Peer-reviewed veterinary case report
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).
- Journal:
- Journal of microbiological methods
- Year:
- 2012
- Authors:
- Mao, Zhijuan et al.
- Affiliation:
- Biological and Environmental College · China
Plain-English summary
This study focuses on a serious problem affecting the large yellow croaker fish, which is being harmed by a bacterial infection caused by Pseudomonas putida. Researchers developed a new test called a visual loop-mediated isothermal amplification (LAMP) method that can quickly detect this bacteria in fish. They created specific primers to target a part of the bacteria's DNA, making the test much more sensitive than older methods, allowing it to detect very low levels of the bacteria. The test was able to identify the bacteria in infected fish tissues and is designed to be simple and cost-effective, which could help in diagnosing this infection more rapidly. Overall, the new LAMP method shows promise for effectively detecting this harmful pathogen in fish.
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.
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Search related cases →Original publication: https://pubmed.ncbi.nlm.nih.gov/22475582/