Method for preparing detection kit based on penicillin-binding protein PBP6 beta-lactam antibiotic receptor assay and detection method thereof

A detection kit and protein-binding technology, applied in the biological field, can solve the problems of β-lactam drug leakage and other problems, and achieve the effects of high sensitivity, overcoming leakage, and easy operation

Active Publication Date: 2014-12-17
北京纳百生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to provide a kind of preparation method of the rapid detection kit of β-lactam antibiotic receptor method based on penicillin-binding protein PBP6, and its use detection method, to solve the problem of traditional ELISA method when detecting β-lactam drugs. Leakage problem

Method used

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  • Method for preparing detection kit based on penicillin-binding protein PBP6 beta-lactam antibiotic receptor assay and detection method thereof
  • Method for preparing detection kit based on penicillin-binding protein PBP6 beta-lactam antibiotic receptor assay and detection method thereof
  • Method for preparing detection kit based on penicillin-binding protein PBP6 beta-lactam antibiotic receptor assay and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1. Preparation method of the β-lactam antibiotic receptor method detection kit for penicillin-binding protein PBP6

[0052] 1) Construction of recombinant expression vector of penicillin-binding protein 6 and protein expression The sequence of penicillin-binding protein 6 was derived from Genebank (No. P08506.2) or other homologous sequences. Primers, introducing enzyme cutting sites NdeI and XhoI in the primers, the primer sequences are as follows:, upstream primer P1: 5'GGAATTC CATATG GCGGAACAAACCGTT 3'

[0053] Downstream primer P2: 5'CCG CTCGAG TTAAGAGAACCAGCTGCCGA 3’

[0054] PCR amplification was performed using the synthesized gene as a template. The amplification procedure was as follows: pre-denaturation at 94°C for 2 minutes, denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 1 minute and 30 seconds, 30 cycles, and extension at 72°C for 5 minutes. The amplified product was electrophoresed on a 1% agarose g...

Embodiment 2

[0081] Example 2. Indirect competitive receptor method detection method for the content of β-lactam drugs in milk

[0082] Taking the milk sample as an example, there is no need for any pretreatment during the detection, and it can be directly spotted on the microtiter plate. Specific steps are as follows:

[0083] (1) Add standard / sample: Use a pipette to draw 50 μl of sample or standard into the microtiter plate in the air, shake gently and place it on a horizontal table.

[0084] (2) Add penicillin-binding protein 6: continue to add 50 μl of penicillin-binding protein 6 solution, shake the microtiter plate gently, and mix the solutions therein evenly;

[0085] (3) Adding enzyme-labeled monoclonal antibody: After adding the enzyme-labeled substance into the wells of the microplate at 50 μl per well, gently shake the microplate to mix the solutions evenly;

[0086] (4) Incubation and plate washing: Cover the microplate with a cover film and place it in a 37°C incubator for ...

Embodiment 3

[0090] Embodiment 3, the accuracy and precision test of kit

[0091] Take 20 blank milk samples, add penicillin G standard to it to make the concentration reach 4 μg / L, use the prepared β-lactam receptor method kit to operate according to the operating instructions, and detect the penicillin G content therein. The sample recovery rate was calculated as the measurement result of accuracy, and the coefficient of variation of the measurement result was used as the precision of the kit. The specific measurement results are shown in the table below.

[0092] Table 1 Measurement results of accuracy and precision

[0093] Sample serial number

[0094] It can be seen from the table that when the kit detects milk samples with the addition of 4 μg / L penicillin G, the recovery rate is 67.5%-107.5%, the average recovery rate is 90.3%, and the coefficient of variation is 13.9%.

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Abstract

The invention aims to provide a method for preparing a rapid detection kit based on penicillin-binding protein PBP6 beta-lactam antibiotic receptor assay as well as a used detection method, aiming at solving the problem of sieve leakage in the process of detecting beta-lactam medicines in a traditional ELISA method. The invention discloses a method for preparing a detection kit based on penicillin-binding protein PBP6 beta-lactam antibiotic receptor assay as well as an indirect competitive receptor assay method for detecting the content of beta-lactam drugs in milk by adopting the detection kit based on penicillin-binding protein PBP6 beta-lactam antibiotic receptor assay. The methods have the beneficial effects that by utilizing the characteristic that the penicillin-binding protein 6 can be specifically bound to the beta-lactam drugs, the drugs can be specifically identified in a buffer liquid system or a sample matrix extracting solution and are not influenced by other antibiotics such as macrolides, quinolones and tetracyclines.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to the field of detection of veterinary drug residues using biotechnology, and relates to a rapid detection kit for beta-lactam antibiotic receptor method based on penicillin-binding protein PBP6. Background technique [0002] β-lactam antibiotics (β-Lactam antibiotics) is a large class of antibiotics containing β-lactam ring structure. According to the differences in the structure of the β-lactam ring, this class of antibiotics mainly includes penicillins, cephalosporins, cephamycins, carbapenems and monocyclic β-lactams, among which the development of penicillins and cephalosporins The fastest and most widely used. [0003] β-lactam drugs mainly inhibit the activity of cell wall mucopeptide synthase, that is, penicillin-binding protein, prevent the synthesis of cell wall peptidoglycan, and rupture the bacterial cells to produce antibacterial effects. Since mammalian cells have no ce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/531
CPCG01N33/566
Inventor 杨春江刘勇朱文壮孟赓秦堃莫勋马孝斌聂艳霞邰宗洋
Owner 北京纳百生物科技有限公司
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