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Updated: Jul 9, 2025
Rapid, Seamless Generation of Recombinant Poxviruses using Host Range and Visual Selection
Published on: May 24, 2020
The establishment of a recombinase polymerase amplification technique for the detection of mouse poxvirus
Yuexiao Lian1, Mengdi Zhang1, Yujun Zhu1
1Guangdong laboratory animals monitoring instituteand Guangdong Provincial Key Laboratory of Laboratory Animals, Guangzhou, 510663, China.
Background:
Ectromelia virus (ECTV) is the causative agent of mousepox in mice. In the past century, ECTV was a serious threat to laboratory mouse colonies worldwide. Recombinase polymerase amplification (RPA), which is widely used in virus detection, is an isothermal amplification method.
Results:
In this study, a probe-based RPA detection method was established for rapid and sensitive detection of ECTV.Primers were designed for the highly conserved region of the crmD gene, the main core protein of recessive poxvirus, and standard plasmids were constructed. The lowest detection limit of the ECTV RT- RPA assay was 100 copies of DNA mol-ecules per reaction. In addition, the method showed high specificity and did not cross-react with other common mouse viruses.Therefore, the practicability of the RPA method in the field was confirmed by the detection of 135 clinical samples. The real-time RPA assay was very similar to the ECTV real-time PCR assay, with 100% agreement.
Conclusions:
In conclusion, this RPA assay offers a novel alternative for the simple, sensitive, and specific identification of ECTV, especially in low-resource settings.
Insights
A new probe-based recombinase polymerase amplification (RPA) method rapidly and sensitively detects Ectromelia virus (ECTV), the cause of mousepox. This simple assay is ideal for identifying ECTV in resource-limited settings.
Area of Science:
- Veterinary Virology
- Molecular Diagnostics
Background:
- Ectromelia virus (ECTV) causes mousepox, posing a significant threat to laboratory mouse colonies.
- Recombinase polymerase amplification (RPA) is a versatile isothermal method for nucleic acid detection.
Purpose of the Study:
- To develop a rapid, sensitive, and specific probe-based RPA assay for ECTV detection.
- To evaluate the assay's performance in clinical samples and compare it with existing methods.
Main Methods:
- Designed primers targeting the conserved crmD gene of ECTV.
- Constructed standard plasmids for assay calibration.
- Validated the assay using clinical samples and compared results with real-time PCR.
Main Results:
- The ECTV RPA assay demonstrated a detection limit of 100 DNA molecules per reaction.
- The method exhibited high specificity, with no cross-reactivity to other common mouse viruses.
- 135 clinical samples were successfully analyzed, confirming the assay's field practicability and showing 100% agreement with real-time PCR.
Conclusions:
- The developed RPA assay is a simple, sensitive, and specific alternative for ECTV identification.
- This method is particularly valuable for ECTV detection in settings with limited resources.
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