Diagnostic application of polymerase chain reaction for detection of Ehrlichia risticii in equine monocytic ehrlichiosis (Potomac horse fever).
Abstract: Genomic amplification by the polymerase chain reaction (PCR) was used to identify a unique genomic sequence of Ehrlichia risticii directly in DNA isolated from peripheral-blood buffy coat cells of E. risticii-infected horses (Potomac horse fever) and from infected cell cultures. A specific primer pair, selected from a cloned, species-specific, 1-kb DNA fragment of the E. risticii genome as a template, was used for the amplification of the target DNA of 247 bp. The optimal number of 40 PCR cycles, determined by analyzing an amplification profile obtained with a constant Taq polymerase concentration, was used to achieve maximum amplification of the E. risticii DNA segment. Efficient amplification of target DNA was achieved with specimens processed by either the phenol extraction or rapid lysis method. The specificity of the amplified DNA product was confirmed by the proper size (247 bp) and appropriate restriction enzyme cleavage pattern of the amplified target DNA, as well as by the specific hybridization signal obtained by using a PCR-amplified 185-bp internal DNA probe. A 10(5)- to 10(6)-fold amplification of target DNA, which allowed detection of E. risticii from as few as two to three infected cells in culture and from a very small volume of buffy coat cells from infected horses, was achieved. This PCR amplification procedure was found to be highly specific and sensitive for the detection of E. risticii for the study of Potomac horse fever.
Publication Date: 1991-10-01 PubMed ID: 1939575PubMed Central: PMC270303DOI: 10.1128/jcm.29.10.2228-2233.1991Google Scholar: Lookup
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- Journal Article
Summary
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The research investigates the use of Polymerase Chain Reaction (PCR) in diagnosing Potomac Horse Fever, a disease caused by the bacterium Ehrlichia risticii, in horses. The PCR techniques were shown to be highly sensitive and specific for detecting the bacterium.
Identification and Amplification of Ehrlichia risticii DNA
- The researchers used PCR, a molecular biology technique, to identify a specific sequence of Ehrlichia risticii DNA in both infected horses and infected cell cultures.
- The team selected a specific primer pair from a cloned, 1-kb DNA fragment of the E. risticii genome to amplify the target DNA of 247 base pairs.
- The amplification was optimized at 40 PCR cycles, which was determined to allow for maximum amplification of the E. risticii DNA segment without degrading the quality of the sample.
Efficiency and Specificity of the PCR Procedure
- The PCR procedure was found to be efficient in amplifying target DNA regardless of whether the samples were prepared through phenol extraction or a rapid lysis method.
- The researchers confirmed the specificity of the amplified DNA product by checking its size (247 base pairs) and its restriction enzyme cleavage pattern, matching what was expected for E. risticii DNA.
- They also used a hybridization signal from a PCR-amplified internal DNA probe to verify the specificity of the amplified product.
Detection Sensitivity and Applications
- The researchers were able to achieve an amplification of target DNA between 10^5 and 10^6 times, which allowed them to detect E. risticii from as few as two to three infected cells in culture.
- They were also able to detect the bacterium using a very small volume of buffy coat cells (the component of blood that contains white blood cells and platelets) from infected horses.
- This discovery suggests that the PCR procedure could be a highly sensitive and specific diagnostic tool for Potomac horse fever, a common and potentially fatal disease in horses.
Cite This Article
APA
Biswas B, Mukherjee D, Mattingly-Napier BL, Dutta SK.
(1991).
Diagnostic application of polymerase chain reaction for detection of Ehrlichia risticii in equine monocytic ehrlichiosis (Potomac horse fever).
J Clin Microbiol, 29(10), 2228-2233.
https://doi.org/10.1128/jcm.29.10.2228-2233.1991 Publication
Researcher Affiliations
- Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park 20742-3711.
MeSH Terms
- Animals
- Base Sequence
- Cells, Cultured
- DNA Probes
- DNA, Bacterial / genetics
- Ehrlichia / genetics
- Ehrlichia / isolation & purification
- Horse Diseases / diagnosis
- Horse Diseases / microbiology
- Horses
- Humans
- Molecular Sequence Data
- Monocytes / microbiology
- Polymerase Chain Reaction / methods
- Polymerase Chain Reaction / statistics & numerical data
- Rickettsiaceae Infections / diagnosis
- Rickettsiaceae Infections / microbiology
- Rickettsiaceae Infections / veterinary
- Sensitivity and Specificity
References
This article includes 14 references
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Citations
This article has been cited 11 times.- Uzal FA, Arroyo LG, Navarro MA, Gomez DE, Asín J, Henderson E. Bacterial and viral enterocolitis in horses: a review. J Vet Diagn Invest 2022 May;34(3):354-375.
- Baird JD, Arroyo LG. Historical aspects of Potomac horse fever in Ontario (1924-2010). Can Vet J 2013 Jun;54(6):565-72.
- Biswas B, Vemulapalli R, Dutta SK. Molecular basis for antigenic variation of a protective strain-specific antigen of Ehrlichia risticii. Infect Immun 1998 Aug;66(8):3682-8.
- Massung RF, Slater K, Owens JH, Nicholson WL, Mather TN, Solberg VB, Olson JG. Nested PCR assay for detection of granulocytic ehrlichiae. J Clin Microbiol 1998 Apr;36(4):1090-5.
- Dutta SK, Vemulapalli R, Biswas B. Association of deficiency in antibody response to vaccine and heterogeneity of Ehrlichia risticii strains with Potomac horse fever vaccine failure in horses. J Clin Microbiol 1998 Feb;36(2):506-12.
- Chang WL, Pan MJ. Specific amplification of Ehrlichia platys DNA from blood specimens by two-step PCR. J Clin Microbiol 1996 Dec;34(12):3142-6.
- Vemulapalli R, Biswas B, Dutta SK. Pathogenic, immunologic, and molecular differences between two Ehrlichia risticii strains. J Clin Microbiol 1995 Nov;33(11):2987-93.
- Pfeffer M, Wiedmann M, Batt CA. Applications of DNA amplification techniques in veterinary diagnostics. Vet Res Commun 1995;19(5):375-407.
- Kakoma I, Hansen RD, Anderson BE, Hanley TA, Sims KG, Liu L, Bellamy C, Long MT, Baek BK. Cultural, molecular, and immunological characterization of the etiologic agent for atypical canine ehrlichiosis. J Clin Microbiol 1994 Jan;32(1):170-5.
- Iqbal Z, Chaichanasiriwithaya W, Rikihisa Y. Comparison of PCR with other tests for early diagnosis of canine ehrlichiosis. J Clin Microbiol 1994 Jul;32(7):1658-62.
- Biswas B, Vemulapalli R, Dutta SK. Detection of Ehrlichia risticii from feces of infected horses by immunomagnetic separation and PCR. J Clin Microbiol 1994 Sep;32(9):2147-51.
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