A detection method of sea urchin pathogen Vibrio fortis
A detection method, pathogenic bacteria technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems that affect the accuracy of detection results, cumbersome operations, false positives, etc., achieve fast and efficient detection, avoid false positives, etc. Negative, ability-enhancing effects
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Embodiment 1
[0036] Design and synthesis of embodiment 1 probe
[0037] 1. Target detection strains and target genes
[0038] The present invention selects Vibrio carteri, an important common pathogenic bacterium in the sea urchin breeding area environment and organisms, as the detection object, and uses membrane protein as the target gene;
[0039] 2. Probe design
[0040] The present invention uses 18-30bp oligonucleotide probes. The basic requirement of probe parameters is specific and efficient hybridization, and the following conditions should be met: ①The Tm value of the probe should remain similar and fluctuate within a range of 10°C; ②The number of consecutive complementary bases that form dimers and hairpin structures Less than 4; ③ The number of consecutive matching bases between the probe and the non-target gene sequence is less than 7 bases; ④ The number of mismatched bases between the probe and the target gene is less than 4 bases.
[0041] The present invention designs 3 p...
Embodiment 2
[0048] The making of embodiment 2 gene chip
[0049] Utilize the probe designed in Example 1 to prepare the important common pathogenic bacteria Vibrio fortis in the sea urchin culture area environment and organisms, the specific steps are as follows:
[0050] 1. Preparation of probe mother solution
[0051] Dilute the synthesized probe (Table 2) into a 100 μmol / L mother solution with double distilled water, and then dilute it to a concentration of 20 μmol / L with a spotting buffer;
[0052] 2. Preparation before sampling
[0053] Transfer 100 μL to wells A1, B1 and C1 of a 96-well plate, and add 100 μL of spotting buffer to well D1;
[0054] 3. Spotting
[0055] Use the AD1500 gene chip spotting instrument of Bio-Dot Company of the United States to carry out non-contact spotting on the aldehyde substrate according to the pre-set program; the spotting volume of each point is 0.5 μL, the spot diameter is 200 μm, and the point spacing is 1.5 μL. mm, the relative humidity of t...
Embodiment 3
[0061] For the detection of the common pathogenic bacteria Vibrio fortis gene chip (prepared in Example 2) in the sea urchin breeding area environment and organisms, the specific steps are as follows:
[0062] 1. Collection and processing of samples to be tested
[0063] Use an 8 μm sterile filter membrane to remove impurities from the obtained water sample, then filter it with a 0.22 μm filter membrane and collect the filter membrane, wrap it in sterile aluminum foil, and store it at -20°C;
[0064] The mud sample is taken from 100g of sediment at the bottom 2-5cm of the breeding environment, and the collected samples are transported to the laboratory in an ice box;
[0065] Sea urchins (animal samples) were cut with sterile scissors, rinsed with sterile water three times, put into cryopreservation tubes, and stored in liquid nitrogen.
[0066] 2. Template DNA preparation
[0067]The DNA of water samples and mud samples was extracted with OMEGA Water DNA Kit (OMEGA Water DN...
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