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Development and evaluation of a Mycobacterium avium subspecies paratuberculosis (MAP) specific multiplex PCR assay


Tasara, T; Hoelzle, L E; Stephan, R (2005). Development and evaluation of a Mycobacterium avium subspecies paratuberculosis (MAP) specific multiplex PCR assay. International Journal of Food Microbiology, 104(3):279-287.

Abstract

There are specificity questions inherent in most of the current PCR systems that amplify the MAP IS900 sequence as an indicator for Mycobacterium avium subspecies paratuberculosis (MAP) presence due to false positives associated with IS900-like sequences that exists in other Mycobacterium species besides MAP. We developed a multiplex PCR system designed to enhance specificity for MAP detection in a single PCR reaction. This PCR assay co-amplifies the mycobacterium species 16S rRNA gene, MAP IS900 and f57 sequences. The assay incorporates an internal PCR amplification control (IC) template system enabling PCR amplification conditions to be assessed in each reaction. The assay's specificity was confirmed by testing 10 isolates of MAP and 59 isolates of other bacterial species. In parallel we also applied on the same samples, one of the established nested PCR systems that targets only the IS900 sequence. The multiplex PCR assay was highly specific for MAP, the nested PCR system was also positive with several other mycobacterium species. In this context, we report for the first time false positive IS900 PCR signals in Mycobacterium chelonae, Mycobacterium terrae and Mycobacterium xenopi strains associated with one of the established PCR systems widely used for MAP detection. Finally, the potential application of this multiplex PCR system in milk analysis is demonstrated through analysis of raw milk samples artificially contaminated with MAP.

There are specificity questions inherent in most of the current PCR systems that amplify the MAP IS900 sequence as an indicator for Mycobacterium avium subspecies paratuberculosis (MAP) presence due to false positives associated with IS900-like sequences that exists in other Mycobacterium species besides MAP. We developed a multiplex PCR system designed to enhance specificity for MAP detection in a single PCR reaction. This PCR assay co-amplifies the mycobacterium species 16S rRNA gene, MAP IS900 and f57 sequences. The assay incorporates an internal PCR amplification control (IC) template system enabling PCR amplification conditions to be assessed in each reaction. The assay's specificity was confirmed by testing 10 isolates of MAP and 59 isolates of other bacterial species. In parallel we also applied on the same samples, one of the established nested PCR systems that targets only the IS900 sequence. The multiplex PCR assay was highly specific for MAP, the nested PCR system was also positive with several other mycobacterium species. In this context, we report for the first time false positive IS900 PCR signals in Mycobacterium chelonae, Mycobacterium terrae and Mycobacterium xenopi strains associated with one of the established PCR systems widely used for MAP detection. Finally, the potential application of this multiplex PCR system in milk analysis is demonstrated through analysis of raw milk samples artificially contaminated with MAP.

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28 citations in Web of Science®
30 citations in Scopus®
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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:05 Vetsuisse Faculty > Institute of Veterinary Bacteriology
05 Vetsuisse Faculty > Institute of Food Safety and Hygiene
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Date:2005
Deposited On:05 Jun 2009 13:11
Last Modified:05 Apr 2016 13:14
Publisher:Elsevier
ISSN:0168-1605
Publisher DOI:https://doi.org/10.1016/j.ijfoodmicro.2005.03.009
PubMed ID:15982769
Permanent URL: https://doi.org/10.5167/uzh-18778

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