Comparison of Real-Time, Quantitative PCR with Molecular Beacons to Nested PCR and Culture Methods for Detection ofMycobacterium aviumsubsp.paratuberculosisin Bovine Fecal Samples

Abstract

An automated PCR with fluorescent probes (molecular beacons) detected Mycobacterium avium subsp. paratuberculosis in bovine feces. When the PCR was compared with culture in testing 41 fecal samples, kappa scores of 0.94 to 0.96, a sensitivity of 93 to 96%, and a specificity of 92% were obtained. Results were quantitated by using a standard curve derived from a plasmid containing IS900. A minimum quantity of 1.7 ؋ 10

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

“…Although PCR-based screening methods performed on (pooled) faecal samples are nowadays offered in some countries, a combination of direct examination of ZN-stained smears, serological methods and culture is still mainly used routinely by veterinary diagnostic laboratories for identifying infected farms (Harris and Barletta, 2001). The rationale for using DNA-based Mapspecific assays on faecal material stems from the expected higher sensitivity and faster performance compared to the culture (Bogli-Stuber et al, 2005;Fang et al, 2002;Huntley et al, 2005;Tasara et al, 2005;Tripathi et al, 2006). Using this experimental Map infection model allowed us to monitor Map excretion over almost 3 years time in the same Map exposed animals and to compare the performance of conventional methods with our DNA-based detection assay on serial faecal samples.…”

“…However, their specificity is compromised by close antigenic similarity between Map and other mycobacteria (Collins, 1996;Manning and Collins, 2001). PCR methods have made the rapid identification of Map in clinical samples possible, by reducing detection time (Englund et al, 1999;Fang et al, 2002;Ikonomopoulos et al, 2004;Motiwala et al, 2003Motiwala et al, , 2004Rajeev et al, 2005). In most studies, the main DNA target has been the insertion sequence IS900, initially considered to be a Map-specific marker (Green et al, 1989).…”

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

“…Pinedo et al (2008) found that when comparing PCR tests with bacteriological culture and ELISA, Kappa values were 0.39 and 0.01, respectively. In contrast, Fang et al (2002), when correlating IS900 Q-PCR with bacteriological culture, a Kappa value of 0.94 was found. In this study, Kappa values in the first, second and fourth samplings ranged from 0.53 to 0.73 indicating a moderate correlation, although the Kappa value in third sampling reached 0.22 indicating a fair correlation.…”

“…Quantification of amplicon copies by q-PCR in DNA samples taken from bovine feces positive to IS900. signs of the disease (Park et al, 2016;Fang et al, 2002). Previously, Khol et al (2010), had followed for 380 days a 2.5 year-old cow that had become naturally infected with Map, sampling feces on 9 occasions.…”

“…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).…”