Gene chip for detecting vibrio harveyi colony and using method of gene chip

A technology of Vibrio Harveylius and gene chip, which is applied in the field of gene chip detection of Vibrio Harveylius, can solve the problems of highly similar probe phenotype and genotype, rapid and accurate identification of Vibrio Harveylius, etc. The time of detection, the effect of improving identification ability and improving specificity

Pending Publication Date: 2016-02-03
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In the existing gene chip and its patent technical reports, such as "a gene chip and its monitoring method that can detect multiple Vibrio in parallel" (invention patent, CN103451310A, 2013) and "a method for detecting 9 A gene chip of a pathogenic Vibrio" (invention patent, CN103820558A, 2014), although the detection of Vibrio using gene chip technology has also been reported, but most of the probes designed in this kind of invention patent are aimed at 16SrRNA gene and hsp60 gene , and this type of probe cannot be effectively used for the Harvey Vibrio group whose phenotype and genotype are highly similar, and cannot be quickly and accurately identified at the species level

Method used

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  • Gene chip for detecting vibrio harveyi colony and using method of gene chip
  • Gene chip for detecting vibrio harveyi colony and using method of gene chip
  • Gene chip for detecting vibrio harveyi colony and using method of gene chip

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Experimental program
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specific Embodiment 1

[0036] Probe Preparation

[0037] 1. Target detection strains and target genes

[0038] The present invention selects Vibrio harvei group, which is an important common pathogenic bacteria in seawater cultured animals, as the detection object, including Vibrio alginolyticus, Vibrio kansaii, Vibrio harveii, Vibrio natrium, Vibrio parahaemolyticus, and rotifers Vibrio, all strains are used toxR gene is the target gene.

[0039] 2. Probe design

[0040] The present invention uses 18-30bp oligonucleotide probes. The basic requirement of probe parameters is specific and efficient hybridization, probe T m The value remains similar and fluctuates within 10°C; the number of consecutive complementary bases forming dimers and hairpin structures is less than 4 (values ​​are less than 4.5kcal / mol); the consecutive matching bases between the probe and the non-target gene sequence The number of bases is less than 7 bases; the number of base mismatches between the probe and the target g...

specific Embodiment 2

[0044] Utilize the probe designed in the above specific embodiment one to prepare a gene chip for detecting Vibrio Harvey group, the specific steps are as follows:

[0045] 1. Preparation of probe mother solution

[0046] Prepare the probe freeze-dried powder with RNAase-free water (i.e. DEPC-treated water) to make a 100 μm / L mother solution. After shaking and mixing, centrifuge at 4°C and 5000rpm / min for 1min to obtain the probe mother solution, which is stored at -20°C;

[0047] 2. Preparation before sampling

[0048] Take a 0.2mL microcentrifuge tube, add 10μL of probe mother solution and 10μL of sampling buffer (SSC) in sequence, shake and mix well, then centrifuge at 4°C and 5000rpm / min for 1min, take the supernatant and load it into a 384-well plate ;

[0049] 3. Spotting

[0050] Confirm the matrix of the spot sheet, and control the humidity (75%) and temperature (23°C) of the spotting instrument. Using high-precision manipulator pointing technology, point the probes...

specific Embodiment 3

[0053] The method for using the gene chip prepared in the above-mentioned specific embodiment 2 for detecting Vibrio harveii group, the specific steps are as follows:

[0054] 1. Sample selection:

[0055] Model strain used: Vibrio alginolyticus (ATCC17749 T ), Vibrio campestris (MCCC1H0050 T ), Vibrio Harvey (MCCC1H0031 T ), Sodium Vibrio (MCCC1H0025 T ), Vibrio parahaemolyticus (MCCC1A02609 T ) and Vibrio rotifers (CAIM577 T ); environmental strains: Vibrio alginolyticus (NBV00022); standard strain samples of seven bacteria, the strain DNA was extracted by phenol chloroform isoamyl alcohol extraction.

[0056] 2. Primer design and synthesis

[0057] Using Primer5.0 against 6 kinds of Vibrio toxR Primers were designed for the conserved regions of the gene. According to the requirements of the primer design principles, each primer was analyzed with Oligo7 software to analyze the parameters of the primers, including the secondary structure, to exclude inappropriate pri...

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Abstract

The invention discloses a gene chip for detecting a vibrio harveyi colony and a using method of the gene chip. The gene chip is characterized in that nucleotide probes for detecting vibrio alginolyticus, V. campbellii, V. harveyi, V. natriegens, V. parahaemolyticus and V. rotiferianus are immobilized on a chip carrier; the probes of various vibrios use a toxR gene as a target gene; the gene sequences of the various probes are as shown in SEQ ID NO: 11-14; and in a step of PCR amplification on a sample, the gene sequences of a PCR amplification forward primer designed for the toxR gene of various strains of the vibrio harveyi colony and a reverse primer with a digoxin marker are as shown in SEQ ID NO. 15-26. The gene chip disclosed by the invention has the advantage that the gene chip is capable of rapidly and effectively detecting 6 pathogenic vibrios of the pathogenic vibrio harveyi colony in a parallel mode, and the gene chip is high in accuracy and sensitivity and is strong in specificity.

Description

technical field [0001] The invention relates to a gene chip for detecting marine pathogenic bacteria, in particular to a gene chip for detecting Harvey Vibrio group and its application method. Background technique [0002] The Harvey Vibrio group is the core group of the genus Vibrio, and is the most common pathogenic bacteria causing marine fish, shrimp and shellfish diseases, including Vibrio alginolyticus ( Vibrio alginolyticus ), Vibrio camporii ( V. campbellii ), Vibrio Harvey ( V. Harvey ), Sodium Vibrio ( V. natriegens ), Vibrio parahaemolyticus ( V. parahaemolyticus ) and Vibrio rotiferus ( V. rotiferianus ). Harvey Vibrio can not only frequently cause outbreaks of aquaculture diseases and cause huge economic losses to the aquaculture industry, but also cause human diseases through contact with seawater, eating raw seafood or eating food contaminated by the bacteria. Harvey Vibrio groups have highly similar phenotypes and genotypes, which cannot be quickly...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/63
CPCC12Q1/689C12Q1/6837C12Q2531/113C12Q2537/143C12Q2565/501
Inventor 张德民郑嘉来唐姝闫永伟熊金波张化俊
Owner NINGBO UNIV
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