Four-color fluorescent PCR kit for detection of vibrios in water body and detection method
A kit and technology for Vibrio, applied in the field of four-color fluorescent PCR kits for detecting Vibrio in water, can solve the problems of long time and time consuming for detection, reduce detection cost, reduce workload, and improve detection. The effect of the rate
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Embodiment 1
[0034]A four-color fluorescent PCR kit for detecting pathogenic Vibrio in water, the kit is composed of multiple fluorescent PCR reaction mother solution, bacteria enrichment reagent (containing PEG8000 with a final concentration of 20% by mass percentage, chlorinated sodium), DNA extraction reagent (containing tris hydrochloride at a final concentration of 20mM, potassium chloride at a final concentration of 100mM, ethylenediaminetetraacetic acid at a final concentration of 2.5mM, and NP40 at a final concentration of 1% by mass) , Taq DNA polymerase with a concentration of 5U / ul, wherein the multiplex fluorescent PCR reaction mother solution is by volume ratio: multiplex 10× fluorescent PCR reaction buffer (containing trishydrochloric acid with a final concentration of 100mM, and a final concentration of 500mM Potassium chloride, glycerol with a final concentration of 50% by mass, dATP, dGTP, dTTP and dGTP with a final concentration of 0.2mM, magnesium chloride with a final co...
Embodiment 2
[0036] 1. Select the following pathogenic microorganisms, the experimental group: O1 group Vibrio cholerae, O139 group Vibrio cholerae, Vibrio parahaemolyticus; control group: Vibrio alginolyticus, Vibrio mimicus, Vibrio river, Vibrio vulnificus, Shigella Bacteria and Salmonella, the above pathogenic strains were isolated and cultured from environmental samples: Vibrio cholerae O1 group, Vibrio cholerae O139 group, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio mimicus, Vibrio river, Vibrio vulnificus, Chi Shigella and Salmonella were collected, isolated and cultivated in various water bodies in Ningbo around March 2011 by Zhou Donggen and other comrades of our research institute. The contact number is 0574-87169626.
[0037] 2. Sample preparation in the simulated environment: each concentration of 0.5ml was 1×10 5 CFU / ml of O1 group Vibrio cholerae, O139 group Vibrio cholerae, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio mimicus, Vibrio river, Vibrio vulnificu...
Embodiment 3
[0044] The 3 samples of drinking water to be tested were tested in the same manner as steps 3, 4, 5, 6, and 7 in Example 2. As a result, the ROX channel of 1 sample of drinking water to be tested showed an S-shaped amplification curve, and the Ct value was between 34 During the period, there was Vibrio parahaemolyticus infection.
[0045] Ningbo Institute of Inspection and Quarantine Science and Technology
[0046] A four-color fluorescent PCR kit and detection method for detecting Vibrio in water
[0047] 12
[0048] PatentIn version 3.5
[0049]
[0050] 1
[0051] 22
[0052] DNA
[0053] Artificial sequence
[0054]
[0055] 1
[0056] CCAGATTGTA AAGCAGGATG GA 22
[0057]
[0058] 2
[0059] 19
[0060] DNA
[0061] Artificial sequence
[0062]
[0063] 2
[0064] GGTCATCTGT AAGTACAAC 19
[0065]
[0066] 3
[0067] 27
[0068] DNA
[0069] Artificial sequence
[0070]
[0071] 3
[0072] CCCGGAGTTT GTAAGCCCAC TACCGGG 27
[0073]
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