Establishment of SYBR Green Ⅰ-based quantitative PCR targeting pseudorabies virus UL35 gene
10.16303/j.cnki.1005-4545.2024.11.04
- VernacularTitle:靶向伪狂犬病病毒UL35基因的SYBR Green Ⅰ荧光定量PCR方法的建立
- Author:
Xinyu ZHANG
1
;
Hongxia WU
;
Yongfeng LI
;
Yuan SUN
;
Qiang FU
;
HuaJi QIU
Author Information
1. 佛山科学技术学院生命科学与工程学院,广东佛山 528231;中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江哈尔滨 150069
- Keywords:
pseudorabies virus;
UL35 gene;
SYBR Green Ⅰ;
real-time PCR
- From:
Chinese Journal of Veterinary Science
2024;44(11):2334-2340
- CountryChina
- Language:Chinese
-
Abstract:
Quantitative analysis of UL35 and gB genes at different time points after PRV infection showed that there were significant differences between the two at 2.5,5.0 and 20.0 h after infec-tion,and the expression of UL35 gene could be observed in the early stage of PRV infection.To de-termine whether the UL 35 gene can be used as a target for the diagnosis of PRV infection,a spe-cific primer pair was designed and synthesized according to the conserved sequence of the UL35 gene of different PRV strains,and used to amplify a fragment of 54 bp in length.After optimizing the reaction conditions and system,the standard curve for the established by SYBR Green Ⅰ real-time PCR detection method showed that it had good repeatability,specificity and a good linear rela-tionship to the template concentration.No amplifications for porcine reproductive and respiratory syndrome virus(PRRSV),classical swine fever virus(CSFV),and African swine fever virus(AS-FV)were detected.Compared with the existing similar methods,the established method showed no difference in sensitivity.The coefficient of variation of inter-and intra-group repeatability for the method was less than 2％.Detection of the samples in mice challenged with PRV revealed a certain amount of viral load in tissue.The results showed that UL35 gene can be used as a target for the diagnosis of PRV infection.
