Liquid-phase chip kit for screening seven virulence genes of streptococcus suis

A liquid-phase chip, virulence gene technology, applied in microorganism-based methods, microbial determination/inspection, microorganisms, etc., can solve problems such as heavy workload, pollution, and low sensitivity, and achieve improved specificity and sensitivity. The effect of high degree and development of application prospects

Inactive Publication Date: 2016-06-08
SHANGHAI ANIMAL EPIDEMIC PREVENTION & CONTROL CENT
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In view of the above-mentioned technical problems in the prior art, the present invention provides a liquid-phase chip kit for screening 7 kinds of virulence genes of Streptococcus suis, and the liquid-phase chip kit for screening 7 kinds of virulence genes of Streptococcus suis needs to solve In the prior art, the screening method for detecting Streptococcus suis virulence factors has a large workload, low sensitivity, and serious technical problems of pollution

Method used

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  • Liquid-phase chip kit for screening seven virulence genes of streptococcus suis
  • Liquid-phase chip kit for screening seven virulence genes of streptococcus suis
  • Liquid-phase chip kit for screening seven virulence genes of streptococcus suis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1 kit design principle

[0028] The present invention first designed specific primers for seven virulence factors including gdh, orf2, mrp, gapdh, fbps, epf, and sly, designed them using DNAstar, and sent them to Shanghai Sunny for synthesis.

[0029] The specific primer sequence is shown in SEQ ID NO: 1-14, and then sent to Shanghai Sunny Biotechnology Co., Ltd. for synthesis. After establishing and optimizing the single-plex PCR detection system for each of the seven primers (gdh, orf2, mrp, gapdh, fbps, epf, sly), they were combined and divided into two groups according to the principle that the annealing temperature is relatively close to the band size and the distance between them is relatively obvious. Groups are group 1 (fbps, gdh, orf2, mrp), group 2 (sly, gapdh, epf), see Figure 5 with Image 6 .

[0030] On the basis of multiple PCR in the early stage, a TAG sequence is added to the 5' end of the specific upstream primer, and 12 carbon bridges a...

Embodiment 2

[0034] Embodiment 2 kit composition

[0035] Multiplex PCR reagent: PCR system 24.5μL

[0036] Combination 1 (contains multiplex PCRMix12.5μL, fbps, gdh, orf2, mrp primers 1μL each, ddH 2 08 μL), combination 2 (including multiplex PCR Mix 12.5 μL, sly, gapdh, epf primers 1 μL each, ddH2O 9 μL).

[0037] Liquid phase chip reagent: 90 μL of reaction system, including 1 μL of each coded reaction microsphere, 1×Tm to 20 μL, 70 μL of SAPE diluent (69.3 μL of 1×Tm and 0.7 μL of SAPE).

Embodiment 3

[0038] Example 3 Kit usage method

[0039] 1. Multiplex PCR

[0040] The reaction system is 25 μL, 24.5 μL multiplex PCR reagent (combination 1, 2) and 0.5 μL nucleic acid to be tested (10 ng / μL).

[0041] Operation steps: After diluting the nucleic acid of the sample to be tested according to the above requirements, add it to the two combined multiplex PCRs in the kit.

[0042] Reaction conditions: 95°C for 5min; 95°C for 30s; 56°C for 30s; 72°C for 1min; 40 cycles, 72°C for 10min.

[0043]2. Hybridization

[0044] 1. Use 2500 microspheres for each coded microsphere to participate in the reaction (ie 1 μL), and make up to 20 μL with 1×Tm;

[0045] 2. Add 2 μL of the above-mentioned PCR amplified product and adjust to a final volume of 25 μL with ddH2O;

[0046] 3. Dilute SAPE to 8 μg / ml with 1×Tm hybridization buffer, add 70 μL to each reaction well, and mix gently;

[0047] 4. Put it into the PCR instrument and hybridize for 35 minutes at 40°C (see Figure 4 ).

[004...

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Abstract

The invention relates to a liquid-phase chip kit for screening seven virulence genes of streptococcus suis. The liquid-phase chip kit comprises specificity forward primers, specificity reverse primer probes, and specificity microspheres. The sequence of the specificity forward primer is shown as SEQ ID NO: 15-21. The sequence of the specificity reverse primer is shown as SEQ ID NO: 2, 4, 6, 8, 10, 12, and 14, and a part of biotin is added at end 5' of any one of the specificity reverse primer. The sequence of the specificity microspheres is shown as SEQ ID NO: 22-28. The liquid-phase chip kit is capable of subjecting seven virulence genes of streptococcus suis to liquid-phase chip screening in the reaction system, and has the advantages of high speed, high flux, high specificity and accuracy, and the like.

Description

technical field [0001] The invention belongs to the field of biological genes, and relates to a liquid-phase chip kit for screening bacterial virulence genes, in particular to a liquid-phase chip kit for screening 7 kinds of virulence genes of Streptococcus suis. Background technique [0002] Streptococcus suis (SS) disease is a zoonotic disease that seriously harms the pig industry and is distributed worldwide. Among them, types 1, 2, 7, and 9 are the most common, and the main virulence factors are glutamate dehydrogenase (gdh), lysozyme-releasing protein (mrp), extracellular factor (epf), hemolysin (sly), Fibronectin-binding protein / fibronectin (fpbs), virulence-associated sequence (orf2), glyceraldehyde-3-phosphate dehydrogenase. Studies have shown that the pathogenicity of SS is closely related to its virulence factors. Among them, mrp and epf are signs of virulent strains, and sly is a thiol-activated toxoid that plays a role in the process of bacterial invasion and l...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/14C12R1/46
CPCC12Q1/689C12Q1/6834C12Q2600/16C12Q2531/113C12Q2537/143C12Q2563/149C12Q2563/107
Inventor 张维谊刘佩红王建宋涛杨显超王晓旭徐锋沈莉萍齐新永
Owner SHANGHAI ANIMAL EPIDEMIC PREVENTION & CONTROL CENT
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