Evaluation of a high-throughput nucleic acid extraction method for the detection of Mycobacterium avium subsp. paratuberculosis in bovine fecal samples by PCR

• Published in: Journal of veterinary diagnostic investigation Vol. 33; no. 2; pp. 375 - 378
• Main Authors: Thirumalapura, Nagaraja R., Feria, Willard, Hue, Eric, Zellers, Corey, Tewari, Deepanker
• Format: Journal Article
• Language: English
• Published: Los Angeles, CA SAGE Publications 01.03.2021
• Subjects: Animals; Bacteriological Techniques - methods; Bacteriological Techniques - veterinary; Brief Reports; Cattle; Cattle Diseases - diagnosis; Feces - microbiology; High-Throughput Nucleotide Sequencing - methods; High-Throughput Nucleotide Sequencing - veterinary; Mycobacterium avium subsp. paratuberculosis - isolation & purification; Paratuberculosis - diagnosis; Polymerase Chain Reaction - veterinary; paratuberculosis; DNA extraction; Mycobacterium avium subsp. paratuberculosis; Johne’s disease; feces; PCR
• ISSN: 1040-6387; 1943-4936
• DOI: 10.1177/1040638721991118

Johne’s disease (paratuberculosis) is an economically important disease of cattle worldwide. The disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), a fastidious gram-positive bacterium. PCR is increasingly used in diagnostic laboratories for the detection of MAP in fecal samples given the rapid test turnaround time and sensitivity and specificity comparable to fecal culture. However, efficient extraction of DNA for sensitive detection of MAP by PCR is affected by the complex lipid-rich cell wall of MAP and the presence of PCR inhibitors in feces. We evaluated a high-throughput nucleic acid extraction method (MagMAX core nucleic acid purification kit with mechanical lysis module) in conjunction with an hspX gene PCR for the detection of MAP from bovine fecal samples, which resulted in correct identification of all negative (13 of 13) and positive (35 of 35) proficiency test samples obtained from the National Veterinary Services Laboratories. In addition, all 6 negative and 50 of 51 positive diagnostic specimens tested were categorized correctly.