Establishment of a fluorescence quantitative PCR assay in detection of Listeria monocytogenes based on internal reference Chang Liyun1,*, Zhang Xiaoli2, Cai Yumei1, Zhao Na1, Li Huawei1, Li Chenghui1, Dong Lixue2,** 1Faculty of Life Science, Tangshan Normal University, Thangshan, Hebei, 063001, China 2Tangshan Animal Disease Prevention and Control Center, Thangshan, Hebei, 063001, China *Corresponding Authors: Liyun Chang, Faculty of Life Science, Tangshan Normal University, Thangshan, Hebei, 063001, China, Email: changliyun2006@163.com
**Lixue Dong, Faculty of Life Science, Tangshan Normal University, Thangshan, Hebei, 063001, China, donglixue1984@126.com
Online Published on 5 January, 2024. Abstract Background Listeria monocytogenes is the only pathogenic bacterium of Listeria that causes disease to humans. The Dairy products, meat and other foods have been proved to be the transmission carriers of Listeria monocytogenes, endangering human health. Therefore, it is necessary to establish a fast, sensitive and specific fluorescent quantitative PCR method for detecting Listeria monocytogenes in milk based on internal reference. Methods According to the genome sequence of Listeria monocytogenes published in Genbank, we screened specific target gene Hly gene, designed specific primers and probes, optimized the reaction system and added internal reference (IAC) to the system. This IAC was detected by TaqMan probes labeled with different fluorophore. 25-100 CFU/25 g artificially contaminated sample was added to evaluate the performance of reaction system. Result The assay was could be used reliably for detection of Listeria monocytogenes with a sensitivity of 10-3ng/uL. For the 10-fold dilutions bacteria DNA extracted by cooking water,the lowest detection limit was 4 × 102 cfu/mL. But for the plasmid with hly, the lowest detection limit can reach 104 cfu/mL. The standard curve of hly and hly-IAC was established and the quantification was linear between Ct and template copy number (r2= 0.999). While the initial sample amount of artificially contaminatedbacteria was 25 CFU/25 g (Fresh milk, milk powder, fermented milk), Listeria monocytogenes could be detected in 14 h. The Hly IAC fluorescent quantitative PCR detection method established in this study, and added internalreference tothe system, can accurately detect Listeria monocytogenesin fresh milk. At the same time, real- time monitoring of the PCR reaction process ensures the reliability of the results, which is suitable for rapid detection of large batches of samples. Top Keywords Fluorescence quantitative PCR, Fresh milk, Internal reference, Listeria monocytogenes, Test method. Top |