title: Multiple Endonuclease Restriction Real-Time Loop-Mediated Isothermal Amplification: A Novel Analytically Rapid, Sensitive, Multiplex Loop-Mediated Isothermal Amplification Detection Technique.
abstract: Loop-mediated isothermal amplification (LAMP) is restricted to detecting a single target, limiting the usefulness of this method. To achieve multiplex LAMP-based detection, we developed a novel approach we called the multiple endonuclease restriction real-time-LAMP assay. In this system, the LAMP forward or backward inner primers contain 5' end short sequences that are recognized by the restriction endonuclease Nb.BsrDI, and the new forward or backward inner primers were modified at the 5' end with a fluorophore and in the middle with a dark quencher. Nb.BsrDI digests the newly synthesized double-stranded terminal sequences (5' end short sequences and their complementary sequences), which releases the quenching, resulting in a gain of signal. The assay permitted real-time detection of single or multiple target sequences in a single tube, and the positive results can be obtained in as short as 12 minutes. The novel methodology is highly efficient and specific, detecting down to 250 fg of DNA per reaction of Listeria DNA tested, and was successful in evaluating raw meat samples. The multiple endonuclease restriction real-time-LAMP technology, which is an extension of LAMP to accommodate robust, target-specific, and multiplex detection, provides a molecular diagnostic tool with less detection time and high sensitivity and specificity compared with those of LAMP and quantitative real-time PCR.
authors.affiliation: State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, 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, Chinese Center for Disease Control and Prevention, 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: School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia.
authors.forename: Ruiting
authors.initials: R
authors.lastname: Lan
authors.affiliation: Department of Microbiology, Guiyang Medical University, Guiyang, China.
authors.forename: Huaqing
authors.initials: H
authors.lastname: Xu
authors.affiliation: State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, 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: Department of Microbiology, 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, Chinese Center for Disease Control and Prevention, Beijing, China; Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China.
authors.forename: Hang
authors.initials: H
authors.lastname: Dai
authors.affiliation: State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China; Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China.
authors.forename: Xuejiao
authors.initials: X
authors.lastname: Yuan
authors.affiliation: State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China; Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China.
authors.forename: Jianguo
authors.initials: J
authors.lastname: Xu
authors.affiliation: State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China; Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China. Electronic address: yechangyun@icdc.cn.
authors.forename: Changyun
authors.initials: C
authors.lastname: Ye
mesh_terms: D001483:Base Sequence; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D005456:Fluorescent Dyes; D006801:Humans; D008089:Listeria monocytogenes / Q000235:genetics / Q000302:isolation & purification*; D008088:Listeriosis / Q000517:prevention & control*; D008460:Meat / Q000032:analysis* / Q000382:microbiology; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D004269:DNA, Bacterial; D005456:Fluorescent Dyes; D004262:DNA Restriction Enzymes