Development of an F57 Sequence-Based Real-Time PCR Assay for Detection ofMycobacterium aviumsubsp.paratuberculosisin Milk

Abstract

A light cycler-based real-time PCR (LC-PCR) assay that amplifies the F57 sequence of Mycobacterium avium subsp. paratuberculosis was developed. This assay also includes an internal amplification control template to monitor the amplification conditions in each reaction. The targeted F57 sequence element is unique for M. avium subsp. paratuberculosis and is not known to exist in any other bacterial species. The assay specificity was demonstrated by evaluation of 10 known M. avium subsp. paratuberculosis isolates and 33 other bacterial strains. The LC-PCR assay has a broad linear range (2 ؋ 10 1 to 2 ؋10 6 copies) for quantitative estimation of the number of M. avium subsp. paratuberculosis F57 target copies in positive samples. To maximize the assay's detection sensitivity, an efficient strategy for isolation of M. avium subsp. paratuberculosis DNA from spiked milk samples was also developed. The integrated procedure combining optimal M. avium subsp. paratuberculosis DNA isolation and real-time PCR detection had a reproducible detection limit of about 10 M. avium subsp. paratuberculosis cells per ml when a starting sample volume of 10 ml of M. avium subsp. paratuberculosis-spiked milk was analyzed. The entire process can be completed within a single working day and is suitable for routine monitoring of milk samples for M. avium subsp. paratuberculosis contamination. The applicability of this protocol for naturally contaminated milk was also demonstrated using milk samples from symptomatic M. avium subsp. paratuberculosis-infected cows, as well as pooled samples from a dairy herd with a confirmed history of paratuberculosis.

Quantification of Mycobacterium avium subsp. paratuberculosis from the tissues of challenged mice using SYBR Green real time PCR assay for the assessment of vaccine efficacy

“…Moreover, F57 gene is reported to be unique for MAP and is not known to exist in any other bacterial species (Poupart et al, 1993;Kralik et al, 2010). The specificity of F57 gene for MAP has been supported by studies with conventional PCR protocols (Vansnick et al, 2004) and in real time PCR assays (Tasara and Stephan, 2005;Herthnek and Bolske, 2006).…”

“…Real time PCR is a sensitive method for detection of MAP and minimizes the risk of false positive results due to amplicon contamination (Herthnek and Bolske, 2006). Several researchers used real time PCR for quantification of MAP in tissues and milk (Nelli et al, 2008;Tasara and Stephan, 2005).…”

Türkiye Trakya Bölgesindeki Süt İşletmelerinden Toplanan Fekal ve Çiğ Süt Örneklerinde Mycobacterium avium subsp. paratuberculosis (MAP) İncelemesi

“…Insertion sequence IS900 is a reference marker for confirmation of MAP whereas it may lead to cross-reactions and possible false positive results [24] . Due to this fact, another alternative genetic element F57 is used [41] . In contrast to IS900, F57 is not similar to genes on other related organisms [42] .…”

Detection of Mycobacterium avium subspecies paratuberculosis excretion in bovine feces, using quantitative real time polymerase chain reaction (Q-PCR)

“…Other genetic markers have been evaluated for detecting Map, such as the SF57, ISMav2 and HspX sequences, but they have the limitation that they are only present in 1 to 6 copies within the Map genome, therefore sensitivity is reduced, although specificity is increased. To increase specificity when using primers for IS900, these should be designed so that they amplify regions that are close to the 5' end of this inserted sequence since it is highly conserved (Cook and Britt, 2007;Tasara and Stephan, 2005;Kim et al, 2002;Fang et al, 2002).…”