Internally Controlled Real-Time PCR Method for Quantitative Species-Specific Detection and vapA Genotyping of Rhodococcus equi

Abstract

We developed a novel quantitative real-time PCR (Q-PCR) method for the soil actinomycete Rhodococcus equi, an important horse pathogen and emerging human pathogen. Species-specific quantification was achieved by targeting the chromosomal monocopy gene choE, universally conserved in R. equi. The choE Q-PCR included an internal amplification control (IAC) for identification of false negatives. A second Q-PCR targeted the virulence plasmid gene vapA, carried by most horse isolates but infrequently found in isolates from other sources. The choE-IAC and vapA assays were 100% sensitive and specific as determined using 178 R. equi isolates, 77 nontarget bacteria, and a panel of 60 R. equi isolates with known vapA ؉ and vapA-negative (including vapB ؉ ) plasmid genotypes. The vapA ؉ frequency among isolate types was as follows: horse, 85%; human, 20%; bovine and pig, 0%; others, 27%. The choE-IAC Q-PCR could detect up to one genome equivalent using R. equi DNA or 100 bacteria/ml using DNA extracted from artificially contaminated horse bronchoalveolar lavage (BAL) fluid. Quantification was linear over a 6-log dynamic range down to Ϸ10 target molecules (or 1,000 CFU/ml BAL fluid) with PCR efficiency E of >0.94. The vapA assay had similar performance but appeared unsuitable for accurate (vapA ؉ ) R. equi quantification due to variability in target gene or plasmid copy number (1 to 9). The dual-reaction Q-PCR system here reported offers a useful tool to both medical and veterinary diagnostic laboratories for the quantitative detection of R. equi and (optional) vapA ؉ "horsepathogenic" genotype determination.

Mycoplasma agalactiae p40 Gene, a Novel Marker for Diagnosis of Contagious Agalactia in Sheep by Real-Time PCR: Assessment of Analytical Performance and In-House Validation Using Naturally Contaminated Milk Samples

“…The linearity of calibration curves represented by the regression coefficient (R 2 ) showed values close to 1 (p40, 0.9921; p40-IAC, 0.9931), indicating that the assay was highly linear. These results are similar to those for other Q-PCR methods used for other bacterial and eukaryotic organisms (10,(19)(20)(21)23).…”

Development of Quantitative Real-Time PCR Assays for Detection and Quantification of Surrogate Biological Warfare Agents in Building Debris and Leachate

“…Standard curves for the five Q-PCR assays developed in this study were constructed (not shown). The limit of detection, corresponding to the smallest amount of template DNA resulting in positive amplification in all replicates (35,40,46), for B. atrophaeus genomic DNA using the ITS and recA Ba Q-PCR assays was 7.5 fg per PCR (2 genome equivalents). The limits of detection of S. marcescens genomic DNA using gyrB, wzm, and recA Sm Q-PCR assays were 7.5 fg (1 genome equivalent), 75 fg (13 genome equivalents), and 7.…”

Identification of Atypical Rhodococcus -Like Clinical Isolates as Dietzia spp. by 16S rRNA Gene Sequencing

“…However, these methods are not useful for human isolates, because they generally do not carry vapA-type virulence plasmids but alternative types, like the pig-associated vapB plasmid and a recently identified new bovine type, or are plasmid-less (16). We therefore developed a PCR method for species-specific R. equi identification based on the amplification of the choE gene (11,20), a chromosomal locus encoding a secreted cholesterol oxidase (14). ChoE is the cytolytic factor responsible for the synergistic hemolysis (CAMP-like) reaction elicited by R. equi in the presence of sphingomyelinase C-producing bacteria, such as Listeria ivanovii, Bacillus cereus, and Staphylococcus aureus (14).…”