Beta-lactamase SHV gene variation detecting chip and application thereof

A lactamase and detection chip technology, which is applied in the field of beta-lactamase SHV gene mutation detection chips, can solve the problems of wasting manpower and material resources, unable to judge whether pathogenic bacteria have drug resistance, time-consuming and the like

Inactive Publication Date: 2007-03-14
SHANGHAI BIOCHIP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In my country, most antibiotics are administered in the first way, but with the use of new antibiotics and the emergence of new drug-resistant strains, the feasibility and scientificity of choosing antibiotics based on experience has been decreasing day by day. If the empirical medication is wrong and then choose method three, it will not only delay the patient's timely and reasonable treatment, aggravate the condition, but also waste manpower and material resources; method two can only roughly divide antibiotics into β-lactam and non-β-lactam administration, It is impossible to judge whether the pathogenic bacteria have drug resistance, and it is not suitable for multiple infections; the third method is the best drug delivery method currently available, but because this method is time-consuming (3 days at the fastest) and laborious, the operation procedure is cumbersome. The experimental results are affected by a variety of uncertain factors and there are large errors. Repeated verification is often required, and the mechanism of drug resistance cannot be clarified. In addition, the drug sensitivity test is limited by the experimental antibiotics, so it

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  • Beta-lactamase SHV gene variation detecting chip and application thereof
  • Beta-lactamase SHV gene variation detecting chip and application thereof
  • Beta-lactamase SHV gene variation detecting chip and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 Reverse hybridization

[0065] 1. Preparation of gene chip

[0066] (1) Probe dissolution

[0067] Each probe was diluted with TE solution to a final concentration of 10 mM. Mix the probe with a concentration of 10mM and the PBS solution with a concentration of 200mM in a medium volume of a 384-well plate, seal the 384-well plate with an adhesive sheet, shake at room temperature for 2 minutes, centrifuge, and store at -20°C for sample application .

[0068] (2) Spotting

[0069] The pre-designed and synthesized probes are loaded onto the solid-phase carrier substrates made of glass slides, silicon wafers, etc. through contact spotting or inkjet spotting. The film base adopts Cell Associates CSS-100 aldehyde base film base, and the Ominigrid 100 model spotting instrument of GeneMachine Company is applied at a humidity of 65-75% (based on the FullMoon film base) and a temperature of 25°C. The format of spotting is 1×3, and each matrix is ​​8×18. After the s...

Embodiment 2

[0078] Example 2 forward hybridization

[0079] 1. Full-length SHV gene amplification and chip preparation

[0080] Primers SHV-F (5'-CTCAAGGATGTATTGTGGTTATGCGT-3', SEQ ID NO: 181) and SHV-R (5'-GGGTTAGCGTTGCCAGTGCTCGAT-3', SEQ ID NO: 182) were used to amplify the full-length SHV gene. PCR amplification was carried out with 100μl reaction system, the reaction system was 0.3mM dNTP, 10mM Tris-HCl, 50mM KCl, 2mM MgCl 2 , 20% Q solution (Qiagen), 0.01 μM SHV-F, 0.2 μM SHV-R, 100 ng genomic DNA, 3U Ex Taq enzyme (Takara). Cycle parameters: denaturation at 94°C for 5 min; denaturation at 94°C for 40 s, annealing at 65°C for 1 min, extension at 72°C for 1 min, a total of 40 cycles; finally extension at 72°C for 5 min. PCR products were purified with QIAquick PCR Purification Kit (Qiagen, Cat. No. 28106). The concentration of the purified PCR product was adjusted to 400 ng / μl mM.

[0081] Mix the probe with a concentration of 400ng / μl and 100% DMSO in a medium volume of a 384-wel...

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Abstract

The present invention discloses one kind of beta-lactamase SHV genovariation detecting chip. The beta-lactamase SHV genovariation detecting chip includes a solid phase carrier and a probe, which is made to hybridize with the nucleotide sequence of the beta-lactamase SHV gene to be detected and/or its complementary sequence. The present invention also discloses the method of using the chip in detecting beta-lactamase SHV genovariation. The chip of the present invention may be used in simultaneous detection of several kinds of beta-lactamase SHV genovariation and in detection of bacterial drug resistance and resisted drug kinds for directing reasonable drug use and avoiding abuse of antibiotics.

Description

technical field [0001] The invention relates to a bacterial drug resistance gene detection chip, in particular to a β-lactamase SHV gene variation detection chip and its application. Background technique [0002] Since the invention of penicillin in 1929, antibiotics have played a huge role in the prevention and treatment of bacterial diseases, greatly improving people's quality of life and prolonging people's life expectancy. However, due to the long-term and extensive use of antibiotics, especially the irregular abuse of antibiotics, the problem of bacterial resistance has become increasingly serious. The most important mechanism of bacterial drug resistance is the production of inactivating enzymes, such as β-lactamases and aminoglycoside inactivating enzymes. β-lactamase can covalently bind to the carboxyl group of the β-lactam ring, hydrolyze its amide bond, and inactivate penicillins and / or cephalosporins. β-lactamase is not only associated with high morbidity and mo...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 盛海辉李红梅肖华胜
Owner SHANGHAI BIOCHIP
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