Objective—To evaluate sensitivity and specificity of a
multiplex polymerase chain reaction (PCR) assay for
simultaneous detection of Rhodococcus equi and differentiation
of strains that contain the virulence-associated
gene (vapA) from strains that do not.
Sample Population—187 isolates of R equi from
equine and nonequine tissue and environmental
specimens and 27 isolates of bacterial species genetically
or morphologically similar to R equi.
Procedure—The multiplex PCR assay included 3
gene targets: a universal 311-bp bacterial 16S ribosomal
RNA amplicon (positive internal control), a 959-bp
R equi-specific target in the cholesterol oxidase gene
( choE ), and a 564-bp amplicon of the vapA gene.
Duplicate multiplex PCR assays for these targets and
confirmatory singleplex PCR assays for vapA and
choE were performed for each R equiisolate. An additional
PCR assay was used to examine isolates for the
Results—Results of duplicate multiplex and singleplex
PCR assays were correlated in all instances, revealing high specificity and reliability (reproducibility)
of the vapA multiplex assay. Of the pulmonary isolates
from horses with suspected R equi pneumonia,
97.4% (76/78) yielded positive results for vapA. Seven
of 50 (14%) human isolates of R equi yielded positive
results for vapA. Six human R equi isolates and 1
porcine isolate yielded positive results for vapB. No
isolates with vapA and vapB genes were detected.
Conclusions and Clinical Relevance—The multiplex
PCR assay is a sensitive and specific method for simultaneous
confirmation of species identity and detection
of the vapA gene. The assay appeared to be a useful
tool for microbiologic and epidemiologic diagnosis and
research. (Am J Vet Res 2005;66:1380–1385)