Real time FQ-PCR detection kit of vibrio parahaemolyticus and detection method
A technology of hemolytic Vibrio and detection kits, which is applied in the determination/testing of microorganisms, biochemical equipment and methods, and resistance to vector-borne diseases, etc. It can solve the problems of cumbersome operations and long time required to achieve repeatability Good, easy to operate, the effect of preventing environmental pollution
Inactive Publication Date: 2009-03-11
ZHEJIANG OCEAN UNIV +1
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[0005] The purpose of the present invention is to provide a real-time FQ-PCR detection kit and detection method of Vibrio parahaemolyticus that can be quantified and qualitative, easy to operate, fast in real time, good in specificity, high in sensitivity and good in repeatability. The detection of hemolytic vibrio requires steps such as bacteria enrichment, isolation and culture, biochemical identification, and serum agglutination, which makes the operation cumbersome and takes a long time, and can quickly detect all vibrio paralyticus
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[0041] The present invention will be further described below in conjunction with the examples, but the protection scope of the invention is not limited thereto. 2×PCR reaction solution, containing 2×PCR reaction solution, MgCl, dNTP, Taq enzyme, etc., such as figure 1 As shown, the real-time FQ-PCR detection kit for Vibrio parahaemolyticus is composed of a carton 1 and a liner 2 to form an empty slot, and a large plastic tube 3 with a cover and a small plastic tube 4 with a cover are arranged in the empty slot , a large plastic tube 3 with a cover and a small plastic tube 4 with a cover are composed of the following, that is, the real-time FQ-PCR detection kit of Vibrio parahaemolyticus is composed of the following test tubes and various parts:
[0042] (1), 2×PCR reaction solution: 10 μM upstream primer whose sequence is 5′—CACGCATTGCGCTCTGAGT—3′, 10 μM downstream primer whose sequence is 5′—CGA GAC ATC CCA GAA CAC AAA—3′, and whose sequence is 5′—FAM — 3 μM TaqMan probe of ...
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The invention relates to a real-time FQ-PCR detection kit for vibrio parahaemolyticus and a detection method thereof. The real-time FQ-PCR detection kit for the vibrio parahaemolyticus comprises a PCR reaction liquid tube, a sample treatment liquid tube, a positive contrast tube, a negative contrast tube, a standard tube and a deionized water tube, wherein the concentration of a 2xPCR reaction liquid is diluted twice; the concentration of an upstream primer is 10 M um, and the concentration of a downstream primer is 10 M um; the concentration of a TaqMan probe is 3 M um; and the volume ratio of various reagents of double distilled water is as follows: the ratio of the 2xPCR reaction liquid to the upstream primer to the downstream primer to the TaqMan probe is 100 to 4 to 4 to 68. The detection method is as follows: in order to quickly detect all the vibrio parahaemolyticus, a sample to be detected is collected and treated at first, and then application of a sample, preparation of the PCR reaction liquid, preparation of a vibrio parahaemolyticus die plate, contrast of a standard product and a negative product, preparation of a negative contrast die plate, and judgment of the standard product, the predeformation and the polymerase chain reaction and the result of the sample to be detected are performed. The real-time FQ-PCR detection kit for the vibrio parahaemolyticus and the detection method have the advantages of qualitative and quantitative property, real time, quickness, simplicity and convenience, good specificity and repeatability, suitability for mass control and prevention of FQ-PCR product pollution.
Description
(1) Field of invention [0001] The invention relates to a composition for microbial measurement, in particular to a real-time FQ-PCR detection kit and a detection method for vibrio parahaemolyticus. FQ-PCR is real-time fluorescent quantitative polymerase chain reaction. (2) Background technology [0002] Vibrio parahaemolyticus (vibrio parahaemolyticus, vp) is a halophilic bacterium belonging to the genus Vibrio, which exists in offshore seawater, seabed sediments, fish, shrimp, shellfish and other seawater. Among the products, it is the main pathogenic bacteria of food poisoning and acute diarrhea in coastal areas of mainland my country. Kanagawa phenomenon (Kanagawa phenomenon, kp) positive strains are pathogenic strains, the strains contain two pathogenic factor genes (tdh and trh), PCR primers designed from the tdh and trh gene sequences can make Kanagawa phenomenon negative strains missed. It cannot fully reflect the details of Vibrio parahaemolyticus contamination in fo...
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IPC IPC(8): C12Q1/68C12Q1/04
CPCY02A50/30
Inventor 王志铮王忠发杨阳王虹玲
Owner ZHEJIANG OCEAN UNIV