J A Donaghy1, J Johnston, M T Rowe. 1. Agri-Food and Biosciences Institute (AFBI), AFESD, Newforge Lane, Belfast, UK. john.donaghy@afbini.gov.uk
Abstract
AIMS: To develop a quantitative PCR assay for sensitive and specific detection of Mycobacterium avium ssp. paratuberculosis (Map) in a range of dairy products. METHODS AND RESULTS: TaqMan(®) assays were designed to target the IS900 and f57 genetic elements of Map. Both real-time PCR assays were integrated with the Adiapure(®) Map DNA extraction kit and assessed separately for the detection/quantification of Map in spiked milk, Cheddar cheese and milk powder. Assays were validated against Cheddar cheese samples containing known concentrations of Map. The IS900 qPCR assay was significantly more sensitive than the assay based on the f57 primer/probe. At a threshold cycle value of 38, limits of detection (LOD) for the IS900 qPCR assay were 0·6 CFU ml(-1), 2·8 CFU g(-1) and 30 CFU g(-1) for artificially contaminated pasteurized milk, whole milk powder and Cheddar cheese, respectively. The respective LOD's for the f57 assay were 6·2 CFU ml(-1), 26·7 CFU g(-1) and 316 CFU g(-1). CONCLUSION: The integrated Adiapure(®) extraction - IS900 real time assay described is a sensitive, quantitative method for the detection of Map in dairy products. This is the first study to consider qPCR as a quantitative estimation of Map-DNA in cheese and whole milk powder. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay developed allows sensitive detection and quantification of Map DNA in a range of dairy products which is valuable for the screening and surveillance of this potential zoonotic organism.
AIMS: To develop a quantitative PCR assay for sensitive and specific detection of Mycobacterium avium ssp. paratuberculosis (Map) in a range of dairy products. METHODS AND RESULTS: TaqMan(®) assays were designed to target the IS900 and f57 genetic elements of Map. Both real-time PCR assays were integrated with the Adiapure(®) Map DNA extraction kit and assessed separately for the detection/quantification of Map in spiked milk, Cheddar cheese and milk powder. Assays were validated against Cheddar cheese samples containing known concentrations of Map. The IS900 qPCR assay was significantly more sensitive than the assay based on the f57 primer/probe. At a threshold cycle value of 38, limits of detection (LOD) for the IS900 qPCR assay were 0·6 CFU ml(-1), 2·8 CFU g(-1) and 30 CFU g(-1) for artificially contaminated pasteurized milk, whole milk powder and Cheddar cheese, respectively. The respective LOD's for the f57 assay were 6·2 CFU ml(-1), 26·7 CFU g(-1) and 316 CFU g(-1). CONCLUSION: The integrated Adiapure(®) extraction - IS900 real time assay described is a sensitive, quantitative method for the detection of Map in dairy products. This is the first study to consider qPCR as a quantitative estimation of Map-DNA in cheese and whole milk powder. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay developed allows sensitive detection and quantification of Map DNA in a range of dairy products which is valuable for the screening and surveillance of this potential zoonotic organism.
Authors: Iker A Sevilla; Elena Molina; Maitane Tello; Natalia Elguezabal; Ramón A Juste; Joseba M Garrido Journal: Front Microbiol Date: 2017-06-09 Impact factor: 5.640