期刊
JOURNAL OF FOOD COMPOSITION AND ANALYSIS
卷 105, 期 -, 页码 -出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.jfca.2021.104224
关键词
Self-quenching fluorogenic approach; Salmo salar; Species identification; Target-specific detection; Loop-mediated isothermal amplification (LAMP)
In this study, Loop-mediated isothermal amplification (LAMP) coupled with a self-quenching approach was developed for accurate species identification in fish. The novel assay showed high tolerance to impurities and produced reliable positive results under different DNA extraction methods and sample treatments. Additionally, using DNA with higher purity improved LAMP amplification.
Loop-mediated isothermal amplification (LAMP) has emerged as an important diagnostic tool for fish authenticity purpose. While the sequence-independent detection methods greatly increased the likelihood of detecting false-positive signals. Instead, target-specific detection of LAMP amplicons can be achieved through the use of target-specific probes or modified primers as biorecognition elements. The present work selected Salmo salar as a case study, and developed a novel LAMP assay coupled with the self-quenching approach for accurate species identification. Specifically, the loop primer (LB-6) was identified as the candidate primer to design the self-quenching element. The fluorophore attached to LB-6 is self-quenched in unbound state. After binding to the dumb bell shaped DNA specifically, the FAM fluorophore is de-quenched, resulting in the fluorescence release. With the novel assay, as little as 5 pg of S. salar DNA could be detected. Moreover, the self-quenching assay embraces a high tolerance to impurities and the positive LAMP results can always be obtained, regardless of the DNA extraction methods and sample treatments. While significant difference with the Ct value according to both the extraction methods and sample treatments, highlighted also an improved LAMP amplification using DNA with higher purity.
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