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Dual-lamp method for simultaneous detection of Vibrio parahaemolyticus and Vibrio vulnificus

A technology for Vibrio vulnificus, Vibrio hemolyticus, applied in the directions of microorganism-based methods, biochemical equipment and methods, microbial determination/inspection, etc.

Active Publication Date: 2018-10-30
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there have been reports of LAMP detection methods for Vibrio vulnificus and Vibrio parahaemolyticus, but these detection methods are all for the detection of single pathogenic bacteria

Method used

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  • Dual-lamp method for simultaneous detection of Vibrio parahaemolyticus and Vibrio vulnificus
  • Dual-lamp method for simultaneous detection of Vibrio parahaemolyticus and Vibrio vulnificus
  • Dual-lamp method for simultaneous detection of Vibrio parahaemolyticus and Vibrio vulnificus

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Experimental program
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Effect test

Embodiment 1

[0025] 1. Establishment of dual LAMP method for Vibrio vulnificus and Vibrio parahaemolyticus

[0026] 1.1 Materials

[0027] dNTPs, BstDNA polymerase (with 10× buffer), PCR grade betaine,

[0028] 1.2 Method

[0029] 1.2.1 Primer design and synthesis

[0030] Design LAMP primers: According to the known V. vulnificus metalloprotease gene (GenBank: U50548.1) and V. parahaemolyticus ompA gene (GenBank: JTGT01000603.1) in GeneBank as target sequences, four specific primers were designed by Primer Explore V4 online design software Sexual primers, for V.vulnificus, add BamHI restriction site between B1c and B2 of BIP, add -TTTT-linker between F1c and F2 of FIP; for V.parahaemolyticus, between B1c and B2 of BIP Add a PstI restriction site, add a -TTTT- linker between F1c and F2 of FIP, and design the following two sets of LAMP primers:

[0031]

[0032]

[0033] 1.2.2 Optimization of LAMP amplification conditions

[0034] Optimizing reaction temperature and reaction time:...

Embodiment 2

[0039] Restriction enzyme digestion analysis

[0040] In order to construct a double LAMP detection method, a BamHI restriction enzyme site and a PstI restriction enzyme were added between the B1 complementary strand (B1c) and B2 of the inner primer BIP of V.vulnificus and V.parahaemolyticus, respectively. site (the two restriction enzyme sites are not contained in the target sequences of the two Vibrio species), and the correctness and specificity of the reaction were determined by restriction analysis. In the double LAMP reaction system, only one template is added, and one kind of restriction enzyme digestion is performed on the amplified products respectively. The result shows: the amplified product of Vibrio vulnificus can be digested by BamHI restriction endonuclease, and the specific band obtained by enzyme digestion is shown in ( image 3 -A 1 swimming lane); Vibrio parahaemolyticus can be digested by PstI restriction endonuclease, the specific band obtained by enzyme ...

Embodiment 3

[0042]Sensitivity determination of dual LAMP detection method for Vibrio vulnificus and Vibrio parahaemolyticus

[0043] 1 Boiling method to extract DNA.

[0044] 2 In order to test the sensitivity of the LAMP method, the extracted genomic DNA of Vibrio vulnificus and Vibrio parahaemolyticus was serially diluted 10 times, a total of 8 gradients, and 1 μL was used as a template for each concentration level, and LAMP amplification after optimized conditions was performed. The products were analyzed by 2% agarose gel electrophoresis.

[0045] 3 Take 1 μl as PCR template, F3 / B3 each 1μl (20mM), Taq enzyme 0.5μl, dNTPs (2.5mM each) 1μl, Taq 10×buffer 2.5μl, ddH2O 18μl, carry out PCR amplification, 94℃ pre-denaturation for 5min; Denaturation at 94°C for 30s, annealing at 57°C for 40s, extension at 72°C for 40s, 25 cycles; extension at 72°C for 5min. The amplified products were analyzed by 2% agarose gel electrophoresis.

[0046] The PCR method was used to detect the sensitivity o...

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Abstract

The invention discloses a double-LAMP (loop-mediated isothermal amplification) detecting method, and belongs to the technical field of molecular biology. By the aid of the method, vibrio parahaemolyticus and vibrio vulnificus can be simultaneously detected. An LAMP detection system comprises primer groups for detecting vibrio parahaemolyticus genes OmpA and vibrio vulnificus metalloprotease genes, and each primer group contains a pair of outer primers F3 and B3 for the OmpA genes and the metalloprotease genes and a pair of inner primers FIP and BIP; PstI and BamHI restriction enzyme cutting sites are respectively arranged on the inner primers BIP. The double-LAMP method has the advantages of high sensitivity and detection speeds, good specificity, simplicity in operation and visual and clear result observation.

Description

technical field [0001] The invention relates to a detection method of pathogenic vibrio, in particular to a double LAMP method capable of simultaneously detecting vibrio parahaemolyticus and vibrio vulnificus. Background technique [0002] Vibrio vulnificus (Vibrio vulnificus) is a Gram-negative halophilic bacterium that is mainly distributed in the offshore marine environment and is the most common, widespread, and most harmful pathogen in aquaculture shrimp, crabs, and oysters. One of the germs: Vibrio parahemolyticus (Vibrio Parahemolyticus), a Gram-negative bacillus, in various shapes such as arc, rod, and filament, widely exists in seawater and seafood, and is a common food in coastal areas of my country Toxic pathogenic bacteria. [0003] Loop-mediated isothermal amplification technology (loop-mediated isothermal amplification, LAMP) is a new pathogenic nucleic acid detection technology invented by Notomi et al. in 2000. This technology designs four specific primers acc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12R1/63
CPCC12Q1/6844C12Q1/689C12Q2600/16C12Q2531/119C12Q2537/143C12Q2563/107
Inventor 周顺高志鑫张敏
Owner QINGDAO AGRI UNIV
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