基于CRISPR系统的禽源多杀性巴氏杆菌、大肠杆菌检测方法的建立  

Establishment of Detection Method for Pasteurella multocida and Escherichia coli from Poultry Based on CRISPR System

作　　者：李燕红 李鋆涛 杨天沐 贾梦言 熊文广 LI Yanhong;LI Juntao;YANG Tianmu;JIA Mengyan;XIONG Wenguang(Guangdong Provincial Key Laboratory of Veterinary Pharmaceutics Development and Safety Evaluation,Guangzhou 510642,China;National Laboratory of Safety Evaluation(Environmental Assessment)of Veterinary Drugs,South China Agricultural University,Guangzhou 510642,China;National Risk Assessment Laboratory for Antimicrobial Resistance of Animal Original Bacteria,Guangzhou 510642,China)

机构地区：[1]广东省兽药研制与安全评价重点实验室,广州510642 [2]国家兽药安全评价(环境评估)实验室(华南农大),广州510642 [3]国家兽医微生物耐药性风险评估实验室,广州510642

出　　处：《中国畜牧兽医》2025年第2期912-921,共10页China Animal Husbandry & Veterinary Medicine

基　　金：广东省基础与应用基础研究基金(2023A1515030137)。

摘　　要：[目的]建立禽源多杀性巴氏杆菌、大肠杆菌的可视化检测方法。[方法]将重组酶聚合酶扩增(RPA)与成簇规律间隔短回文重复序列及其相关蛋白(CRISPR-Cas)系统相结合,针对两种病原菌设计特异性CRISPR相关RNA(crRNA)序列,筛选RPA引物,并对CRISPR检测体系进行优化,探究单管双靶标扩增体系,评估检测方法的特异性及灵敏度,验证临床样品检测的可行性。[结果]本研究建立的检测方法最优组合为:Buffer中Mg^(2+)浓度20 mmol/L、Cas12a蛋白80 nmol/L、crRNA 400 nmol/L、ROX荧光探针14μmol/L。特异性及灵敏度检测结果显示,本研究建立的检测方法特异性良好,在蓝光下观察,灵敏度与实时荧光定量PCR一致,比普通PCR高100倍。临床样品检测结果显示,巴氏杆菌、大肠杆菌均被有效检出,本研究所建立方法的临床符合率为100%。[结论]本研究成功建立了禽源多杀性巴氏杆菌、大肠杆菌的可视化检测方法,其特异性及灵敏度较高,可以准确检测临床样品中的目标病原菌。试验结果为监测与防控禽源细菌性疾病提供了新方法。[Objective]The objective of this experiment was to establish a visual detection method for Pasteurella multocida and Escherichia coli of avian origin.[Method]Recombinant enzyme polymerase amplification(RPA)was combined with clusters of regularly spaced short palindromic repeats and their associated proteins(CRISPR-Cas)system to design specific CRISPR-associated RNA(crRNA)sequences,and screen RPA primers.The CRISPR detection system was optimized to explore the single-tube double-target amplification system,evaluate the specificity and sensitivity of the detection method,and verify the feasibility of clinical sample detection.[Result]The optimal combination of detection methods established in this study was:20 mmol/L Mg^(2+) in buffer,80 nmol/L Cas12a protein,400 nmol/L crRNA and 14μmol/L ROX fluorescent probe.The specificity and sensitivity test results showed that the detection method established in this study had good specificity,and the sensitivity was consistent with Real-time quantitative PCR when observed under blue light,and 100 times higher than ordinary PCR.The results of the clinical samples showed that Pasteurella multocida and Escherichia coli were effectively detected in the samples,and the clinical compliance rate of the method established in this study was 100%.[Conclusion]This study successfully established a visual detection method for Pasteurella multocida and Escherichia coli of avian origin,which had high specificity and sensitivity,and could accurately detect target pathogens in clinical samples.The results provided a new method for monitoring and controlling avian bacterial diseases.

关 键 词：多杀性巴氏杆菌 大肠杆菌 重组酶聚合酶扩增(RPA) CRISPR-Cas12a 可视化检测 

分 类 号：S858.292[农业科学—临床兽医学]