Improved molecular detection of Neorickettsia risticii with a duplex real-time PCR assay in the diagnosis of Potomac horse fever.
Abstract: Neorickettsia risticii, an obligate intracellular bacterium, is the causative agent of Potomac horse fever (PHF). Diagnosis of PHF is based on demonstration of serum antibodies, isolation of N. risticii, and/or detection of nucleic acid by a PCR assay. An existing real-time PCR assay targeting the N. risticii 16S rRNA has been validated using blood samples from horses with colitis, and snails; to our knowledge, the performance of the assay for other sample types has not been reported. We describe here a modification of the 16S rRNA gene assay by the addition of a set of primers and probe targeting the N. risticii p51 gene to form a duplex assay. We validated the new assay using diagnostic specimens from 56 horses with suspected PHF. The assay consistently detected down to 5 copies of synthetic targets, and did not show any cross-reaction with common equine enteric pathogens. Although we did not establish the diagnostic sensitivity and specificity of the duplex assay, results for both gene targets were in complete agreement, with the exception of 4 fecal samples that tested positive for the 16S rRNA gene only. Further analysis indicated that testing of fecal samples using our 16S rRNA gene assay alone can produce a false-positive result.
Publication Date: 2022-11-13 PubMed ID: 36373552PubMed Central: PMC9751466DOI: 10.1177/10406387221135184Google Scholar: Lookup
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- Journal Article
Summary
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This research focuses on an enhanced molecular detection method with a duplex real-time PCR assay for diagnosing Potomac horse fever, caused by Neorickettsia risticii. A new duplex assay was validated and displayed consistent detection with no cross-reaction with similar equine pathogens.
Understanding the Context
- Neorickettsia risticii is an obligate intracellular bacterium known to cause Potomac horse fever (PHF), a disease prevalent among horses. Traditional methods of diagnosing this illness involve performing serum antibody tests, isolating the bacterium, or utilizing a real-time PCR assay to detect the bacterium’s nucleic acid.
- The real-time PCR assay used traditionally targets the 16S rRNA of the bacterium. Until now, the validation of its effectiveness was conducted predominantly on blood samples from horses affected with colitis and snails. The performance of this assay in other types of samples remained unreported. This research details an advanced version of this assay.
The Duplex Assay Modification
- This research describes a modification to the existing 16S rRNA assay. In the improved version, a set of primers and a probe that target the groEL gene of the bacteria are introduced. Together, these form a duplex assay.
- The new duplex assay was tested on diagnostic specimens extracted from 56 horses suspected of suffering from PHF. It revealed promising results with consistent detection down to just 5 copies of synthetic targets. Importantly, the modified assay did not indicate any cross-reaction with common equine enteric pathogens, pointing to its specificity.
- While the diagnostic sensitivity and specificity of the duplex assay were not established in this study, the results for the 16S rRNA gene and the groEL gene were in complete agreement, except for four fecal samples that were positive only for the 16S rRNA gene.
Implications and Potential Limitations
- A critical find through this research points to the limitation of using the 16S rRNA gene assay alone for testing fecal samples. It suggests this can lead to false-positive results. This emphasizes the need for the new duplex assay which includes the groEL gene to mitigate this problem.
- Despite offering promising results, the duplex assay is still in preliminary stages as the research did not investigate its diagnostic sensitivity and specificity. Further studies and validations would be required to understand the potentials and limits of this improved diagnostic tool.
Cite This Article
APA
Thirumalapura NR, Livengood J, Beeby J, Wang W, Goodrich EL, Goodman LB, Erol E, Tewari D.
(2022).
Improved molecular detection of Neorickettsia risticii with a duplex real-time PCR assay in the diagnosis of Potomac horse fever.
J Vet Diagn Invest, 35(1), 62-66.
https://doi.org/10.1177/10406387221135184 Publication
Researcher Affiliations
- Pennsylvania Veterinary Laboratory, Pennsylvania Department of Agriculture, Harrisburg, PA, USA.
- Pennsylvania Veterinary Laboratory, Pennsylvania Department of Agriculture, Harrisburg, PA, USA.
- Animal Health Diagnostic Center, Departments of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA.
- Animal Health Diagnostic Center, Departments of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA.
- Animal Health Diagnostic Center, Departments of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA.
- Public & Ecosystem Health, Cornell Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA.
- Veterinary Diagnostic Laboratory, Department of Veterinary Science, University of Kentucky, Lexington, KY, USA.
- Pennsylvania Veterinary Laboratory, Pennsylvania Department of Agriculture, Harrisburg, PA, USA.
MeSH Terms
- Horses / genetics
- Animals
- Neorickettsia risticii / genetics
- RNA, Ribosomal, 16S / genetics
- Real-Time Polymerase Chain Reaction / veterinary
- Anaplasmataceae Infections / diagnosis
- Anaplasmataceae Infections / veterinary
- Anaplasmataceae Infections / microbiology
- Horse Diseases / microbiology
Conflict of Interest Statement
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
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This article includes 20 references
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