Development and evaluation of a real-time quantitative PCR for the detection of equine infectious anemia virus.
Abstract: Equine infectious anemia (EIA) has a worldwide distribution and causes severe economic losses to the equine industry. The EIA virus (EIAV) genome sequences from different countries are highly diverse, which poses a great challenge for pathogen identification with PCR. Phylogenetic analysis showed that although gag is the most conserved structural gene, it still has great genome variability. Currently, most existing PCR methods are designed based on the gag gene sequence and therefore do not cover all the viral strains, especially Asian EIAV strains. In this study, we developed a tat-gag-based real-time quantitative PCR (TG-qPCR) for the detection of EIAV by targeting the fragment between the tat and gag genes, which was relatively conserved in all the known EIAV strains. The performance of the TG-qPCR was evaluated against that of the standard qPCR (recommended by WOAH) by testing viral RNA extracted from viral supernatants of EIAV and EIAV, proviral DNA from peripheral blood mononuclear cells of artificially immunized horses, and virus nucleic acid from EIAV positive serum samples. The TG-qPCR assay had high specificity, sensitivity, and reproducibility. The detection limit of the TG-qPCR assay was 1 copy/reaction for both viral RNA and proviral DNA based on the Poisson distribution. Compared to the qPCR, the TG-qPCR has better inclusivity and can detect not only Asian EIAV strains but also almost all the representative EIAV strains from other continents. The above results show that the TG-qPCR assay could serve as an effective tool for the early diagnosis of clinical EIA disease.IMPORTANCEEquine infectious anemia (EIA) has a worldwide distribution and causes significant losses to the equine industry worldwide. A reliable detection method is necessary to control the transmission of EIA virus (EIAV). Currently, most of the available real-time PCR assays, including the qPCR of recommended by WOAH, are developed according to the sequences of European or American EIAV strains; however, the primers and probe sequences have low homology with Asian EIAV strains. To the best of our knowledge, no qPCR method capable of the well detection of Asian EIAV strains, especially Chinese EIAV strains, has been published to date. The development of a sensitive, specific, and rapid qPCR assay for the detection of the EIAV strains is therefore of great importance.
Publication Date: 2023-10-09 PubMed ID: 37811976DOI: 10.1128/spectrum.02599-23Google Scholar: Lookup
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Summary
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The research article presents a study where a new method for detecting the Equine Infectious Anemia Virus (EIAV) was developed and evaluated. This method, a real-time quantitative PCR, is an improvement over previous diagnostic techniques as it can identify virus strains from different regions, specifically those from Asia which have been difficult to detect with existing methods.
Objective and Importance of the Research
- This research aims to provide an effective, reliable, and comprehensive method to detect the EIAV. The virus causes Equine Infectious Anemia, which is responsible for massive economic losses in the equine industry globally.
- Existing testing methods, mostly derived from the sequence of the virus’ gag gene, have a gap— they fail to cover all EIAV strains, particularly those from Asia. This situation necessitates the development of an improved detection system.
- Controlling the transmission of EIAV is essential, hence, having a dependable detection technique is critical. A method that accounts for the viral strains’ diversity, including Asian variants, is of immense importance.
Development of the New Method
- The investigators developed a tat-gag-based real-time quantitative PCR (TG-qPCR) by targeting the fragment between the tat and gag genes of the EIAV. This region of the genome maintains relative consistency across different EIAV strains.
- The new method’s performance was evaluated against the standard qPCR, which is currently recommended by WOAH. The scientists tested viral RNA from EIAV, proviral DNA from horse’s blood cells, and virus nucleic acid from EIAV positive serum samples.
Evaluation of the New Method
- The TG-qPCR displayed high sensitivity, specificity, and reproducibility, marking its reliability. It could detect as limited as 1 copy/reaction for both the viral RNA and proviral DNA.
- The TG-qPCR outperforms the standard qPCR regarding inclusivity, since it can detect a wide range of EIAV strains from different continents, not just Asia.
- The results suggest that TG-qPCR could serve as an efficient early diagnostic tool for clinical EIA disease.
Conclusion
- The research introduces a novel tool that helps in the early diagnosis of EIAV, especially with its ability to detect diverse strains of the virus. The high sensitivity, specificity, and reproducibility of the TG-qPCR makes it a potentially valuable tool in managing and controlling the EIAV transmission, thereby mitigating its economic impacts on the equine industry.
Cite This Article
APA
Li S, Guo K, Wang X, Lin Y, Wang J, Wang Y, Du C, Hu Z, Wang X.
(2023).
Development and evaluation of a real-time quantitative PCR for the detection of equine infectious anemia virus.
Microbiol Spectr, e0259923.
https://doi.org/10.1128/spectrum.02599-23 Publication
Researcher Affiliations
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agriculture Sciences, Harbin, China.
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agriculture Sciences, Harbin, China.
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agriculture Sciences, Harbin, China.
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agriculture Sciences, Harbin, China.
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agriculture Sciences, Harbin, China.
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agriculture Sciences, Harbin, China.
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agriculture Sciences, Harbin, China.
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agriculture Sciences, Harbin, China.
- WOAH Reference Laboratory for Equine Infectious Anemia, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin, China.
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agriculture Sciences, Harbin, China.
- WOAH Reference Laboratory for Equine Infectious Anemia, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin, China.
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