Development and validation of a direct real-time PCR assay for Mycobacterium bovis and implementation into the United States national surveillance program
- Subject Areas
- Microbiology, Molecular Biology, Veterinary Medicine, Infectious Diseases
- bovine tuberculosis, PCR, abattoir surveillance, Mycobacterium bovis
- © 2016 Dykema et al.
- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ PrePrints) and either DOI or URL of the article must be cited.
- Cite this article
- 2016. Development and validation of a direct real-time PCR assay for Mycobacterium bovis and implementation into the United States national surveillance program. PeerJ PrePrints 4:e1703v1 https://doi.org/10.7287/peerj.preprints.1703v1
Abattoir surveillance for bovine tuberculosis, which consists of identifying and submitting granulomas for histopathology and mycobacterial culture was the primary means for detecting new cases in the United States. Mycobacterial culture is expensive, labor intensive and identifies cases weeks after slaughter, hampering trace back efforts. To address this inefficiency, the United States Department of Agriculture replaced culture with real-time PCR for screening granulomas. The objectives of this paper were to describe the development and validation of this PCR as well as the performance of the assay during the first year of implementation. Using archived culture and histologically positive tissue, the sensitivity was 0.96 (95% CI: 0.89, 0.99) for the Mycobacterium tuberculosis complex primer-probe set and 0.89 (95% CI: 0.80, 0.95) for the Mycobacterium bovis specific primer-probe set. Specificity, estimated during by side by side testing was 0.998 (95% CI: 0.994, 1.000). After implementation, 6124 samples over 54 weeks were tested and all 36 histopathology positive samples were detected including 2 additional cases initially misclassified by histopathology. It appeared that specificity may have declined during post validation testing with 47/6086 signaling positive but not confirmed by either histopathology or culture. While PCR implementation has significantly improved the efficiency of the US slaughter surveillance program, careful attention must be paid to prevent and address cross contamination in the laboratory.
This is a submission to PeerJ for review.
Location of MTBC primer-probes on the insertional elements, IS1081 and IS6110
The location of primer-probe sets for Mycobacterium tuberculosis complex insertional elements A) IS1081. B) IS6110. Primers designated “Miller” refer to Miller et al. 1997.
Primers and probes
Primers and probes used for real-time PCR identification of M. tuberculosis complex and M. bovis.
Initial performance of the primer and probe sets
The initial performance of the selected primer and probes designed to detect M. tuberculosis complex and M. bovis. A) Analytical sensitivity using M. bovis DNA. B) Calculated slope, R2 value, efficiency, and lowest detectable DNA concentration for each primer-probe set
Comparison of 3 DNA isolation methods using purification method A (magnetic bead) or method B (spin column)
Three DNA isolation methods, NaOH, phenol/chloroform and TE were evaluated using 3 replicates of 5 different samples. If all 3 replicates signaled positive, a mean and standard deviation of the Cτ were calculated. All 3 isolation methods were compared using 2 DNA purification methods; method A (magnetic bead) or method B (spin column).
Comparing DNA isolation and purification methods
Estimates and 95% confidence intervals comparing differences in means of two DNA isolation methods and two DNA purification methods. Using a linear model, the means differences in Cτ values per sample were generated and a 95% confidence interval calculated for each comparison. A result was considered statistically significant if the 95% CI did not span zero. A, B: Comparison of DNA Isolation methods using either DNA Purification method A or B. C, D: Comparison of DNA Purification Methods A and B using either DNA Isolation method 2 or 3.