Establishment of Droplet Digital PCR Method for Detection of Listeria monocytogenes and Its Application in the Development of Reference Materials
XU Jia-Wei1,2,*, XIN Chang-Wei3,*, LI Tie-Shan2, ZHAO Ge1, QU Zhi-Na1, ZHAO Jian-Mei1, GAO Yu-Bin1, ZHANG Xi-Yue1,**, WANG Jun-Wei1,**
1 Pathogenic Microorganism Monitoring Room, China Animal Health and Epidemiology Center, Qingdao 266032, China; 2 Department of Rehabilitation Medicine, The Affiliated Hospital of Qingdao University, Qingdao 266003, China; 3 Bo'ai County Vocational Secondary Vocational School, Bo'ai 454450, China
Abstract：Listeria monocytogenes is one of the most serious foodborne pathogens. The aim of this study is to establish a rapid, sensitive, and accurate droplet digital PCR (ddPCR) method for detecting L. monocytogenes, and apply the method to develop the reference materials. In the study, the ddPCR method was established with the invasion associated protein gene (iap) as the target gene, the reaction conditions were optimized, the specificity, sensitivity, and repeatability of the method were tested, and the established method was used to detect clinical samples and the standard material was developed by using the method. The results showed that the ddPCR method had the best amplification effect when the amount of 10 μmol/L primer was 1.5 μL, the amount of 10 μmol/L probe was 0.45 μL and the annealing temperature was 60 ℃. Finally, the ddPCR method for L. monocytogenes was established. The established ddPCR method had good specificity and did not cross-react with other non-specific strains. Repeatability test results showed that the coefficient of variation between groups was 1.57%~4.32%, which proved that the method had good repeatability. The method had high sensitivity, and the lower limit of detection was 6.65 copies/μL. The results of clinical samples showed that the ddPCR method and fluorescence quantitative PCR method were 100% consistent with the results of 47 clinical positive samples stored in the laboratory. This method was used to measure the uniformity and stability of the reference materials, and 9 laboratories determined the reference materials. The fixed value was 5.79×103 copies/μL, and the uncertainty was 0.47×103 copies/μL. The ddPCR method established in this study can be used for the detection of L. monocytogenes in the laboratory and the development of reference materials, etc., and can be a technical means to monitor L. monocytogenes infection.
徐佳微, 辛长卫, 李铁山, 赵格, 曲志娜, 赵建梅, 高玉斌, 张喜悦, 王君玮. 单增李斯特菌微滴式数字PCR检测方法的建立及在标准物质研制方面的应用[J]. 农业生物技术学报, 2024, 32(10): 2413-2423.
XU Jia-Wei, XIN Chang-Wei, LI Tie-Shan, ZHAO Ge, QU Zhi-Na, ZHAO Jian-Mei, GAO Yu-Bin, ZHANG Xi-Yue, WANG Jun-Wei. Establishment of Droplet Digital PCR Method for Detection of Listeria monocytogenes and Its Application in the Development of Reference Materials. 农业生物技术学报, 2024, 32(10): 2413-2423.
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