Submitted to: Journal of Microbiological Methods
Publication Type: Peer reviewed journal
Publication Acceptance Date: 4/6/2005
Publication Date: 5/31/2005
Citation: Ravva, S.V., Stanker, L.H. 2005. Real-time quantitative pcr detection of mycobacterium avium subsp. paratuberculosis and differentiation from other mycobacteria using sybr green and taqman assays. Journal of Microbiological Methods. 63(2005) 305-317. Interpretive Summary: Mycobacterium avium subsp. paratuberculosis (Map), the causative agent of Johne's disease in cattle, is responsible for a $250 million annual loss to the US dairy industry. Such losses can be minimized by early detection and culling of infected cattle and by preventing the oral-fecal routes of transmission, as the pathogen survives in feces for longer than a year. Early detection of infection is difficult since the fecal culture methods, the current gold standard, can take as long as 4 to 6 months to confirm the presence of pathogens. Lack of rapid and sensitive methods hitherto hindered the efforts to monitor the environmental fate and transfer of Map from animal manure to crops. We have developed sensitive real-time sequence detection (qPCR) methods (SYBR Green and TaqMan) for the detection of Map. These methods take only few hours to identify Map as compared to 6 months by culture methods. Map cells can be quantified directly from growth media (7H9 broth) by using the SYBR Green assay. This method is sensitive to detect 0.016 cells. Both SYBR Green and TaqMan assays are highly specific for the detection of Map. They did not detect other mycobacteria that are likely to be present in environmental samples. The rapid qPCR methods developed represent significant contribution for sensitive detection of environmental strains of Map and are suitable for monitoring the fate and transfer of Map in dairy and agricultural environments.
Technical Abstract: Sensitive real-time sequence detection methods based on two different chemistries were developed for Mycobacterium avium subsp. paratuberculosis (Map), the causative agent of Johne's disease in cattle. One is based on the detection of SYBR Green bound to PCR products and the second method is more specific, detecting the cleavage of a fluorogenic (TaqMan) probe bound to a target sequence during primer extension phase. Novel primers and probes that amplify small fragments (<80 bp) of the Map specific insertion sequence, IS900 were designed. Both the SYBR green and TaqMan assays are sensitive, able to detect 3.5 fg of DNA extracted from Map strain ATCC19698. This amount of DNA corresponds to the detection of 0.17 cells. Map cells were quantified directly from 7H9 broth using the SYBR Green assay and compared to dilutions of DNA extracted from an equivalent number of cells. The SYBR Green assay of 7H9 broth resulted in a minimum detectable limit of 0.016 cells (equivalent to 0.33 fg of DNA). Media ingredients were not observed to interfere with the assay. Since no extraction step was necessary in the direct cell measurements, direct detection was ten fold more sensitive than detection of extracted DNA. Both SYBR Green and TaqMan assays are highly specific for the detection of Map. They did not detect any closely related members of the avium complex, other species of mycobacteria, or related genera that are likely to be present in environmental samples. No reporter signal was detected during TaqMan assays performed with 100 pg of template DNA from the non-Map organisms.