Improved DNA amplification of the hallmark IS900 element in Mycobacterium avium subspecies paratuberculosis: a re-examination based on whole genome sequence analysis

Authors: Bannantine, John; Stabel, Judith; Bayles, Darrell; BIET, FRANCK - Inrae

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/8/2023
Publication Date: 1/31/2023
Citation: Bannantine, J.P., Stabel, J.R., Bayles, D.O., Biet, F. 2023. Improved DNA amplification of the hallmark IS900 element in Mycobacterium avium subspecies paratuberculosis: a re-examination based on whole genome sequence analysis. Applied and Environmental Microbiology. 89(2). https://doi.org/10.1128/aem.01682-22.
DOI: https://doi.org/10.1128/aem.01682-22

Interpretive Summary: This paper is an example of how applied genomic studies can aid diagnostic test development. The focus of this study is on the hallmark IS900 insertion sequence that in present in the Mycobacterium avium subspecies paratuberculosis genome at least 16 times. Old legacy primers that have been used for PCR tests to detect this bacterial pathogen that causes Johne's disease were designed using sequence data from only a single copy of IS900 and not considering all copies collectively as a group. This has resulted in errors in the primers used which can be easily corrected to improve test sensitivities. We tested legacy primers that contain these errors and their corrected versions by real time PCR and showed improved sensitivity on purified genomic DNA was well as real-world samples. These findings will help animal producers, veterinarians, but especially researchers and state lab diagnosticians.

Technical Abstract: Amplification of the IS900 repetitive element is a hallmark nucleic acid-based diagnostic test for Mycobacterium avium subspecies paratuberculosis (Map), which causes Johne’s disease in ruminants. This assay is most frequently used to determine if presence of the bacterium in feces of infected cattle, sheep or other ruminants. This multi-copy insertion element is also widely used for strain typing. However, some commonly used primers to amplify IS900 were developed prior to the availability of complete genome sequences or using sequences that contain errors. Two primer sets developed in the 1990s were widely used for decades and their use has continued in current studies. Genome sequence analysis of the binding locations of one primer pair (P90+P91) identified some errors and inefficiencies that can be modified to further increase sensitivity of detection. The P90 primer has two missing nucleotides near the 3’end. Likewise, this same primer does not bind all copies of IS900 due to polymorphisms located in the 5’ end of the insertion sequence. Another primer set (150C+921) typically used for IS900 DNA amplification is not affected by these problems. These IS900 primer pairs, along with newly developed primers, were tested on purified genomic DNA to determine how primer design errors might improve amplification efficiencies. Real-time PCR (qPCR) assays were performed using four primer sets. The newly designed PCR primer set (JB5) showed increased sensitivity by 2-3 quantification cycles using purified genomic DNA and was similar in efficiency to 150C+921. These results were extended with a small sample of DNA extracted from the feces and tissues of infected cows, which showed similar increased efficiencies to that obtained with purified genomic DNA. Although P90 and P91 successfully amplify Map DNA, its use should be discontinued in favor of more efficient primer pairs in future studies.