Development and validation of a triplex real-time PCR for rapid detection and specific identification of M. avium sub sp. paratuberculosis in faecal samples

Irenge, Léonid M.; Walravens, Karl; Govaerts, Marc; Godfroid, Jacques; Rosseels, Valérie; Huygen, Kris; Gala, Jean‐Luc

doi:10.1016/j.vetmic.2008.09.087

Cited by 45 publications

(36 citation statements)

References 37 publications

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“…A extração de DNA do fragmento foi realizada por Kit comercial da Qiagen (QIAamp ® DNA Mini Kit para tecidos). A reação de qPCR foi realizada usando os Primers e sondas para o gene is900 (F-5'-TGCTGATCGCCTTGCTCA-3' e R-5'-GGGCCTGA-TCGGCGATGAT-3'; S-5'-FAM-CCG GGC AGC GGC TGC TTT ATA TTC-3'-BHQ1) (Sigma ® ); e os Primers e sondas para f57 (F-5'-TTCATC-GATACCCAAACTCAGAGA-30 e R-5'-GTTCGCCGCTTGAATGGT-3'; S-5'Cy5-5'-TGCCAGCCGCCCACTCGTG-3'-BHQ2) (Sigma ® ) (Irenge et al 2009). Na reação foram utilizados 2µl de solução de DNA, 12,5µl do Mix (RealQ-PCR-RT dUTP-UNG Master Mix kit) (Ampliqon, Dinamarca), Primers na concentração de 0,6 pmol/µl e sondas na concentração de 0,4 pmol/µl, 0,5µl de H 2 O e 2µl de MgCl 2 (50nM) (Invitrogen ® ), num volume total de reação de 25µl.…”

Section: Methodsunclassified

Diagnóstico de paratuberculose por biópsia retal em búfalos

et al. 2015

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Paratuberculosis in a herd of buffaloes was studied in the municipality of São Mateus, Maranhão, Brazil. Rectal biopsies were performed in 140 male and female Murrah, Mediterranean and crossbreed buffaloes older than 3 years. Postmortem examination of 11 buffaloes was performed to compare the rectal biopsies with possible lesions in mesenteric nodes and the intestine. The history of the herd and clinical examination revealed progressive weight loss and non-responsive antimicrobial diarrhea, dehydration and submandibular edema. Rectal biopsies showed in six buffaloes microscopically suggestive lesions for paratuberculosis through hematoxilin-eosin staining (HE), characterized by moderate multifocal granulomatous enteritis with epithelioid cell infiltration. In four buffaloes Langhans giant cells were found. In 15 buffaloes lymphocytic infiltrate was observed in the lamina propria of the large intestine. Ziehl-Neelsen staining (ZN) revealed in 4.3% (6/140) acid-fast bacilli in the rectal mucosa. Real time PCR amplified to 5.71% (7/140) Mycobacterium avium subsp. paratuberculosis (Map) DNA. 11 buffalos were submitted to postmortem examination, gross examination revealed augmented mesenteric nodes with whitish areas in the cut surface. The mucosa of the small intestine was irregular and thickened, with evident traverse folds and Peyer plates. The brownish intestinal content was fluid, the ileocecal valve area thickened and edematous with evident lymphatic vessels. Histological lesions in the mesenteric lymph node and small intestine four buffalo were compatible with those already described in the literature, and presented acid-fast bacilli by ZN staining and amplification of Map genetic material in qPCR. The concordance between the rectal biopsy and the postmortem samples was in agreement with the Kappa test (K=0.792) and was considered substantial or high. The rectal biopsy showed to be promising and can be used by practitioners, together with other techniques, for antemortem diagnosis in buffalo herds suspected to be affected by paratuberculosis. Samples obtained by rectal biopsy were suitable for ZN staining and real time PCR. Rectal biopsy can be used as a tool for diagnosis and control of Johne´s disease in buffaloes to select and eliminate positive animals within the herd and decrease gradually the spread of Map.

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Section: Methodsunclassified

Diagnóstico de paratuberculose por biópsia retal em búfalos

et al. 2015

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“…의 잠복기를 거친 후 발현된다 [14]. 임상 증상이 나타나기 전 증상이 없는 상태에서 감염된 개체는 분변을 통해 MAP 를 지속해서 배출하게 되는데, 이를 통해 축사 환경을 오염 시키고 fecal-oral route를 통해 다른 개체의 감염을 유발한 다.…”

Section: 박홍태·박현의·신민경·조용일·유한상unclassified

Discovery of antigens for early detection of Mycobacterium avium subsp. paratuberculosis and analysis of characteristics using bioinformatics tools

Park¹,

Park²,

Shin³

et al. 2015

Korean Journal of Veterinary Research

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: Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is one of the most widespread and economically important diseases in cattle. Current diagnostic methods are based on the detection of anti-MAP antibodies in serum or isolation of the causative agent. However, these techniques are often not applicable for cases of subclinical infection due to relatively low sensitivity. Therefore, finding new antigen candidates that strongly react with the host immune system had been attempted. To effectively detect infection during the subclinical stage, several antigen candidates were selected based on previous researches. Characteristics of the selected antigen candidates were analyzed using bioinformatics-based prediction tools. A total of nine antigens were selected (MAP0862, MAP3817c, MAP2077c, MAP0860c, MAP3954, MAP3155c, MAP1204, MAP1087, and MAP2963c) that have MAPspecific and/or high immune responses to infected animals. Using a transmembrane prediction tool, five of the nine antigen candidates were predicted to be membrane protein (MAP3817c, MAP3954, MAP3155c, MAP1087, and MAP1204). Some of the predicted protein structures identified using the I-TASSER server shared similarities with known proteins found in the Protein Data Bank database (MAP0862, MAP1204, and MAP2077c). In future studies, the characteristics and diagnostic efficiency of the selected antigen candidates will be evaluated.

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“…The quantification of fluorescence emitted during each PCR cycle is proportional to the amount of DNA. Other variants are the PCR amplification system called loop-mediated isothermal amplification (LAMP), which does not require the use of a thermocycler (Enosawa et al, 2003), and the triple real-time PCR (TRT-PCR), designed by Irenge et al (2009).…”

Section: Detection Of Map-specific Gene Segmentmentioning

confidence: 99%