Mistaken identity of a PCR target proposed for identification of Mycoplasma bovis and the effect of sequence variation on assay performance

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

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 4/3/2018
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Background. Mycoplasma bovis is an important cause of disease in cattle and has recently emerged as a primary disease agent in bison. Because the bacterium requires specialized growth conditions many diagnostic laboratories use PCR to replace or complement traditional isolation and identification methods. The uvrC gene, highly conserved among M. bovis isolates, is a frequent target of these assays. Among PCRs reported to target uvrC is a real-time assay using primers Mbov F2024 (5’-TCTAATTTTTTCATCATCGCTAATGC-3’) and Mbov R2135 (5’-TCAGGCCTTTGCTACAATGAAC-3’). Methods and Results. Our laboratory routinely uses Mbov F2024 and Mbov R2135 in a conventional PCR format for identification of M. bovis. However, we have encountered several isolates that test negatively or give inconsistent results, all confirmed to be M. bovis based on 16SrRNA gene sequence, including 3 from which an unexpectedly large amplicon of >1.5 Kb is obtained. Further investigation revealed that Mbov F2024 and Mbov R2135 do not amplify a portion of the uvrC gene. Rather, they are positioned within a 405 bp open reading frame immediately downstream that is annotated as a “putative lipoprotein” in the type strain, PG45. Upon noting the presence of polymorphisms in corresponding sequences for other isolates found in GenBank, and considering our prior false negative results, these observations raise concern as to whether this PCR method is suitable for identification of all strains. To examine sequence heterogeneity within the Mbov F2024 / Mbov R2135 amplicon and, in particular, the probe- and primer-binding sites, we used primers uvrC-1 (5’-CTATGACGCTTCAGTTGAAGA-3’) and 0313-1 (5’-GCTTTTGTCAATTGCTTCAA-3’) to amplify and sequence a 486 bp region encompassing the Mbov F2024 / Mbov R2135 amplicon from 54 M. bovis isolates (37 from cattle and 17 from bison). DNA sequences from this region were also retrieved from privately held genome sequences for an additional 131 isolates (117 from cattle, 13 from bison and one from a white-tailed deer) and from publically available GenBank submissions for a further 26 cattle isolates. Among these 211 isolates we identified 6 variants with a large insertion and/or 2-8 nucleotide substitutions as compared to PG45. All variants have sequence identical to the real-time PCR probe and to primer Mbov R2135, but 5 of the variants have a single substitution in the 3’ portion of primer Mbov F2024. One variant additionally has an insertion of 1,658 bp positioned between the primers. Top-scoring BLAST matches with the insertion are 99% identical to a transposase found in M. bovis isolates PG45 and CQ-W70. The effect of the insertion and Mbov F2024 substitution on the kinetics and performance of the real-time PCR was analyzed by comparing standard curves and Ct values obtained using isolates with or without those features. The amplification efficiency and limit of detection appear unaffected by the Mbov F2024 substitution but isolates with the insertion consistently test negatively, both in our laboratory and in an AAVLD-accredited diagnostic laboratory in which this method is used. Conclusion. We found no evidence that the Mbov F2024 substitution affects assay sensitivity, performance or limit of detection. Nonetheless, laboratories using this method for identification of M. bovis should be aware that the amplified fragment lies within an open reading frame that may be less well-conserved than the intended target, the uvrC gene, and that occasional false negative results may be obtained.