Establishment of a double fluorescence RT-LAMP method for detection of chicken astrovirus and avian nephritis virus
10.16303/j.cnki.1005-4545.2025.02.05
- VernacularTitle:鸡星状病毒和禽肾炎病毒二重荧光RT-LAMP检测方法的建立
- Author:
Junke ZHAO
1
;
Dan YU
;
Wenqiao YIN
;
Yanfang ZHANG
;
Zhiqin XIE
;
Qing FAN
;
Huaying YU
;
Zhixun XIE
Author Information
1. 广西大学动物科学技术学院,广西南宁 530004;广西壮族自治区兽医研究所农业农村部中国(广西)—东盟跨境动物疫病防控重点实验室/广西兽医生物技术重点实验室,广西南宁 530001
- Publication Type:Journal Article
- Keywords:
chicken astrovirus;
avian nephritis virus;
visualization;
RT-LAMP;
differential diagnosis
- From:
Chinese Journal of Veterinary Science
2025;45(2):204-211,218
- CountryChina
- Language:Chinese
-
Abstract:
To establish a double fluorescent RT-LAMP that can distinguish between chicken astro-virus(CAstV)and avian nephritis virus(ANV),two sets of specific primers and probes labeled with fluorescent groups were designed by comparing the conserved sequences of CAstV ORF1b gene and ANV ORF1b gene downloaded from GenBank.The CAstV probe was labeled with CY3 fluorophore at the 3'end and the quencher BHQ2 at the 5'end.The ANV probe was labeled with CY3 fluorophore at the 3'end and the quencher BHQ2 at the 5'end.The reaction system of the method was optimized,and specificity,repeatability,interference,sensitivity,and clinical sample detection were performed.The results were compared with RT-qPCR and RT-PCR to verify the ef-fectiveness of the method.The experimental results showed that the established double fluorescence RT-LAMP method could complete the reaction within 60 min,and the optimal reac-tion temperature was 65 ℃.This method only detects ANV and CAstV,with no cross-reactivity to other common avian pathogens.The minimum detection limit of CAstV was 1.4 ×10 2 copies/μL,and the minimum detection limit of ANV was 1.5×10 2 copies/μL.The interference and reproduc-ibility tests were good.Compared with RT-qPCR,the coincidence rates of CAstV and ANV were 97.97％and 98.85％,respectively.In conclusion,the double fluorescence RT-LAMP method estab-lished in this study has the characteristics of rapidity,accuracy,good specificity,high sensitivity and good reproducibility,which is suitable for clinical diagnosis and provides technical support for the epidemiological investigation of CAstV and ANV.
