Development and Evaluation of a Novel Multicopy-Element-Targeting Triplex PCR for Detection of Mycobacterium avium subsp. paratuberculosis in Feces

Abstract

The enteropathy called paratuberculosis (PTB), which mainly affects ruminants and has a worldwide distribution, is caused by Mycobacterium avium subsp. paratuberculosis. This disease significantly reduces the cost-effectiveness of ruminant farms, and therefore, reliable and rapid detection methods are needed to control the spread of the bacterium in livestock and in the environment. The aim of this study was to identify a specific and sensitive combination of DNA extraction and amplification to detect M. avium subsp. paratuberculosis in feces. Negative bovine fecal samples were inoculated with increasing concentrations of two different bacterial strains (field and reference) to compare the performance of four extraction and five amplification protocols. The best results were obtained using the JohnePrep and MagMax extraction kits combined with an in-house triplex real-time PCR designed to detect IS900, ISMap02 (an insertion sequence of M. avium subsp. paratuberculosis present in 6 copies per genome), and an internal amplification control DNA simultaneously. These combinations detected 10 M. avium subsp. paratuberculosis cells/g of spiked feces. The triplex PCR detected 1 fg of genomic DNA extracted from the reference strain K10. The performance of the robotized version of the MagMax extraction kit combined with the IS900 and ISMap02 PCR was further evaluated using 615 archival fecal samples from the first sampling of nine Friesian cattle herds included in a PTB control program and followed up for at least 4 years. The analysis of the results obtained in this survey demonstrated that the diagnostic method was highly specific and sensitive for the detection of M. avium subsp. paratuberculosis in fecal samples from cattle and a very valuable tool to be used in PTB control programs.

Characteristics and Epidemiological Investigation of Paratuberculosis in Dairy Cattle in Tai’an, China

“…It indicates that type C is the predominant type in cattle in Shandong Province, China. It was consistent with most other previous studies, and type C is the predominant type in the world [24,25,[32][33][34][35][36][37][38][39][40].…”

Detection of Mycobacteria, Mycobacterium avium Subspecies, and Mycobacterium tuberculosis Complex by a Novel Tetraplex Real-Time PCR Assay

“…Identification of M. avium subsp. paratuberculosis isolates was achieved using a triplex real-time PCR (30). The remaining M. avium subspecies were identified by PCR amplification of IS1245, IS901, and IS901-flanking region (FR300) genomic targets (33).…”

A comparative study evaluating the efficacy of IS_MAP04 with IS900 and IS_MAP02 as a new diagnostic target for the detection of Mycobacterium avium subspecies paratuberculosis from bovine faeces

“…Detection limits of 10 3 to 10 2 CFU/g for IS_MAP02 were previously observed (Stabel and Bannantine, 2005;Sevilla et al, 2014), indicating that IS_MAP02 was able to achieve detection sensitivities comparable to IS900. One study recorded a detection limit of 10 1 CFU/g for both IS900 and IS_MAP02 when probes were used, but this detection limit was not achieved consistently (Sevilla et al, 2014). A weak fluorescent FAM signal was recorded for MAP detection limit of 10 1 CFU/g for IS_MAP04 TaqMan qPCR, but this detection limit was not achieved consistently.…”

“…A weak fluorescent FAM signal was recorded for MAP detection limit of 10 1 CFU/g for IS_MAP04 TaqMan qPCR, but this detection limit was not achieved consistently. Studies that used the three-copy ISMav2 element recorded MAP detection limits of 10 3 CFU/g Sevilla et al, 2014). Even after MAP DNA extraction optimizations, a detection limit of 10 2 CFU/g was recorded .…”

“…Multiplex PCR assays that incorporated singlecopy or multi-copy targets were developed that aide MAP diagnostic tests in sensitivity and specificity (Tasara et al, 2005;Glanemann et al, 2008;Schönenbrücher et al, 2008;Sevilla et al, 2014). The potential value of implementing the multi-copy IS_MAP04 element in such a duplex or multiplex qPCR assay could be of significant value and warrant further investigation, especially when additional validation is required with a MAP diagnostic test.…”