中国兽医杂志2025，Vol.61Issue(11)：70-76,7.DOI:10.20157/j.cnki.zgsyzz.2025.11.010
牛轮状病毒RAA-CRISPR/Cas13a快速检测方法的建立
Establishment of RAA-CRISPR/Cas13a Rapid Detection Method for Bovine Rotavirus
摘要
Abstract
To develop a rapid detection method for bovine rotavirus(BRV),this study combined recombinase-aided amplification(RAA)technology with the clustered regularly interspaced short palindromic repeats and associated protein 13a(CRISPR/Cas13a)system.Target RNA,RAA primers,and crRNA were designed based on the conserved VP6 gene sequence of BRV.Optimal RAA primers and reaction conditions were screened to establish a BRV RAA-CRISPR/Cas13a detection method.The specificity,sensitivity,and repeatability of the method were validated,and the results were compared with those obtained by reverse transcription-real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)in clinical samples.The results showed that the RAA-CRISPR/Cas13a method exhibited strong specificity,with no cross-reactivity observed with bovine viral diarrhea virus(BVDV),bovine coronavirus(BCoV),bovine astrovirus(BoAstV),or Escherichia coli.The detection limit was 1.9×10² copies/µL,indicating high sensitivity.Intra-and inter-assay coefficients of variation were 3.9%and 7.4%,respectively,both below the thresholds of 5%and 10%,demonstrating good repeatability.Among 108 bovine fecal samples tested,the positive detection rate of both the RAA-CRISPR/Cas13a and RT-qPCR methods was 44%(47/108),with a 100%concordance rate between the two methods.In conclusion,the BRV RAA-CRISPR/Cas13a detection method established in this study is highly sensitive and specific,providing a robust tool for rapid clinical detection of BRV.关键词
牛轮状病毒(BRV)/重组酶介导等温扩增(RAA)/CRISPR/Cas13a/实时荧光定量聚合酶链式反应(qPCR)/病毒检测Key words
bovine rotavirus(BRV)/recombinase-aided amplification(RAA)/CRISPR/Cas13a/real-time fluorescence quantitative polymerase chain reaction(qPCR)/virus detection分类
畜牧业引用本文复制引用
袁炫帅,赵琳琳,龙淼..牛轮状病毒RAA-CRISPR/Cas13a快速检测方法的建立[J].中国兽医杂志,2025,61(11):70-76,7.基金项目
辽宁省教育厅基本科研项目(JYTYB2024027) （JYTYB2024027）
