminants

Development and validation of a loop‐mediated isothermal amplification assay—a rapid and sensitive detection tool forMycobacterium aviumsubsp.paratuberculosisin small ruminants

DNA Amplification Technique for Detection of Bovine Brucellosis

Validation of Loop-Mediated Isothermal Amplification (LAMP) Field Tool for Rapid and Sensitive Diagnosis of Contagious Agalactia in Small Ruminants

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