Primer and probe for detecting acidovorax citrulli and detection method for acidovorax citrulli
A kind of melon fruit spot bacteria and probe technology, which can be used in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., and can solve the problems of specificity, sensitivity and broad spectrum.
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Embodiment 1
[0015] Design and synthesis of embodiment 1 primers and probes
[0016] In this study, through the analysis of the genome sequence of the reported Melon fruit spot bacteria, the primers and probes were designed using the software primer Express 3.0, and finally the primer sequences determined by comparison and screening were as follows:
[0017] Seq1: 5′-CTGATAATCCTCGGCTCAACAA-3′,
[0018] Seq2: 5'-TGAGCGCATTTCTGACGAG-3',
[0019] The probe sequence is: Seq3: 5′-AAGAAATACGCCCTCGCCAATCTCC-3′,
[0020] The 5' end of the probe is labeled with a reporter fluorescent group, and the 3' end is labeled with a non-fluorescent quencher group.
Embodiment 2
[0021] Embodiment 2 Establishment of real-time fluorescent PCR detection method
[0022] Add 12.5 μL 2× fluorescent PCR reaction master mix, 1 μL primer Seq1 (10 μmol / L), 1 μL primer Seq2 (10 μmol / L), 0.5 μL TaqMan probe Seq3 (10 μmol / L), 1 μL DNA, supplement Ultrapure water to 25 μl. After putting the sample tube into the ABI 7900 fluorescent PCR instrument, set the following conditions for the reaction: the first cycle is 95°C for 10 minutes; the next 40 cycles are 94°C for 15s and 58°C for 1min. Fluorescence signals are collected in each cycle, and the results are judged according to the amplification curve after the reaction.
Embodiment 3
[0023] The specific experiment of embodiment 3 real-time fluorescent PCR method
[0024] The specificity of the method was tested with Pseudomonas cucurbita and its relatives. The related species used included A.avenae subsp.aveane, A.avenae subsp.cattleyae, A.konjaci and other genera, 23 strains of plant pathogens and 10 strains of Melon fruit blotch from different sources. In the experiment, directly take the concentration as 10 6 1 μl of bacterial solution with about CFU / mL was used as a template for real-time fluorescent PCR reaction, and the results showed that all melon fruit spot disease strains were positive ( figure 1 The curve marked by A) other non-cucurbit fruit blotches ( figure 1 The curve marked by B in the middle) are all negative results. It was proved that the method has good specificity.
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