Method and detection kit for quantitatively detecting ralstonia solanacearum in soil
A technology for quantitative detection and bacterial wilt bacteria, which is used in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problem of not being able to distinguish dead bacteria and live bacteria well, and the complexity and time-consuming quantitative detection of pathogenic microorganisms. Problems such as labor consumption and trouble, to achieve good economic and social benefits, high sensitivity and good stability
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Embodiment 1
[0031] Taking representative R. solanacearum as the experimental strain, prepare a series of R. solanacearum concentration gradients and add them to the sterilized soil for correlation analysis between plate colony counting method and MPN counting method. Bacterial colonies were inoculated into 100mL SMSA enrichment medium, and incubated at 30°C with shaking at 200r / min for 13h. Then serial dilutions of different concentrations were prepared by ten-fold dilution and added to the sterilized soil, and the number of R. solanacearum was counted by plate colony counting method and MPN counting method, respectively.
[0032] Plate colony count method refers to the literature [Shen Ping, Fan Xiurong, Li Guangwu. Microbiology Experiments [M]. Beijing: Higher Education Press, 1999, 92-94. ], firstly carry out gradient dilution on the soil bacterial suspension prepared above, take 0.1mL of the appropriate dilution of the bacterial suspension and spread it on the SMSA enrichment medium...
Embodiment 2
[0046] Taking representative R. solanacearum as the experimental strain, prepare a series of R. solanacearum concentration gradients and add them to the sterilized soil for correlation analysis between plate colony counting method and MPN-PCR counting method counting results. Ralstonia solanacearum colonies were inoculated into 100mL SMSA enrichment medium, cultured at 30°C with shaking at 200r / min for 13h. Then serial dilutions of different concentrations were prepared by ten-fold dilution and added to the sterilized soil, and the number of R. solanacearum was counted by plate colony counting method and MPN-PCR counting method.
[0047] Plate colony count method refers to the literature [Shen Ping, Fan Xiurong, Li Guangwu. Microbiology Experiments [M]. Beijing: Higher Education Press, 1999, 92-94. ], firstly carry out gradient dilution on the soil bacterial suspension prepared above, take 0.1mL of the appropriate dilution of the bacterial suspension and spread it on the SMS...
Embodiment 3
[0062] The specificity of the PCR detection method mainly depends on the specificity of the primers, so the selection of the target gene should have species or genus specificity in order to avoid the occurrence of false positives. According to the hrpB gene sequence (AF295618.1) of R. solanacearum, the primer design software Primer 5.0 was used to design primers, and the specific primers P1 and P2 for detecting R. solanacearum were designed. The amplified fragment of the primers was 146bp (SEQ ID NO. 1); the above two pairs of primers were synthesized by GenScript. The specific sequence is as follows:
[0063] P1: 5'-CGGTTTTCGCCGCTGATTTCG-3',
[0064] P2: 5'-CTTCTTCCGCTTTCTTCATCGCACT-3'.
[0065] Using hrpB-specific primers from R. solanacearum for Bacillus subtilis 168, Pseudomonas putida KT2440, Ralstonia solanacearum, Brevibacterium, Microbacterium cellulosus, Microbacterium, Pseudomonas, Rhodococcus, Salmonella, Escherichia coli DH5α, etc. 10 bacterial strains were subj...
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