Establishment and application of RT-RAA-CRISPR/Cas13a diagnostic method for porcine Senecavirus
10.16303/j.cnki.1005-4545.2025.02.04
- VernacularTitle:猪塞内卡病毒RT-RAA-CRISPR Cas13a检测方法的建立及应用
- Author:
Chenyu LI
1
;
Zhou SHA
;
Hui ZHENG
;
Jin CUI
;
Tianying CHI
;
Feng CHEN
;
Zhenshan CAO
;
Hui ZHANG
;
Shengqiang GE
;
Rong WEI
;
Fulong NAN
;
Shaopeng GU
;
Bo NI
Author Information
1. 山西农业大学动物医学学院,山西晋中 030801
- Publication Type:Journal Article
- Keywords:
RT-RAA;
CRISPR/Cas13a;
SVA
- From:
Chinese Journal of Veterinary Science
2025;45(2):195-203
- CountryChina
- Language:Chinese
-
Abstract:
The objective of this study was to develop a rapid and precise detection technique for por-cine Senecavirus A(SVA)employing reverse transcription recombinase polymerase amplification(RT-RAA)in conjunction with CRISPR Cas13a technology.Additionally,the study aimed to opti-mize the assay's reaction conditions to enhance amplification efficiency.Eight RT-RAA primer sets were designed based on the conserved gene sequence of porcine SVA,and a series of reaction condi-tions were evaluated to refine the RT-RAA reaction system.Subsequently,CRISPR-derived RNA(crRNA)sequences were developed and selected to construct the RT-RAA-CRISPR reaction sys-tem.The method's specificity was determined by examining six prevalent porcine pathogenic nucleic acids,while its sensitivity was assessed using SVA cRNA standards quantified by digital PCR.The method's stability and the consistency of clinical sample analysis were also evaluated.The findings revealed that the optimized RT-RAA and CRISPR reaction systems exhibited the highest amplifi-cation efficiency at a reaction temperature of 37 ℃.Among the eight crRNAs,five were identified as exhibiting the strongest detection signals.The formulated RT-RAA-CRISPR Cas13a method demonstrated exceptional specificity,showing no cross-reactivity with other common porcine disea-ses,including ASFV,PRRSV,PEDV,PCV2,CSFV,and PRV.The method achieved high sensitivi-ty,detecting as low as 0.86 copies/μL of SVA,and exhibited stable fluorescence output,robust re-producibility,and the ability to complete clinical sample analysis within 50 minutes.Consistency e-valuation with six positive and 58 negative samples indicated 100％agreement in outcomes.These results substantiate that the study successfully established a rapid and specific RT-RAA-CRISPR Cas13a detection method for the on-site identification of porcine Senecavirus A,demonstrating high specificity and sensitivity,and holds promise for application in SVA monitoring and control initia-tives.
