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

doi:10.16431/j.cnki.1671-7236.2025.02.040

Abstract

Abstract: 【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 Mg²⁺ in buffer,80 nmol/L Cas12a protein,400 nmol/L crRNA and 14 μmol/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.

Key words: Pasteurella multocida; Escherichia coli; recombinant enzyme polymerase amplification (RPA); CRISPR-Cas12a; visual detection

CLC Number:

S858.292

LI Yanhong, LI Juntao, YANG Tianmu, JIA Mengyan, XIONG Wenguang. Establishment of Detection Method for Pasteurella multocida and Escherichia coli from Poultry Based on CRISPR System[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(2): 912-921.

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

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