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

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. We discovered that a previously described 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, 1 of which falls within a primer-binding sequence. Additionally, 3 isolates from this group were found to have a 1,658-bp transposase gene insertion within the amplified region that leads to a false-negative result. The insertion was not detected in a further 164 isolates. 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 region amplified may be prone to nucleotide substitutions and/or insertions relative to the sequence used for its design and that occasional false-negative results may be obtained.

Keywords: Mycoplasma bovis; PCR; uvrC gene.

Figure 1.

Schematic representation of the uvrC gene and the predicted lipoprotein gene immediately adjacent (locus tag MBOVPG45_0313), as found in the type strain, PG45, with the positions of PCR primers used in our study indicated by vertical bars. Primers Mbov F2024 and Mbov R2135 are those used for the rtPCR of reference 2. Arrows specify the direction of transcription (for genes) or amplification (for primers). The position at which a transposase insertion occurs in some isolates is indicated (bp 282 of the lipoprotein gene open reading frame, relative to the first nucleotide of the start codon).

Figure 2.

Nucleotide alignment of M. bovis uvrC-1/Mb 0313-1 amplicon variants identified in our study. Variant 1 is found in the type strain, PG45, used to design the PCR assay reported by reference 2. PCR primer sequences and the rtPCR probe are in bold and underlined; open reading frames (ORFs) are shaded in gray. Arrows specify the direction of amplification (for primers) or transcription (for ORFs). The position at which a transposase insertion occurs in some isolates is indicated by an arrowhead. Coordinates for the uvrC and lipoprotein gene ORFs are relative to the first nucleotide of their start codons.