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Loop-mediated isothermal amplification detection primer group, detection method and kit of vibrio parahaemolyticus

A hemolytic vibrio and loop-mediated isothermal technology, applied in biochemical equipment and methods, microbial measurement/testing, recombinant DNA technology, etc., can solve the problems of tlh and other genes that are not specific enough, and achieve strong specificity and easy operation Simple process and good accuracy

Inactive Publication Date: 2015-04-15
GUANGDONG INST OF MICROORGANISM +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although there has been a patent report on the establishment of a LAMP method for the tlh gene and some other sequences of Vibrio parahaemolyticus (patent application number: 2007100304416), due to the lack of specificity of tlh and other genes (Croci et al., 2007), the established LAMP The method has some limitations in practical application

Method used

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  • Loop-mediated isothermal amplification detection primer group, detection method and kit of vibrio parahaemolyticus
  • Loop-mediated isothermal amplification detection primer group, detection method and kit of vibrio parahaemolyticus
  • Loop-mediated isothermal amplification detection primer group, detection method and kit of vibrio parahaemolyticus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Detection of Vibrio parahaemolyticus in shrimp

[0029] 1. Sample enrichment

[0030] Homogenize shrimp purchased in the market, weigh 25 g of the homogenized sample, put it into 225 mL of 3% NaCl alkaline peptone water, and incubate at 36°C±1°C for 10 hours to obtain a bacterial culture.

[0031] 2. Bacterial DNA extraction

[0032]Take 1 mL of bacterial culture and centrifuge at 12000r / min for 5min, discard the supernatant, collect the bacteria, add 50uLTE to fully suspend and mix, put in a water bath at 100°C for 10min, ice bath for 3min, centrifuge at 12000r / min for 5min, take the supernatant for later use, and obtain bacterial DNA.

[0033] 3. LAMP amplification

[0034] The loop-mediated isothermal amplification system has a total volume of 25 μL, including 2.5 μL 10× Thermopol reaction buffer, 0.4 μL 10 mmol / L dNTPs, 0.5 μL 10 μmol / L upstream outer primer F3, 0.5 μL 10 μmol / L downstream outer primer B3, 1 μL 40 μmol / L upstream internal primer FIP,...

Embodiment 2

[0043] Example 2: Detection of Vibrio parahaemolyticus in yellow croaker

[0044] The yellow croaker purchased in the market was homogenized, and the homogenized sample was weighed to perform LAMP detection according to the method of Example 1, except that the reaction temperature of LAMP was 65° C. and incubated for 45 minutes, and the others were the same as Example 1.

[0045] The result is: the color of the reaction tube turns orange (the negative control (the genomic DNA of Vibrio vulnificus ATCC27562) is orange, the positive control (the genomic DNA of the positive strain Vibrio parahaemolyticus ATCC33847) is green), indicating that the sample does not have contamination by Vibrio parahaemolyticus . This is consistent with the detection results of traditional biochemical methods, which proves that the data of this method is reliable.

Embodiment 3

[0046] Example 3: Specificity of the LAMP method

[0047] Collect 110 strains of Vibrio parahaemolyticus and non-Vibrio parahaemolyticus, culture the strains in tryptone broth TSB (Vibrio parahaemolyticus in 3% Nacl TSB) for 24 hours at 37°C, take 1mL of the bacterial culture at 12000r Centrifuge at 100°C for 5 minutes, discard the supernatant, collect the bacteria, add 50 μL TE to fully suspend and mix, place in 100°C water bath for 10 minutes, ice bath for 3 minutes, centrifuge at 12000 r / min for 5 minutes, take the supernatant for later use, and thus obtain 110 strains of bacteria DNA.

[0048] LAMP amplification: The loop-mediated isothermal amplification (LAMP) reaction system is 25 μL, including: 2.5 μL 10× Thermopol reaction buffer, 0.4 μL 10 mmol / L dNTPs, 0.5 μL 10 μmol / L upstream external primer F3, 0.5 μL 10 μmol / L Downstream outer primer B3, 1 μL 40 μmol / L upstream inner primer FIP, 1 μL 40 μmol / L downstream inner primer BIP, 0.6 μL 100 mmol / L MgSO 4 , 12.5 μL 2mo...

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Abstract

The invention discloses a loop-mediated isothermal amplification detection primer group, a detection method and a kit of vibrio parahaemolyticus. The detection primer group is as follows: an upstream outer primer F3: 5'-GCAAAGAAACGCTTGGCG-3', a downstream outer primer B3: 5'-TGCATAGCAATGTTGTCGCT-3', an upstream inner primer FIP: 5'-TCTCTCGGGTGGTGGATGGGTTTCGTTACACTCCGTTCGC-3'; a downstream inner primer BIP: 5'-ATGGTTTGCTACTCTCGCACCCTCGGCTGACAAATGGCTCTA-3'. The detection method disclosed by the invention has the advantages of high sensitivity, high specificity, good accuracy rate and short detection time, only 12 hours are taken from sample treatment to result report, no PCR instrument or electrophoresis instrument is needed, the operation process is simple, and compared with other PCR techniques, the loop-mediated isothermal amplification detection primer group is relatively high in specificity and is applicable to detection of primary testing organizations and food companies.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and in particular relates to a loop-mediated isothermal amplification detection primer set, detection method and kit for Vibrio parahaemolyticus. Background technique: [0002] Vibrio parahaemolyticus (Vibrio parahaemolyticus) is recognized worldwide as an important food-borne pathogen, and it is also the number one pathogen of microbial food poisoning in my country in recent years. Food poisoning caused by Vibrio parahaemolyticus can cause acute gastroenteritis, and in a few cases, it can also cause septicemia, which is life-threatening. The high pollution rate of Vibrio parahaemolyticus in aquatic products has led to cases of food poisoning caused by this bacteria occurring from time to time around the world. In the summer of 2006 alone, more than 900 cases of VP-related diarrhea were reported in a certain place in Chile; from August 25 to September 25, 2007, two consecutive cases of VP food pois...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6844C12Q1/689C12Q2531/119Y02A50/30
Inventor 徐晓可吴清平张菊梅程健恒张淑红邓梅清
Owner GUANGDONG INST OF MICROORGANISM
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