Establishment and evaluation of a dual fluorescent RT-LAMP assay for PEDV and TGEV detection
10.16303/j.cnki.1005-4545.2024.08.04
- VernacularTitle:PEDV与TGEV双重荧光RT-LAMP检测方法的建立与评价
- Author:
Ran ZANG
1
;
Feifei XU
;
Danyang ZHENG
;
Zhiqian ZHAO
;
Mi ZHAO
;
Hui WANG
;
Jie GAO
;
Yang MU
Author Information
1. 西北农林科技大学动物医学院,陕西杨凌 712100
- Keywords:
porcine epidemic diarrhea virus;
transmissible gastroenteritis virus;
dual fluorescent RT-LAMP;
multicolor imaging system
- From:
Chinese Journal of Veterinary Science
2024;44(8):1600-1610
- CountryChina
- Language:Chinese
-
Abstract:
To develop a rapid differential detection method for porcine epidemic diarrhea virus(PEDV)and transmissible gastroenteritis virus(PEDV),M gene sequences of PEDV and TGEV were compared,the inner and outer primer pairs and probes were designed according to the highly conserved region.PEDV-Probe was labeled with FAM at5'end and BHQ1 at 3'end.TGEV-Probe was labeled with CY5.5 at the 5'end and BHQ2 at the 3'end.After optimizing the reaction condi-tions and system,a dual fluorescent RT-LAMP assay for PEDV and TGEV rapid identification was established.The amplification could be completed within 60 min in a 63 ℃ water bath and then stopped at 85 ℃ for 10 min.Then the tubes were placed in a multicolor imaging system,and the re-sults could be observed under 520 nm and 690 nm dual channels.There was no cross-reaction when other common swine viral pathogens were detected by this method.The sensitivity of the assay was evaluated with a 10-fold diluted recombinant plasmid as templates.The lowest detection limit was 102 copies/μL recombinant plasmid,which was 10 times more sensitive than the conventional RT-PCR method.Seventy-two PEDV-positive samples,49 TGEV-positive samples,and 40 PEDV and TGEV co-infected samples were detected from 175 anal swab samples of diarrheic piglets by the established method,which were all higher than the detection rates of the conventional RT-PCR method.The dual fluorescent RT-LAMP method established in this study can be used to amplify the target gene in an ordinary water bath without gel electrophoresis,which provides technical sup-port for rapid and convenient differential diagnosis of PED and TGE and simultaneous detection of PEDV and TGEV co-infection.
