Detection reagent kit and detection method of aeromonas bacteria
A detection kit and technology for Aeromonas, applied in botany equipment and methods, microbiological-based methods, biochemical equipment and methods, etc., can solve the problem that whether the detection sensitivity is consistent cannot be proved, and the detection sensitivity and operation are not provided. Complicated problems, to achieve good application prospects, prevent fish diseases, and high sensitivity
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Embodiment 1
[0030] Example 1 Primer Design
[0031] 1. Experimental method
[0032] 1. PCR primer design and synthesis
[0033] According to experience, the common glycerophospholipid cholesterol acyltransferase gene (gene number: HM245310.1, HM245308.1, GQ856318.1, CP000644.1, AF268080.1) and 16s rRNA (gene number: HM245310 .1, HM245308.1, GQ856318.1, CP000644.1, AF268080.1) designed two pairs of primers respectively, and sent them to Shanghai Sangong for synthesis after Blast comparison.
[0034] Table 1PCR primer information
[0035]
[0036] 2. Template preparation
[0037] Streak inoculate Aeromonas hydrophila TW-YYS-26 and Aeromonas caviae ATCC15468 stored at -20°C on LB solid medium plate, culture at 28°C, pick a single colony after growing a single colony and inoculate it into LB Liquid culture medium, cultivate overnight at 28°C in a shaker to activate the bacteria. The bacterial culture solution was taken, and DNA was extracted using a genomic DNA extraction kit (TIANGEN...
Embodiment 2
[0049] Embodiment 2 primer specificity test
[0050] 1. Test method
[0051] 1. PCR primers
[0052] Take the primer pair 1 in Example 1.
[0053] 2. Template preparation:
[0054] The bacteria listed in Table 1 were respectively activated with culture medium before the test. After the bacteria were activated, DNA was extracted according to the operating instructions of the Bacterial Genomic DNA Extraction Kit (TIANGEN) and used as a template for PCR detection.
[0055] 3. PCR amplification
[0056] Take the genomic DNA obtained in step 2 as a template, and the template for the negative control is ddH 2 O, amplify according to the amplification procedure in Example 1.
[0057] 4. Result detection
[0058] Take 5 μL of the amplified product, use 1% agarose gel, electrophoresis at a constant voltage of 80V for 60 min, and place it in a gel imaging system to observe the PCR amplification results.
[0059] 2. Results
[0060] The result is as figure 2 As shown, only Aer...
Embodiment 3
[0062] Embodiment 3 primer sensitivity test
[0063] 1. Test method
[0064] 1. PCR primers
[0065] Take the primer pair 1 in Example 1.
[0066] 2. Template preparation:
[0067] Aeromonas hydrophila TW-YYS-26 and Aeromonas caviae ATCC15468 were respectively activated with medium. After the bacteria were activated, DNA was extracted according to the operating instructions of the Bacterial Genomic DNA Extraction Kit (TIANGEN);
[0068] Determine the concentration of the DNA sample, and follow the steps of 10 for the DNA sample -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 、10 -9 Perform serial dilutions.
[0069] 3. PCR amplification
[0070] The genomic DNA of Aeromonas hydrophila and Aeromonas caviae in a gradient dilution was used as a template, and amplified according to the amplification procedure in Example 1.
[0071] 4. Result detection
[0072] Take 5 μL of the amplified product, use 1% agarose gel, electrophoresis at a constant voltage of 80V f...
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