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Method, special kit and test paper strip for detecting beta-lactam antibiotics based on penicillin-binding protein

A technology for lactams and antibiotics, applied in the field of detecting beta-lactam antibiotics, can solve the problems of high cost, long cycle, complicated process and the like, and achieve the effects of simple storage, low cost and simple operation

Active Publication Date: 2011-11-23
北京维德维康生物技术有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The basis of the immunoassay method is the antibody. The conventional antibody preparation method is mainly to immunize animals to obtain polyclonal serum and use hybridoma technology to obtain monoclonal antibody. The cycle is long, the process is complicated, and the cost is high.

Method used

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  • Method, special kit and test paper strip for detecting beta-lactam antibiotics based on penicillin-binding protein
  • Method, special kit and test paper strip for detecting beta-lactam antibiotics based on penicillin-binding protein
  • Method, special kit and test paper strip for detecting beta-lactam antibiotics based on penicillin-binding protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1, the preparation of recombinant bacteria

[0052] The Streptococcus pneumoniae R6 genome was used as a template, and the following primers were used for PCR amplification to obtain PCR products; the PCR products were digested with restriction endonucleases NdeI and XhoI, and the PCR products were subjected to 1% agarose gel electrophoresis to recover about 2250bp Target gene fragment (i.e. PBP2x target gene); electrophoresis results are as follows figure 1 As shown, lanes 1-6 are the target genes, and lane 7 is the marker.

[0053] Upstream primer: 5'-TT CATATG ATGAAGTGGACAAAAAGA-3' (5' end contains NdeI restriction site and protective base)

[0054] Downstream primer: 5'-TT CTCGAG TTAGTCTCCTAAAGTTAAT-3' (3' end contains XhoI restriction site and protective base)

[0055] Recover the target gene fragment, connect it to the PMD19-T vector, transform the ligated product into Escherichia coli DH5α, screen the positive single clone through resistance, cul...

Embodiment 2

[0059] Embodiment 2, preparation and purification of protein

[0060] 1. Protein preparation

[0061] Inoculate the recombinant strain BL21-pET28b-PBP2x into LB liquid medium containing 30ug / mL kanamycin, shake at 250rpm at 37°C, and culture overnight (12h) to obtain seed liquid; take 1mL seed liquid and inoculate 100mL kanamycin-containing Namycin 30ug / mL LB liquid medium, shake at 250rpm at 37°C, to the A of the culture system 600 When it is 0.6, take out 1mL of the bacterial liquid as the uninduced control, add IPTG to the rest of the bacterial liquid to a final concentration of 1mM, shake the bacteria at 200pm 30°C, and start from the 3rd hour of induction (recorded as the 0th hour when adding IPTG), every Take out 1mL of bacterial liquid every hour, and the induction time is 3h, 4h, 5h, 6h, 7h, 8h and overnight (12h); centrifuge the collected bacterial liquid at 10000rpm at 4°C for 5min to collect the bacterial cells; use Tris-HCL (10mM Tris pH7.0) to resuspend the bact...

Embodiment 3

[0080] Embodiment 3, the kit that detects β-lactam antibiotics

[0081] 1. The kit consists of the following materials:

[0082] 1. ELISA plate coated with penicillin-binding protein: the purified penicillin-binding protein prepared in Example 2 was configured with coating buffer to 1 ug / mL; 100 uL per well.

[0083] 2. Enzyme standard substance working solution: HRP-labeled ampicillin working solution, its working concentration is 1ug / mL, obtained by diluting HRP-labeled ampicillin with sample diluent.

[0084] 3. Standard product of β-lactam antibiotics: the standard product is penicillin G freeze-dried powder, which was purchased from Sigma-Aldrich Company in the United States; the product catalog number is 46616; the penicillin G was dissolved in the sample diluent to obtain the following different concentrations Standard solution: 8.1μg / L, 2.7μg / L, 0.9μg / L, 0.3μg / L, 0.1μg / L.

[0085]4. Substrate chromogenic solution: made up of A liquid and B liquid, A liquid is the aqu...

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Abstract

The invention discloses a method, a special kit and test paper strips for detecting beta-lactam antibiotics based on penicillin-binding protein. The receptor kit used for detecting beta-lactam antibiotics comprises: protein represented by SEQ ID NO: 1, enzyme labeled ampicillin and a standard substance solution, wherein the standard substance is penicillin G. The kit and colloidal gold test paper strips provided by the invention have advantages of high sensitivity, high accuracy, high precision, low cost, simple operation, short detection period, simple storage, and long shelf-life. The kit and colloidal gold test paper strips are suitable for various work units. With the kit and colloidal gold test paper strips, simultaneous and rapid detections of large batches of samples can be realized, and on-site high flux rapid detections can be realized. Therefore, the kit, the colloidal gold test paper strips and the method provided by the invention play an important role in the detections of beta-lactam antibiotics.

Description

technical field [0001] The invention relates to a method for detecting β-lactam antibiotics based on penicillin binding protein, a special kit and a test strip. Background technique [0002] β-lactam antibiotics are a general term for a class of antibiotics containing a quaternary β-lactam ring structure. They are currently the most widely used class of antibiotics in the pharmaceutical and veterinary industries. They are widely used in the treatment and prevention of staphylococcus, pneumococcus, streptococcus, Escherichia coli, Haemophilus, Salmonella caused animal urinary tract, gastrointestinal tract, respiratory tract infection and cow mastitis and other diseases. Long-term intake of food containing β-lactam antibiotic residues can lead to increased bacterial resistance, dysbiosis in the digestive system, and even induce allergic reactions. [0003] As people pay more and more attention to food safety, the European Union, China, the United States, Japan and other count...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/577G01N33/558
Inventor 沈建忠江海洋吴小平徐飞王战辉赵宁杨丽丽李向梅李艳伟
Owner 北京维德维康生物技术有限公司
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