Detection of Mycobacterium avium subsp. paratuberculosis in ovine faeces by direct quantitative PCR has similar or greater sensitivity compared to radiometric culture

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

“…The fact that, MAP being detected in tissues from vaccinated challenged mice by qPCR assay suggests that the vaccine is not able to prevent the animals from infection, but only could limit its spread and hence progression of disease.It was suggested that the real time quantitative PCR has very high analytical sensitivity for MAP and detects even non viable MAP (Kawaji et al, 2007). In conclusion, this study supports the use of real time PCR for the rapid detection and quantification of MAP from tissues of challenge models and hence useful for testing the vaccines/ drug efficacy in limiting the MAP infection and progression of disease.…”

“…Real time PCR quantification of MAP in the tissues: Real time qPCR for MAP F57 gene was standardized with the above mentioned primers and SYBR green chemistry using Eppendorf Real Plex Master cycler following the previously published method with modifications (Kawaji et al, 2007). Absolute quantification of MAP in the tissues was performed based on the calibration curve derived with a series of 10-fold diluted standards ranging from 10 9 to 1 copy of recombinant plasmid.…”

“…MAP load in the specific organs (liver, spleen and mesenteric lymph node) estimated by culture and histopathology lesion score are good indicators of infection status of mice following challenge with MAP (Veazey et al, 1995;Chen et al, 2008;Park et al, 2008) but are time taking. Kawaji et al (2007) reported that qPCR assay has similar sensitivity to a gold standard radiometric culture assay. 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).…”

Fecal culture and two fecal-PCR methods for the diagnosis of Mycobacterium avium subsp. paratuberculosis in a seropositive herd

“…Some PCR systems that target IS900 also can give false-positive results with DNA from mycobacteria other than MAP and with DNA from other types of organisms (Möbius et al, 2008a;2008b). Due to this, new protocols avoiding cross-reactions have been reported (Bull et al, 2003;Herthnek and Bölske, 2006;Kawaji et al, 2007). In response to the uncertainty about the specificity of PCR systems that target IS900 for the identification of MAP, the use of several other target sequences for MAP identification systems have been proposed: ISMap02, ISMav2, hspX, locus 255, and F57 (Stabel and Bannantine, 2005;Slana et al, 2009;Kralik et al, 2010;Sidoti et al, 2011;Keller et al, 2014).…”

The diagnostic performance of an antibody enzyme-linked immunosorbent assay using serum and colostrum to determine the disease status of a Jersey dairy herd infected with Mycobacterium avium subspecies paratuberculosis

“…The HT-J PCR was originally based on research performed using direct quantitative real-time (q)PCR, where 8 out of 13 experimentally infected FC-positive sheep, 24 out of 40 MAP-exposed, FC-negative sheep, and 68 out of 69 MAP-exposed, FCpositive sheep tested positive by PCR. 5 A 2014 study that investigated the HT-J PCR using Australian cattle and sheep populations found that in 870 cattle, 111 were FC positive and 124 were HT-J PCR positive, while in 507 sheep, 111 were FC positive and 117 were HT-J PCR positive. 7 However, that study 7 also attempted to identify fecal samples that were known to contain very low numbers of MAP, as the identification of subclinically infected animals is of particular importance with regard to maintenance of infection.…”