Development of Nested PCR and Duplex Real-Time Fluorescence Quantitative PCR Assay for the Simultaneous Detection of Theileria equi and Babesia caballi.
Abstract: Equine piroplasmosis (EP) is a type of blood protozoan disease caused by tick-borne parasites, (), () and . While many studies have been conducted on EP diagnosis, diagnostic methods exhibiting high sensitivity and specificity remain lacking. Therefore, nested PCR (nPCR) and duplex real-time fluorescence quantitative PCR (qPCR) that can simultaneously detect both and causing agents were established and compared. The two techniques were used to analyze 36 horse blood samples for EP. This set of samples was also detected by a multinested PCR (mnPCR) targeting the gene of and the gene of . By nPCR, duplex real-time fluorescence qPCR and mnPCR, infections with were detected in 16.67% (6/36), 2.78% (1/36), 19.44% (7/36) of the horses, respectively. The prevalence was 58.33% (21/36) by the nPCR, 33.33% (12/36) by the duplex real-time fluorescence qPCR and 2.78% (1/36) by the mnPCR. The overall prevalence of infection with mixed parasites by nPCR was 5.56% (2/36), by duplex real-time fluorescence qPCR was 2.78% (1/36) and by mnPCR 0% (0/36). Results suggest that nPCR can detect and positive samples with good specificity and sensitivity, although distinguishing between the two parasites requires an electrophoresis with 4% agarose gels. The duplex real-time fluorescence qPCR can readily distinguish between and infection, but with low sensitivity.
Copyright © 2022 Lv, Zhang, Yang, Liu and Deng.
Publication Date: 2022-05-18 PubMed ID: 35664851PubMed Central: PMC9158504DOI: 10.3389/fvets.2022.873190Google Scholar: Lookup
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Summary
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The research article focuses on the development of nested Polymerase Chain Reactions (nPCR) and duplex real time fluorescence quantitative PCR (qPCR) for the simultaneous detection of Theileria equi and Babesia caballi, which cause Equine piroplasmosis, a blood protozoan disease. The study found that nPCR has good sensitivity and specificity, while duplex real-time fluorescence qPCR can distinguish between the two parasites.
Objective of the Research
- The main purpose of this research is to develop nPCR and duplex real-time fluorescence qPCR that can simultaneously detect both Theileria equi and Babesia caballi.
- The comparative effectiveness and efficiency of these tests were assessed. This was done by detecting the level of sensitivity and specificity of both on a set of horse blood samples.
- The objective was also to overcome the gap left by lack of diagnostic methods exhibiting high sensitivity and specificity for Equine piroplasmosis (EP).
Methodology
- The researchers used nPCR and duplex real-time fluorescence qPCR techniques to analyze 36 horse blood samples for EP.
- These samples were also tested by a multinested PCR (mnPCR) targeting specific genes of Theileria equi and Babesia caballi.
- The detection rates were compared across all three methods.
Results
- nPCR detected infections with Theileria equi in 16.67% (6/36) of the horses, while duplex real-time fluorescence qPCR detected infections in 2.78% (1/36), and mnPCR detected infections in 19.44% (7/36).
- Babesia caballi prevalence was 58.33% (21/36) by the nPCR, 33.33% (12/36) by the duplex real-time fluorescence qPCR and 2.78% (1/36) by the mnPCR.
- The overall prevalence of infection with mixed parasites by nPCR was 5.56% (2/36), by duplex real-time fluorescence qPCR was 2.78% (1/36) and by mnPCR 0% (0/36).
- nPCR demonstrated good specificity and sensitivity, although distinguishing between the two parasites required electrophoresis with 4% agarose gels.
- The duplex real-time fluorescence qPCR method was able to distinguish between Theileria equi and Babesia caballi infection. However, it demonstrated a low level of sensitivity.
Conclusion
- The research demonstrates that nPCR can accurately and specifically detect Theileria equi and Babesia caballi in horse blood samples although an additional process (electrophoresis with 4% agarose gels) is required to differentiate between the two.
- The duplex real-time fluorescence qPCR, despite its lower sensitivity, could readily distinguish between these two infections, indicating the method has potential for further development and refinement.
Cite This Article
APA
Lv K, Zhang Y, Yang Y, Liu Z, Deng L.
(2022).
Development of Nested PCR and Duplex Real-Time Fluorescence Quantitative PCR Assay for the Simultaneous Detection of Theileria equi and Babesia caballi.
Front Vet Sci, 9, 873190.
https://doi.org/10.3389/fvets.2022.873190 Publication
Researcher Affiliations
- Department of Animal Genetics, Breeding and Reproduction, College of Animal Science and Veterinary Medicine, Shenyang Agricultural University, Shenyang, China.
- Key Laboratory of Livestock Infectious Diseases in Northeast China, Ministry of Education, Key Laboratory of Zoonosis, College of Animal Science and Veterinary Medicine, Shenyang Agricultural University, Shenyang, China.
- Department of Animal Genetics, Breeding and Reproduction, College of Animal Science and Veterinary Medicine, Shenyang Agricultural University, Shenyang, China.
- Key Laboratory of Livestock Infectious Diseases in Northeast China, Ministry of Education, Key Laboratory of Zoonosis, College of Animal Science and Veterinary Medicine, Shenyang Agricultural University, Shenyang, China.
- Department of Animal Genetics, Breeding and Reproduction, College of Animal Science and Veterinary Medicine, Shenyang Agricultural University, Shenyang, China.
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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