Evaluation of a multiplex polymerase chain reaction assay for simultaneous detection of Rhodococcus equi and the vapA gene.
Abstract: 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.
Publication Date: 2005-09-22 PubMed ID: 16173481DOI: 10.2460/ajvr.2005.66.1380Google Scholar: Lookup
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- Evaluation Study
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research focused on testing the effectiveness of a multiplex PCR assay in detecting Rhodococcus equi bacteria and its virulence-associated gene (vapA). The study confirmed that this method showed high sensitivity and specificity, making it a valuable tool for diagnoses and epidemiological research.
Objectives and Methods of the Study
- The primary purpose of the study was to evaluate the sensitivity and specificity of a multiplex Polymerase Chain Reaction (PCR) assay. This assay is designed to simultaneously detect Rhodococcus equi and distinguish the strains that contain the virulence-associated gene (vapA) from those that do not.
- The researchers used 187 isolates of R equi from both equine and nonequine tissues, as well as environmental samples, and 27 isolates of bacterial species that are genetically or morphologically similar to R equi.
- The multiplex PCR assay involved three gene targets: a 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.
- For each R equi isolate, duplicate multiplex PCR assays for these targets and confirmatory singleplex PCR assays for vapA and choE were performed.
Main Findings
- The study found that the results of duplicate multiplex and singleplex PCR assays were all consistent, which indicates high reliability (reproducibility) of the vapA multiplex assay.
- Among the lung samples from horses suspected of having R equi pneumonia, 97.4% (76/78) yielded positive results for vapA.
- Out of 50 human isolates of R equi, seven (14%) rendered positive results for vapA.
- A vapB gene PCR assay revealed that six human R equi isolates and one pig isolate were positive for vapB. However, none of the isolates contained both vapA and vapB genes.
Conclusions
- The researchers concluded that the multiplex PCR assay is a sensitive and specific method for simultaneously confirming species identity and detecting the vapA gene.
- Due to its high sensitivity and specificity, the assay could be a useful tool for microbiologic and epidemiological diagnosis and research.
Cite This Article
APA
Halbert ND, Reitzel RA, Martens RJ, Cohen ND.
(2005).
Evaluation of a multiplex polymerase chain reaction assay for simultaneous detection of Rhodococcus equi and the vapA gene.
Am J Vet Res, 66(8), 1380-1385.
https://doi.org/10.2460/ajvr.2005.66.1380 Publication
Researcher Affiliations
- Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843-4475, USA.
MeSH Terms
- Actinomycetales Infections / diagnosis
- Actinomycetales Infections / veterinary
- Animals
- Bacterial Proteins / genetics
- Horse Diseases / diagnosis
- Horse Diseases / microbiology
- Horses
- Humans
- Polymerase Chain Reaction / methods
- Reproducibility of Results
- Rhodococcus equi / genetics
- Rhodococcus equi / isolation & purification
- Sensitivity and Specificity
- Species Specificity
- Virulence Factors / genetics
Citations
This article has been cited 19 times.- Narváez SÁ, Fernández I, Patel NV, Sánchez S. Novel Quantitative PCR for Rhodococcus equi and Macrolide Resistance Detection in Equine Respiratory Samples. Animals (Basel) 2022 May 3;12(9).
- Cohen ND, Kahn SK, Cywes-Bentley C, Ramirez-Cortez S, Schuckert AE, Vinacur M, Bordin AI, Pier GB. Serum Antibody Activity against Poly-N-Acetyl Glucosamine (PNAG), but Not PNAG Vaccination Status, Is Associated with Protecting Newborn Foals against Intrabronchial Infection with Rhodococcus equi. Microbiol Spectr 2021 Sep 3;9(1):e0063821.
- Bordin AI, Cohen ND, Giguère S, Bray JM, Berghaus LJ, Scott B, Johnson R, Hook M. Host-directed therapy in foals can enhance functional innate immunity and reduce severity of Rhodococcus equi pneumonia. Sci Rep 2021 Jan 28;11(1):2483.
- Mallicote M, House AM, Sanchez LC. A review of foal diarrhoea from birth to weaning. Equine Vet Educ 2012 Apr;24(4):206-214.
- Bujold AR, Lani NR, Sanz MG. Strain-to-strain variation of Rhodococcus equi growth and biofilm formation in vitro. BMC Res Notes 2019 Aug 19;12(1):519.
- Stefańska I, Witkowski L, Rzewuska M, Dzieciątkowski T. Development and evaluation of the internal-controlled real-time PCR assay for Rhodococcus equi detection in various clinical specimens. J Vet Med Sci 2016 May 3;78(4):543-9.
- Shaw SD, Cohen ND, Chaffin MK, Blodgett GP, Syndergaard M, Hurych D. Estimating the Sensitivity and Specificity of Real-Time Quantitative PCR of Fecal Samples for Diagnosis of Rhodococcus equi Pneumonia in Foals. J Vet Intern Med 2015 Nov-Dec;29(6):1712-7.
- Berghaus LJ, Giguère S, Guldbech K, Warner E, Ugorji U, Berghaus RD. Comparison of Etest, disk diffusion, and broth macrodilution for in vitro susceptibility testing of Rhodococcus equi. J Clin Microbiol 2015 Jan;53(1):314-8.
- Liu H, Wang Y, Yan J, Wang C, He H. Appearance of multidrug-resistant virulent Rhodococcus equi clinical isolates obtained in China. J Clin Microbiol 2014 Feb;52(2):703.
- Krewer Cda C, Spricigo DA, de Avila Botton S, da Costa MM, Schrank I, de Vargas AC. Molecular characterization of Rhodococcus equi Isolates of horse breeding farms from an endemic region in South of Brazil by multiplex PCR. Braz J Microbiol 2008 Jan;39(1):188-93.
- Bordin AI, Suchodolski JS, Markel ME, Weaver KB, Steiner JM, Dowd SE, Pillai S, Cohen ND. Effects of administration of live or inactivated virulent Rhodococccus equi and age on the fecal microbiome of neonatal foals. PLoS One 2013;8(6):e66640.
- Burton AJ, Giguère S, Sturgill TL, Berghaus LJ, Slovis NM, Whitman JL, Levering C, Kuskie KR, Cohen ND. Macrolide- and rifampin-resistant Rhodococcus equi on a horse breeding farm, Kentucky, USA. Emerg Infect Dis 2013 Feb;19(2):282-5.
- Padmavathy B, Vinoth Kumar R, Patel A, Deepika Swarnam S, Vaidehi T, Jaffar Ali BM. Rapid and sensitive detection of major uropathogens in a single-pot multiplex PCR assay. Curr Microbiol 2012 Jul;65(1):44-53.
- Al Akhrass F, Al Wohoush I, Chaftari AM, Reitzel R, Jiang Y, Ghannoum M, Tarrand J, Hachem R, Raad I. Rhodococcus bacteremia in cancer patients is mostly catheter related and associated with biofilm formation. PLoS One 2012;7(3):e32945.
- Monego F, Maboni F, Krewer C, Vargas A, Costa M, Loreto E. Molecular characterization of Rhodococcus equi from horse-breeding farms by means of multiplex PCR for the vap gene family. Curr Microbiol 2009 Apr;58(4):399-403.
- Rodríguez-Lázaro D, Lewis DA, Ocampo-Sosa AA, Fogarty U, Makrai L, Navas J, Scortti M, Hernández M, Vázquez-Boland JA. Internally controlled real-time PCR method for quantitative species-specific detection and vapA genotyping of Rhodococcus equi. Appl Environ Microbiol 2006 Jun;72(6):4256-63.
- Vazquez-Boland JA, Val-Calvo J, Duquesne F, Decorosi F, Viti C, Petry S, Scortti M. Rhodococcus parequi sp. nov., a new species isolated from equine farm soil closely related to the pathogen Rhodococcus equi. Int J Syst Evol Microbiol 2025 Mar;75(3).
- Higgins C, Cohen ND, Slovis N, Boersma M, Gaonkar PP, Golden DR, Huber L. Antimicrobial Residue Accumulation Contributes to Higher Levels of Rhodococcus equi Carrying Resistance Genes in the Environment of Horse-Breeding Farms. Vet Sci 2024 Feb 17;11(2).
- da Silveira BP, Barhoumi R, Bray JM, Cole-Pfeiffer HM, Mabry CJ, Burghardt RC, Cohen ND, Bordin AI. Impact of surface receptors TLR2, CR3, and FcγRIII on Rhodococcus equi phagocytosis and intracellular survival in macrophages. Infect Immun 2024 Jan 16;92(1):e0038323.
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