title: Development of a visual loop-mediated isothermal amplification method for rapid detection of the bacterial pathogen Pseudomonas putida of the large yellow croaker (Pseudosciaena crocea).
abstract: In recent years, the large yellow croaker (Pseudosciaena crocea), an important marine fish farmed in the coastal areas of Zhejiang province, east China, has become severely endangered as a result of the bacterial pathogen Pseudomonas putida. This paper reports the development of a visual loop-mediated isothermal amplification (LAMP) assay for rapid detection of the pathogen. Four primers, F3, B3, FIP and BIP, were designed on the basis of DNA sequence of the rpoN gene of P. putida. After optimization of the reaction conditions, the detection limit of LAMP assay was 4.8cfu per reaction, 10-fold higher than that of conventional PCR. The assay showed high specificity to discriminate all P. putida isolates from nine other Gram-negative bacteria. The assay also successfully detected the pathogen DNA in the tissues of infected fish. For visual LAMP without cross-contamination, SYBR Green I was embedded in a microcrystalline wax capsule and preset in the reaction tubes; after the reaction the wax was melted at 85°C to release the dye and allow intercalation with the amplicons. The simple, highly sensitive, highly specific and cost-effective characteristics of visual LAMP may encourage its application in the rapid diagnosis of this pathogen.
authors.affiliation: Biological and Environmental College, Zhejiang Wanli University, Ningbo 315100, China. mzjjane@hotmail.com
authors.forename: Zhijuan
authors.initials: Z
authors.lastname: Mao
authors.forename: Yangyu
authors.initials: Y
authors.lastname: Qiu
authors.forename: Lei
authors.initials: L
authors.lastname: Zheng
authors.forename: Jigang
authors.initials: J
authors.lastname: Chen
authors.forename: Jifang
authors.initials: J
authors.lastname: Yang
mesh_terms: D000818:Animals; D001431:Bacteriological Techniques / Q000379:methods*; D002681:China; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D005393:Fish Diseases / Q000175:diagnosis* / Q000382:microbiology; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010473:Perciformes / Q000382:microbiology*; D011552:Pseudomonas Infections / Q000175:diagnosis / Q000662:veterinary*; D016958:Pseudomonas putida / Q000235:genetics* / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D014730:Veterinary Medicine / Q000379:methods
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial