DHFR-based multi-residue fluorescence polarization immunoassay method for sulfonamide synergist drugs

A technology of sulfonamide synergist and fluorescence polarization, which is applied in the direction of material analysis, material analysis, fluorescence/phosphorescence, etc. through optical means, which can solve the problems of time-consuming and complicated operation, achieve the effect of no cross-reaction and expand the scope of application

Inactive Publication Date: 2021-04-13
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The present invention aims at the shortcomings of the above-mentioned existing methods for detecting sulfa synergist drugs such as complicated o

Method used

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  • DHFR-based multi-residue fluorescence polarization immunoassay method for sulfonamide synergist drugs
  • DHFR-based multi-residue fluorescence polarization immunoassay method for sulfonamide synergist drugs
  • DHFR-based multi-residue fluorescence polarization immunoassay method for sulfonamide synergist drugs

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Preparation of dihydrofolate reductase DHFR derived from sulfa synergist drug-sensitive strains

[0039] The method of the invention is applicable to sulfa synergist drug-sensitive bacterial strains including Staphylococcus aureus, Streptococcus pneumoniae, Mycobacterium tuberculosis and the like. Hereinafter, the method of the present invention will be described in detail by taking Staphylococcus aureus ATCC 29213 as an example. This strain was obtained commercially.

[0040] 1. Primer Design

[0041] According to the dhfr gene sequence of Staphylococcus aureus ATCC 29213 on GeenBank in NCBI, primers were designed according to the principle of homology arms, in which the upstream primer was inserted into the NheI restriction site, and the downstream primer was inserted into the XhoI restriction site.

[0042] Table 1.1 Sequence of primers for amplification of Staphylococcus aureus ATCC 29213dhfr gene

[0043]

[0044] The primer used in the present inve...

Embodiment 2

[0075] Example 2. Establishment of multi-residue fluorescence polarization detection method based on receptor DHFR sulfonamide synergist drugs

[0076] 1. Main reagents

[0077] Sulfa synergist drugs trimethoprim (TMP), fluorescein isothiocyanate BDF, N-hydroxysuccinimide (NHS), ethyldimethylaminocarbodiimide hydrochloride (EDC·HCl) , N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 2-(N-morpholine)ethanesulfonic acid MES, Tris, etc. are all commercially available.

[0078] Dihydrofolate reductase DHFR was obtained by the method in Example 1. The method of the present invention is applicable to the recombinant dihydrofolate reductase of sulfonamide synergist drug-sensitive strains including Staphylococcus aureus, Streptococcus pneumoniae, Mycobacterium tuberculosis, etc. Staphylococcus-DHFR is taken as an example to illustrate the method of the present invention in detail.

[0079] TMP hapten (TMP-CS) comes from China Agricultural University. The TMP hapten (represen...

Embodiment 3

[0143] Example 3. Application of the multi-residual fluorescence polarization detection method based on DHFR sulfa synergist drugs in milk

[0144] 1. Milk sample pretreatment

[0145] After research, the inventors found that after extraction with 55% saturated ammonium sulfate, the matrix effect can be eliminated by diluting 5 times.

[0146] Take 4mL of pure milk and skim milk and 4mL of 55% saturated ammonium sulfate solution in a 10mL centrifuge tube, vortex for 2min, sonicate for 5min, centrifuge at 4°C and 8000rpm for 10min, extract the supernatant, take 2mL and mix with 8mL reaction buffer, Check the FP value.

[0147] 2. FPIA detection of sulfa synergist drugs based on DHFR

[0148] Five ASGs S The spiking curves in whole milk and skim milk are as follows Figure 8 As shown, the specific test results are shown in Table 3.1 and Table 3.2. Five ASGs in Pure Milk S The limit of detection was 10.1-40.7 μg / L, and the detection range was 22.7-832.6 μg / L. Five ASGs in ...

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Abstract

The invention provides a DHFR-based multi-residue fluorescence polarization immunoassay method for sulfonamide synergist drugs, which is characterized in that dihydrofolate reductase DHFR is derived from sulfanilamide synergist drug sensitive strains, including staphylococcus aureus, streptococcus pneumoniae and mycobacterium tuberculosis. According to the invention, a sulfanilamide synergist drug multi-residue fluorescence polarization immunoassay method based on the sulfanilamide synergist drug receptor protein dihydrofolate reductase is established for the first time, the established method is applied to multi-residue detection of sulfanilamide synergist drugs in milk, and a method technical support is provided for screening of sulfanilamide synergist drugs in food. According to the invention, DHFR from sulfanilamide synergist drug sensitive strains is creatively adopted, and the method is initiated in the field. The method breaks through the limitation of the conventional method, expands the application range, and has the advantages of rapidness, simplicity, accuracy and the like.

Description

technical field [0001] The invention belongs to the technical field of veterinary drug residue detection, and in particular relates to a DHFR-based multi-residue fluorescence polarization immunoassay method for sulfa synergist drugs. Background technique [0002] Antibacterial synergists (ASGs) are 2,4-diaminopyrimidine compounds, which are dihydrofolate reductase (DHFR) inhibitors that block bacterial folic acid metabolism. The common structure of sulfonamide synergist drugs is diaminopyrimidine, among which, common drugs mainly include trimethoprim (Trimethoprim, TMP), dimethoprim (Diaveridine, DVD), ometoprim (Ormetoprim, OMP), Brodimoprim (BDP), Baquiloprim (BQP), etc. There are many kinds of sulfa synergist drugs, and they can be used in combination with other antibiotics to expand the antibacterial spectrum. If it is used in combination with sulfonamides, because sulfonamides can inhibit dihydrofolate synthase, thereby double blocking bacterial folic acid metabolism,...

Claims

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Application Information

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IPC IPC(8): G01N33/533G01N33/573G01N21/64
CPCG01N33/533G01N33/573G01N21/6486
Inventor 梁晓于万鹏
Owner QINGDAO AGRI UNIV
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