Mistaken identity of an open reading frame proposed for PCR-based identification of Mycoplasma bovis and the effect of polymorphisms and insertions on assay performance

Authors: Register, Karen; Boatwright, Jr, William; Thacker, Tyler; JELINSKI, MURRAY - University Of Saskatchewan

Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/15/2017
Publication Date: 1/10/2018
Citation: Register, K.B., Boatwright Jr, W.D., Gesy, K.M., Thacker, T.C., Jelinski, M.D. 2018. Mistaken identity of an open reading frame proposed for PCR-based identification of Mycoplasma bovis and the effect of polymorphisms and insertions on assay performance. Journal of Veterinary Diagnostic Investigation. 30(4):637-641. https://doi.org/10.1177/1040638718764799.
DOI: https://doi.org/10.1177/1040638718764799

Interpretive Summary: Mycoplasma bovis is an important cause of disease in cattle and bison. Because the bacterium requires specialized growth conditions many diagnostic laboratories routinely use PCR-based testing to replace or complement conventional isolation and identification methods. A commonly used PCR target is the uvrC gene, which is found only in M. bovis and has a sequence that is highly conserved among different strains. Here we detail our discovery that a previously reported diagnostic PCR putatively targeting the uvrC gene actually amplifies a fragment from a gene that is immediately adjacent and predicted to encode a lipoprotein. Comparison of the lipoprotein gene sequence from 211 isolates representing the United States, Canada, Europe, Israel, China and Australia revealed it is less well-conserved than the uvrC gene and that the DNA sequence variation found in some isolates leads to false negative results. Laboratories utilizing this PCR for identification of M. bovis should be aware that it does not target the intended gene and that occasional false negative results may be obtained.

Technical Abstract: Mycoplasma bovis is an important cause of disease in cattle and bison. Because the bacterium requires specialized growth conditions many diagnostic laboratories routinely use PCR to replace or complement conventional isolation and identification methods. A frequently used target of such assays is the uvrC gene, which has been shown to be highly conserved among isolates. Here we detail our discovery that a previously described diagnostic PCR putatively targeting the uvrC gene amplifies a fragment from an adjacent gene predicted to encode a lipoprotein. Comparison of the lipoprotein gene sequence from 211 isolates revealed several single nucleotide polymorphisms, one of which falls within a primer-binding sequence. Additionally, 3 isolates were found to have a 1658 bp transposase gene insertion within the amplified region that leads to a false negative result. We found no evidence that the nucleotide substitution within the primer-binding region affects the assay sensitivity, performance or limit of detection. Nonetheless, laboratories utilizing this method for identification of M. bovis should be aware that the amplified fragment lies within an open reading frame that appears to be less well-conserved than the intended target, the uvrC gene, and that occasional false negative results may be obtained.