Biosensor and method for detecting salmonella
A biosensor and detection method technology, applied in the field of molecular biology, to achieve the effect of detection at the single-cell level
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Embodiment 1
[0062] 1. Experimental materials
[0063] 1.1 Test strains
[0064] Salmonella (CGMCC 1.0090), Enterobacter sakazakii (CICC 21560), Listeria monocytogenes (CMCC55004), Staphylococcus aureus (ATCC 25923), Vibrio parahaemolyticus (CMCC20001), Escherichia coli (ATCC43889) were all imported from China Provided by the Food Safety Laboratory of the School of Food Science and Nutritional Engineering, Agricultural University.
[0065] 1.2 Main reagents
[0066] Yeast extract powder, peptone, yeast extract, beef extract, glucose, dipotassium hydrogen phosphate, ammonium citrate, anhydrous sodium acetate, magnesium sulfate heptahydrate, manganese sulfate tetrahydrate, Tween 80, sodium chloride, agar, ethyl alcohol Diaminetetraacetic acid (EDTA), sodium hydroxide, tris(Tris), glacial acetic acid, hemin (Hemin), agarose, 3,3',5,5'-tetramethylbis Aniline (TMB), RPA reaction kit, sulfuric acid, terminal deoxynucleotidyl transferase (TdT), double-strand specific nuclease (DSN), dGTP, dATP...
Embodiment 2
[0091] 1. Optimization of RPA reaction conditions
[0092]In the present invention, the RPA reaction is first carried out, and the reaction conditions are 39° C. and 20 minutes. The RPA reaction was carried out with different ratios of primers, and the optimal ratio of primers was found for constant temperature amplification. After the reaction was completed, the amplified products were analyzed by 2% agarose gel electrophoresis. It can be obtained that all the products amplified by RPA with the newly designed primers are longer than the DNA fragments amplified without the universal sequence (comparison sequence RPA-R: CCTCAATACTGAG CGGCTGCTCGCCTTTGCTGG, see Table 1); and when the upstream primer: downstream Primer: Universal Primer=3:1:2, the brightness of the electrophoresis band is brighter than other bands (swimming lane 8), indicating that the amount of RPA product is also the largest ( figure 1 ).
[0093] 2. Optimization of Lambda exonuclease reaction conditions
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