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Detecting kit for five kinds of pathogenic vibrio in aquatic product and detecting method thereof

A pathogenic Vibrio and detection kit technology, which is applied in the detection of pathogenic Vibrio in aquatic products by using mPCR-DHPLC technology, the composition used for detection, and the field of kits, which can solve economic losses and affect the analysis of results , false positives and other problems, to achieve the effect of short inspection time, simple method, labor and financial saving

Inactive Publication Date: 2010-01-27
曹际娟 +4
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Among the existing detection methods, conventional microbial detection methods are still used for the detection of pathogenic Vibrio in aquatic products. These methods are time-consuming and laborious, and cannot meet the requirements of rapid and high-throughput detection. Analysis of the results may result in greater economic losses
Moreover, existing microbiological testing methods cannot provide accurate typing information

Method used

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  • Detecting kit for five kinds of pathogenic vibrio in aquatic product and detecting method thereof
  • Detecting kit for five kinds of pathogenic vibrio in aquatic product and detecting method thereof
  • Detecting kit for five kinds of pathogenic vibrio in aquatic product and detecting method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1, the establishment of 5 kinds of pathogenic Vibrio detection kits and detection methods thereof in aquatic products

[0048] The target genes and their primers determined in this example for detection are shown in the following table:

[0049]

[0050]

[0051] On this basis, a detection kit for 5 pathogenic Vibrio species in aquatic products for PCR amplification was designed: 1 mL of detection solution contained 10 mM Tris Cl, 50 mM KCl, 25 mM MgCl, dNTPs (dATP, dGTP, dCTP and dTTP). ) 2.5mM each, Taq DNA polymerase 5U / μL and 10μM each of the 5 pathogenic Vibrio primer pairs in the above table.

[0052] The mPCR-DHPLC method for detecting 5 pathogenic Vibrio species in samples using this kit includes the following steps:

[0053] ① Preparation of DNA solution of the sample to be tested:

[0054] a. Aquatic product samples: Aseptically weigh 25 grams of samples and culture them in 225 mL of 3% sodium chloride nutrient broth for 18 to 24 hours. Use ...

Embodiment 2

[0071] Example 2. Specificity test

[0072] Take the test strains listed in Table 1, extract genomic DNA after culture, and establish template library. Then, using these genomic DNAs as templates, the collagenase gene (vcc) of Vibrio cholerae, the hemolysin gene (vmh) of Vibrio mimicus, the thermolabile hemolytic toxin gene (tlh) of Vibrio parahaemolyticus, the Vibrio alginolyticus The collagenase gene (vac) of Vibrio vulnificus and the cytolysin gene (vvh) of Vibrio vulnificus were detected by mPCR-DHPLC using the kit and detection method established in Example 1. The DHPLC-specific detection patterns of 5 types of pathogenic Vibrio are attached Figures 1 to 6 shown.

[0073] The results showed that in the mPCR reaction system,

[0074] a. The vmh target gene fragments were amplified in all Vibrio mimicus, while all other non-mimicking Vibrio strains were negative (attached). figure 1 );

[0075] b. Vibrio vulnificus all amplified the vvh target gene fragments, while n...

Embodiment 3

[0080] Embodiment 3, detect the mPCR-DHPLC detection test of 5 kinds of pathogenic Vibrio simultaneously

[0081] Take the standard strains of Vibrio mimicus ATCC33653, Vibrio vulnificus ATCC27562, Vibrio parahaemolyticus ATCC17803, Vibrio alginolyticus ATCC17749, and Vibrio cholerae ATCC14035 for 18 hours at 36 °C, measure their OD value, estimate the number of bacteria, and then press 10 -1 , 10 -2 , 10 -3 Equal multiples of gradient dilution, take several suitable gradients of bacterial liquid for plate count, repeat twice, take the average value to determine the bacterial concentration.

[0082] Take several bacterial liquids with suitable gradient, prepare genomic DNA by kit extraction method, prepare DNA solution of the sample to be tested, and carry out mPCR-DHPLC analysis and detection under the above conditions. The DHPLC spectrum of the measurement results is attached Image 6 shown.

[0083] It can be seen from the results that the mPCR-DHPLC behaviors of the 5 t...

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Abstract

The invention discloses a detecting kit for five kinds of pathogenic vibrio in aquatic product and a detecting method thereof, which is used for food microbial contamination detection and monitoring. In the kit, 1 mL of detecting solution contains 10 mM of Tris.Cl, 50mM of KCl, 25mM of MgCl2, 2.5mM of dNTP, 5U / muL of Taq DNA polyase and five kinds of 10 mu M pathogenic vibrio primers. The kit and the detecting method of the invention can detect 5 kinds of pathogenic vibrio in aquatic products once; the detection lower limit is lower than 100cfu / mL, so that the method of the invention can detect when the number of each pathogenic vibrio is only 100 in each mL of bacteria solution. In addition, the method has shorter detection time than the traditional method, is more convenient, can save great labor and money and is suitable for quick detecting requirements.

Description

technical field [0001] The invention relates to a method for detecting pathogenic microorganisms in food, in particular to a method for detecting pathogenic Vibrio bacteria in aquatic products by using mPCR-DHPLC technology. It also relates to compositions used in the assay, ie kits. Background technique [0002] With the improvement of living standards and the gradual opening up after my country's entry into the WTO, people's demand for aquatic products is increasing, and the import and export trade of aquatic products is also expanding. However, the monitoring and detection of pathogenic microorganisms, especially pathogenic Vibrio, in aquatic products has always been one of the important issues restricting the trade of aquatic products in my country. In 1998 and 2000, my country's cod fillets exported to France and aquatic products exported to Sweden were detected with Vibrio vulnificus and Vibrio mimicus, and their code names were cancelled. Since 2001, all aquatic pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N30/02C12R1/63
CPCY02A50/30
Inventor 邵碧英郑晶黄晓蓉
Owner 曹际娟
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