Evaluation of a multiplex polymerase chain reaction assay for simultaneous detection of Rhodococcus equi and the vapA gene.
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.


METHODS
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.


METHODS
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 equi isolate. An additional PCR assay was used to examine isolates for the vapB gene.


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
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.
Halbert
Natalie D
ND
Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843-4475, USA.
Reitzel
Ruth A
RA
Martens
Ronald J
RJ
Cohen
Noah D
ND
D000193:Actinomycetales Infections / Q000175:diagnosis / Q000662:veterinary; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D006734:Horse Diseases / Q000175:diagnosis / Q000382:microbiology; D006736:Horses; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D016929:Rhodococcus equi / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013045:Species Specificity; D037521:Virulence Factors / Q000235:genetics*
D001426:Bacterial Proteins; D037521:Virulence Factors