Establishment of RAA detection method for infectious laryngotracheitis virus
10.16303/j.cnki.1005-4545.2025.02.06
- VernacularTitle:传染性喉气管炎病毒RAA检测方法的建立
- Author:
Wanying FENG
1
;
Zhuanzhuan WANG
;
Yining LIU
;
Guangming CHEN
;
Xiaohui GUO
;
Weixin LI
;
Weiqing LI
;
Zhiqiang ZHANG
;
Peiguo LI
;
Zhaoxing ZHANG
;
Tonglei WU
;
Qinghui JIA
Author Information
1. 河北科技师范学院河北省预防兽医学重点实验室,河北秦皇岛 066604
- Publication Type:Journal Article
- Keywords:
infectious laryngotracheitis;
recombinase;
RAA
- From:
Chinese Journal of Veterinary Science
2025;45(2):212-218
- CountryChina
- Language:Chinese
-
Abstract:
The aim of this study is to establish a rapid,efficient,and sensitive method for detecting the infectious laryngotracheitis virus(ILTV).The DNA of ILTV was extracted and used as a tem-plate to develop a recombinant enzyme-mediated isothermal amplification(RAA)fluorescence de-tection method for ILTV through optimization of conditions,sensitivity analysis,and repeatability assessment.Additionally,the nucleic acids of avian influenza virus(AIV),IBV,and Newcastle dis-ease virus(NDV)were detected to verify the specificity of this method.Finally,this method was applied to analyze 59 clinical samples collected from multiple large-scale chicken farms in Hebei Province,and the results were compared with those obtained from real-time fluorescence quantifi-cation(qPCR)and PCR methods according to national standards.The results showed that the RAA detection method established in this study had a reaction system of 25.0 μL buffer,2.1 μL primer,0.6 μL probe,5.0 μL magnesium acetate,and 5.0 μL template.The reaction temperature was 39 ℃ and the amplification time was within 20 minutes.The sensitivity of this method was 101 copies/μL,and the specificity detection was 100％.Testing of 59 clinical samples showed that 17 were detected positive by both RAA fluorescence and qPCR,and 12 were detected by PCR,and the detection rate of RAA(fluorescence)was consistent with real-time fluorescence quantification and qPCR,which was higher than that of the PCR assay.The research results indicate that the RAA fluorescence method has a short detection time,good specificity and sensitivity,and can be used for rapid detection of ILTV.
