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Updated: Jun 2, 2025
Author Spotlight: Advancements in Multiplex Detection of Respiratory Viruses
Published on: November 10, 2023
Multiplex one-step RT‒qPCR assays for simultaneous detection of AMDV, MEV and CDV
Zhi Cao1,2, Hang Xu1, Xinru Zhao1
1College of Veterinary Medicine, Qingdao Agricultural University, Qingdao, 266109, China.
Background:
Aleutian mink disease, mink viral enteritis and canine distemper are known as the three most serious diseases that cause great economic loss in the mink industry. In clinical practice, aleutian mink disease virus (AMDV), mink enteritis virus (MEV) and canine distemper virus (CDV) are common mixed infections, and they have similar clinical clinical signs, such as diarrhoea. Therefore, a rapid and accurate differential diagnosis method for use on mink ranches is essential for the control of these three pathogens. Here, we developed multiplex one-step real-time quantitative PCR (RT‒qPCR) assays for the simultaneous detection and quantification of AMDV, MEV and CDV by using three primers and probes based on the conserved NS1, VP2 and N genes, respectively.
Results:
The results showed that the established method can not cross-react with other mink pathogens, with a detection sensitivity of 25 copies/µL and a coefficient of variation less than 3.51%. Moreover, the interference experiment showed that the presence of AMDV, MEV and CDV templates at different concentrations would not interfere with the detection results. Furthermore, two hundred clinical samples of mink with diarrhoea were simultaneously analysed using multiplex RT‒qPCR and single RT‒qPCR, the Kappa values were all greater than 0.921, indicating that there was a high degree of coincidence between the two detection methods.
Conclusions:
In conclusion, multiplex RT‒qPCR exhibited high specificity, sensitivity, and reproducibility, indicating that this method can be used as a reliable and specific tool for the differential detection and quantification of AMDV, MEV and CDV.
Insights
A new multiplex real-time quantitative PCR (RT‒qPCR) assay allows for the simultaneous detection of aleutian mink disease virus (AMDV), mink enteritis virus (MEV), and canine distemper virus (CDV). This rapid diagnostic tool is crucial for controlling these economically significant mink diseases.
Area of Science:
- Veterinary Virology
- Molecular Diagnostics
- Animal Health Economics
Background:
- Aleutian mink disease, mink viral enteritis, and canine distemper cause significant economic losses in the mink industry.
- Mixed infections of aleutian mink disease virus (AMDV), mink enteritis virus (MEV), and canine distemper virus (CDV) present similar clinical signs, complicating diagnosis.
- A rapid and accurate differential diagnosis method is essential for managing these prevalent mink pathogens.
Purpose of the Study:
- To develop a multiplex one-step real-time quantitative PCR (RT‒qPCR) assay.
- To enable simultaneous detection and quantification of AMDV, MEV, and CDV.
Main Methods:
- Utilized multiplex one-step RT‒qPCR targeting conserved NS1, VP2, and N genes.
- Developed specific primers and probes for simultaneous detection of the three viruses.
- Validated the assay using clinical samples from mink with diarrhea.
Main Results:
- The assay demonstrated high specificity, with no cross-reactivity with other mink pathogens.
- Sensitivity was determined to be 25 copies/µL, with a coefficient of variation below 3.51%.
- High agreement (Kappa > 0.921) was observed between multiplex RT‒qPCR and single RT‒qPCR methods in analyzing 200 clinical samples.
Conclusions:
- Multiplex RT‒qPCR is a highly specific, sensitive, and reproducible method.
- This assay serves as a reliable tool for differential diagnosis and quantification of AMDV, MEV, and CDV.
- The developed method is suitable for practical application on mink ranches for disease control.
