Establishment and application of LAMP-Taqman detection method of porcine pseudorabies virus
10.16303/j.cnki.1005-4545.2024.10.04
- VernacularTitle:猪伪狂犬病病毒LAMP-Taqman检测方法的建立与应用
- Author:
Yu LI
1
;
Lei SHI
;
Guoqiang SHI
;
Yinglu ZHANG
;
Zhenguo DONG
Author Information
1. 河北三狮生物科技有限公司,河北石家庄 050035
- Keywords:
pseudorabies virus;
LAMP;
Taqman;
detection method
- From:
Chinese Journal of Veterinary Science
2024;44(10):2110-2115
- CountryChina
- Language:Chinese
-
Abstract:
A LAMP-Taqman rapid detection system for porcine pseudorabies virus(PRV)was de-veloped based on LAMP and quantitative PCR.LAMP primers were designed for PRV conserved sequences,and the loop primer modified by the fluorescent quenching group was used as the Taq-man probe.The composition optimization,specificity,sensitivity and repeatability of the LAMP-Taqman system were tested using positive samples and recombinant plasmid as templates.Thirty-eight samples of pork swabs were tested with the commercial LAMP detection kit in parallel to verify the actual detection effect of the LAMP-Taqman detection system.The results showed that the optimal final concentration of each component was as follows:PRV-FIP/BIP 0.8 μmol/L,Bst DNA polymerase 0.7 U/μL,Taq DNA polymerase 0.24 U/μL,dNTPs 1.6 mmol/L,MgSO47.2 mmol/L.This system had good specificity and did not cross-react with other virus samples.The linear correlation coefficient of gradient samples was 0.995,the coefficient of variation of repeatable tests was less than 3.000％,and the minimum detection limit could reach 2.81 ×102 copies/μL.The test results of the actual swab samples were consistent with the commercial isothermal fluores-cence detection reagents.In conclusion,the PRV detection system established by LAMP-Taqman method in this study is specific,sensitive,stable and accurate,and is a reliable technical method suitable for the accurate detection of porcine PRV.
