Detection of Mycobacterium avium subsp paratuberculosis in faeces using different procedures of pre-treatment for real-time PCR in comparison to culture

作者：Sting R^*; Hrubenja M; Mandl J; Seemann G; Salditt A; Waibel S

来源：Veterinary Journal, 2014, 199(1): 138-142.

DOI：10.1016/j.tvjl.2013.08.033

• Johne's disease
• Paratuberculosis
• Cattle
• Faeces
• Real-time PCR
• Culture

One of the most relevant aspects in the diagnosis of paratuberculosis (Johne's disease) in cattle is the availability of a method for the rapid and sensitive detection of Mycobacterium avium subsp. paratuberculosis (MAP) in order to facilitate the prompt removal of pathogen-shedding animals from a herd. To meet this requirement, methods for pre-treatment of bovine faecal samples and subsequent extraction of DNA for detection of MAP by real-time PCR were compared with MAP culture results. A total of 116 bovine faecal samples that showed weak (64.7%), moderate (18.1%) or strong (17.2%) growth of MAP on solid HEY medium were investigated.
For PCR, supernatants, sediments or bacterial pellets were obtained from faecal samples by pre-treatment before extraction of MAP DNA based on silica membranes or magnetic particles. Samples then were tested by MAP IS900 and ISMav2 real-time PCR with an analytical sensitivity of 6 and 28 genome equivalents (GE) per mL, respectively.
The best results were obtained by including a microfiltration step in the sample pre-treatment in combination with silica membrane-based mini-columns or magnetic particles for DNA extraction. This approach enhanced the detection rate of MAP in IS900 real-time PCR from 58.6% to 84.5% using silica membrane mini-columns and from 61.2% to 64.7% using magnetic particles.

• 出版日期2014-1