Frontiers in cellular and infection microbiology · 2025
A CRISPR/cas13a-assisted precise and portable test for Brucella nucleic acid detection.
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Liu Haiwen, Xu Ling, Xiu Ying, Ta Na, Xu Qingqing, Fan Yu, Li Kun, Zhao Hongyan, Piao Dongri, Ren Feng, Jiang Hai
Аннотация
INTRODUCTION: Brucella infection in humans or animals can lead to brucellosis, which has the potential to significantly impact both the economy and public health. Currently, molecular biological methods for diagnosing brucellosis are either complex or have low sensitivity, and it is difficult to apply them in real-life settings in the field. Therefore, this study aims to establish a rapid and convenient nucleic acid-based molecular biology method for on-site rapid detection of Brucella and early clinical screening of brucellosis.
METHODS: Based on the conserved sequence of the Brucella Bcsp31 gene, we designed CRISPR RNA (crRNA) and RAA primers. We developed a fluorescence detection method and a paper strip detection method by integrating RAA amplification with CRISPR/Cas13a detection. We applied these methods to analyze 100 samples of suspected brucellosis-infected milk, 123 samples of human whole blood, and 100 samples of sheep vaginal swabs in order to validate their practical utility.
RESULTS: The RAA-CRISPR/Cas13a Brucella fluorescence detection method and the strip test method had detection limits of 100 copies/μL and 101 copies/μL, respectively, and both methods had a specificity of 100%. The positivity rate of the RAA-CRISPR/Cas13a fluorescence detection method for the milk, human whole blood, and sheep vaginal swab samples was 93% (93/100), 82.12% (101/123), and 91% (91/100), respectively; the strip test method, 87% (87/100), 64.23% (79/123), and 76% (76/100), respectively.
CONCLUSION: In this study, we have developed a RAA-CRISPR detection method based on the Brucella BCSP31 gene, with potential applications in the identification of Brucella nucleic acid and implications for clinical diagnosis of brucellosis.
METHODS: Based on the conserved sequence of the Brucella Bcsp31 gene, we designed CRISPR RNA (crRNA) and RAA primers. We developed a fluorescence detection method and a paper strip detection method by integrating RAA amplification with CRISPR/Cas13a detection. We applied these methods to analyze 100 samples of suspected brucellosis-infected milk, 123 samples of human whole blood, and 100 samples of sheep vaginal swabs in order to validate their practical utility.
RESULTS: The RAA-CRISPR/Cas13a Brucella fluorescence detection method and the strip test method had detection limits of 100 copies/μL and 101 copies/μL, respectively, and both methods had a specificity of 100%. The positivity rate of the RAA-CRISPR/Cas13a fluorescence detection method for the milk, human whole blood, and sheep vaginal swab samples was 93% (93/100), 82.12% (101/123), and 91% (91/100), respectively; the strip test method, 87% (87/100), 64.23% (79/123), and 76% (76/100), respectively.
CONCLUSION: In this study, we have developed a RAA-CRISPR detection method based on the Brucella BCSP31 gene, with potential applications in the identification of Brucella nucleic acid and implications for clinical diagnosis of brucellosis.