Detection of <i>Mycobacterium Avium</i> Subsp. <i>Paratuberculosis</i> in Bovine Fecal Samples: Comparison of Three Polymerase Chain Reaction—Based Diagnostic Tests with a Conventional Culture Method

Taddei, Simone; Robbi, C.; Cesena, C.; Rossi, Ilenia; Schiano, Emiliana; Arrigoni, N.; Vicenzoni, G.; Cavirani, S.

doi:10.1177/104063870401600603

Cited by 39 publications

(33 citation statements)

References 27 publications

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“…Isolation of MAP DNA from fecal samples was performed using a MAP DNA extraction and purification commercial kit (QIAamp DNA Blood minikit, Qiagen), and the purified DNA sequences were tested by real-time PCR (Adiagene, Saint Brieuc, France), which amplified a Map IS900. The sensitivity for the fecal PCR is highly dependent on the fecal shedding status (32% for light shedders to 91% for heavy shedders) but the overall sensitivity of the test is 60% and specificity is 100% (Taddei et al, 2004). The blood samples were analyzed by using a paratuberculosis indirect ELISA (Institut Pourquier, Montpellier, France) that used PPA3 as antigen.…”

Section: Genetic Association Studymentioning

confidence: 99%

Identification of single nucleotide polymorphisms in the bovine solute carrier family 11 member 1 (SLC11A1) gene and their association with infection by Mycobacterium avium subspecies paratuberculosis

Ruiz-Larrañaga

Garrido

Manzano

et al. 2010

Journal of Dairy Science

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Johne's disease is a chronic enteritis caused by Mycobacterium avium ssp. paratuberculosis (MAP) that causes substantial financial losses for the cattle industry. Susceptibility to MAP infection is reported to be determined in part by genetic factors, so markerassisted selection could help to obtain bovine populations that are increasingly resistant to MAP infection. Solute carrier family 11 member 1 (SLC11A1) was adjudged to be a potential candidate gene because of its role in innate immunity, its involvement in susceptibility to numerous intracellular infections, and its previous association with bovine MAP infection. The objectives of this study were to carry out an exhaustive process of discovery and compilation of polymorphisms in SLC11A1 gene, and to perform a population-based genetic association study to test its implication in susceptibility to MAP infection in cattle. In all, 57 single nucleotide polymorphisms (SNP) were detected, 25 of which are newly described in Bos taurus. Twenty-four SNP and two 3′-untranslated region polymorphisms, previously analyzed, were selected for a subsequent association study in 558 European Holstein-Friesian animals. The SNP c.1067C > G and c.1157-91A > T and a haplotype formed by these 2 SNP yielded significant association with susceptibility to MAP infection. The c.1067C > G is a nonsynonymous SNP that causes an amino acid change in codon 356 from proline to alanine (P356A) that could alter SLC11A1 protein function. This association study supports the involvement of SLC11A1 gene in susceptibility to MAP infection in cattle. Our results suggest that SNP c.1067C > G may be a potential causal variant, although functional studies are needed to assure this point.

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Section: Genetic Association Studymentioning

confidence: 99%

Identification of single nucleotide polymorphisms in the bovine solute carrier family 11 member 1 (SLC11A1) gene and their association with infection by Mycobacterium avium subspecies paratuberculosis

Ruiz-Larrañaga

Garrido

Manzano

et al. 2010

Journal of Dairy Science

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“…According to numerous authors, PCR analysis was unable to match the sensitivity of fecal culture for identifying minute quantities of M. avium subsp. paratuberculosis (49,56). An increased sensitivity of the PCR analysis can be achieved by improved DNA extraction protocols guaranteeing the efficient removal of PCR inhibitors such as phytic acid, polyphenolics, polysaccharides, and hemin (4,5,18,37,52).…”

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confidence: 99%

New Triplex Real-Time PCR Assay for Detection ofMycobacterium aviumsubsp.paratuberculosisin Bovine Feces

Schönenbrücher

Abdulmawjood

Failing

et al. 2008

Appl Environ Microbiol

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In the present study, a robust TaqMan real-time PCR amplifying the F57 and the ISMav2 sequences of Mycobacterium avium subsp. paratuberculosis from bovine fecal samples was developed and validated. The validation was based on the recommendations of International Organization for Standardization protocols for PCR and real-time PCR methods. For specificity testing, 205 bacterial strains were selected, including 105 M. avium subsp. paratuberculosis strains of bovine, ovine, and human origin and 100 non-M. avium subsp. paratuberculosis strains. Diagnostic quality assurance was obtained by use of an internal amplification control. By investigating six TaqMan reagents from different suppliers, the 100% detection probability was assessed to be 0.1 picogram M. avium subsp. paratuberculosis DNA per PCR. The amplification efficiency was 98.2% for the single-copy gene F57 and 97.8% for the three-copy insertion sequence ISMav2. The analytical method was not limited due to instrument specificity. The triplex real-time PCR allowed the reliable detection of M. avium subsp. paratuberculosis DNA using the ABI Prism 7000 sequence detection system, and the LightCycler 1.0. TaqMan mgb and locked nucleic acid fluorogenic probes were suitable for fluorescent signal detection. To improve the detection of M. avium subsp. paratuberculosis from bovine fecal samples, a more efficient DNA extraction method was developed, which offers the potential for automated sample processing. The 70% limit of detection was assessed to be 10 2 CFU per gram of spiked bovine feces. Comparative analysis of 108 naturally contaminated samples of unknown M. avium subsp. paratuberculosis status resulted in a relative accuracy of 98.9% and a sensitivity of 94.4% for fecal samples containing <10 CFU/g feces compared to the traditional culture method.Mycobacterium avium subsp. paratuberculosis is the causative agent of ruminant paratuberculosis (Johne's disease), which has become a worldwide problem. There is controversy regarding its zoonotic capacity and potential role in the human Crohn's disease (14). Because of these reasons, a rapid, costeffective, and automated diagnosis of this pathogen is a high priority task not only for animal breeders but also for the food production industry and for public health institutions. Culturebased detection of M. avium subsp. paratuberculosis is timeconsuming, labor-intensive, and therefore not suitable. The PCR has been shown to be a powerful tool in microbiological diagnostics (12,43). Guidelines for diagnostic quality assurance have been set by the International Organization for Standardization (7,8). Standardized PCR and real-time PCR methods should fulfill numerous criteria, including a high detection probability with regard to the investigated matrix, the sample preparation, and DNA extraction as well as high specificity, robustness, and user-friendly protocols. In this context the real-time PCR technology offers the possibility for a onestep and closed-tube reaction (13).As a molecular reference marker for the confirm...

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“…Application of real-time, nested, and TaqMan PCR methods, along with immunomagnetic bead separation of the microorganism and dot blot hybridization of PCR products, to assays for the detection of M. avium subsp. paratuberculosis have improved the sensitivity of detection and have made it possible to perform semiquantitative analysis (3,8,14,15,18,28). However, the majority of these assays have been based upon the amplification of the M. avium subsp.…”

mentioning

confidence: 99%

Development of a Nested PCR Method Targeting a Unique Multicopy Element, ISMap02, for Detection ofMycobacterium aviumsubsp.paratuberculosisin Fecal Samples

Stabel

Bannantine

2005

J Clin Microbiol

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This study describes the development of a nested PCR assay that uses a unique element (ISMap02) for Mycobacterium avium subsp. paratuberculosis that is present at six copies within the genome. In addition, the sensitivity of the assay with this element was compared to the sensitivity of detection of the IS900 element in both conventional and real-time PCR assays. The specificity of the ISMap02 element was evaluated by PCR of the DNA extracted from isolates of M. avium subsp. paratuberculosis and M. avium subsp. avium, as well as DNA from M. fortuitum, M. scofulaceum, M. phlei, M. smegmatis, and M. gordonae. Only M. avium subsp. paratuberculosis DNA was detectable after amplification with the ISMap02 primers. The sensitivity of detection for the ISMap02 element in either a conventional or a real-time PCR format was less than 100 fg DNA or 10 2 CFU/ml in serial titration curves with pure bacteria. These results were comparable to those obtained for the IS900 element. Experimental spiking of a negative fecal sample followed by M. avium subsp. paratuberculosis DNA extraction resulted in detection thresholds of 10 2 CFU/g for the IS900 element and 10 3 CFU/g for the ISMap02 element by using a real-time PCR format, but this sensitivity dropped 10-fold for both elements in a conventional PCR format. Analyses of fecal samples obtained from naturally infected animals demonstrated a sensitivity for the detection of M. avium subsp. paratuberculosis DNA by use of the ISMap02 element similar to that achieved by use of the IS900 element when it was used in a conventional PCR format. The real-time PCR format improved the levels of detection of both elements, but not to a significant degree. In conclusion, the ISMap02 element provides a very sensitive and specific alternative as a diagnostic reagent for use in PCR assays for the detection of paratuberculosis.

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Detection of Mycobacterium Avium Subsp. Paratuberculosis in Bovine Fecal Samples: Comparison of Three Polymerase Chain Reaction—Based Diagnostic Tests with a Conventional Culture Method

Cited by 39 publications

References 27 publications

Identification of single nucleotide polymorphisms in the bovine solute carrier family 11 member 1 (SLC11A1) gene and their association with infection by Mycobacterium avium subspecies paratuberculosis

Identification of single nucleotide polymorphisms in the bovine solute carrier family 11 member 1 (SLC11A1) gene and their association with infection by Mycobacterium avium subspecies paratuberculosis

New Triplex Real-Time PCR Assay for Detection ofMycobacterium aviumsubsp.paratuberculosisin Bovine Feces

Development of a Nested PCR Method Targeting a Unique Multicopy Element, ISMap02, for Detection ofMycobacterium aviumsubsp.paratuberculosisin Fecal Samples

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