title: Rapid and sensitive detection of Listeria monocytogenes by cross-priming amplification of lmo0733 gene.
abstract: Listeria monocytogenes is a food-borne pathogen that causes severe opportunistic infection in humans and animals. This study reports the development of single cross-priming amplification (S-CPA) and double CPA (D-CPA) assays targeting species-specific gene lmo0733 for identifying L. monocytogenes strains. The CPA assays were performed at a constant temperature 64 °C using seven specific primers and evaluated for specificity and sensitivity. The color change of positive amplification was directly observed by Loopamp®Fluorescent Detection Reagent (FD), and the DNA products were visualized as a ladder-like banding pattern on 2.5% gel electrophoresis. Moreover, the positive reactions were also detected by real-time measurement of turbidity. 50 L. monocytogenes and 46 non-L. monocytogenes strains were used for the method verification, and the specificity was 100%. The limit of detection (LoD) of the S-CPA and D-CPA assays was 2.5 pg DNA per reaction and 10-fold more sensitive than PCR. A total of 60 pork samples were tested for L. monocytogenes using the S-CPA assay developed in the study, and the accuracy of the S-CPA and the culture-biotechnical method was 100% identical. The results suggested that the S-CPA assay was a rapid, sensitive, and valuable tool for detection of L. monocytogenes in food products.
authors.affiliation: State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, China CDC, Changping, Beijing, China.|Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Wang
authors.affiliation: State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, China CDC, Changping, Beijing, China.|Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, China CDC, Changping, Beijing, China.|Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China.
authors.forename: Aijing
authors.initials: A
authors.lastname: Ma
authors.affiliation: Guiyang Medical University, Guiyang, China.
authors.forename: Dongxun
authors.initials: D
authors.lastname: Li
authors.affiliation: State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, China CDC, Changping, Beijing, China yechangyun@icdc.cn.|Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China.
authors.forename: Changyun
authors.initials: C
authors.lastname: Ye
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D005516:Food Microbiology; D005784:Gene Amplification; D006801:Humans; D057230:Limit of Detection; D008089:Listeria monocytogenes / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D000069466:Red Meat / Q000382:microbiology*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D013552:Swine; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers
keywords: CPA; L. monocytogenes detection; Lmo0733 gene amplification; LoD