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PCR (Polymerase Chain Reaction) synchronization detection kit for A and B genes of staphylococcus aureus enterotoxin

A technology for simultaneous detection of staphylococcus gut, applied in the field of microbial detection, can solve the problems of long detection time of the two-way agar diffusion method, unfavorable promotion and use of basic units, long detection time, etc., and achieves low sample detection cost, long storage time, and no Effects of radioactive contamination

Active Publication Date: 2012-07-04
BEIJING SANYUAN FOOD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of ELISA is that it is easily affected by food ingredients and staphylococcal protein A, the detection time is long, and the cost is high, which is not conducive to the promotion and use in grassroots units.
The two-way agar diffusion method has a long detection time and low sensitivity

Method used

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  • PCR (Polymerase Chain Reaction) synchronization detection kit for A and B genes of staphylococcus aureus enterotoxin
  • PCR (Polymerase Chain Reaction) synchronization detection kit for A and B genes of staphylococcus aureus enterotoxin
  • PCR (Polymerase Chain Reaction) synchronization detection kit for A and B genes of staphylococcus aureus enterotoxin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Construction of the PCR kit for detecting Staphylococcus aureus enterotoxin A and B with degenerate primers

[0027] (1) Degenerate primer SEAB upstream and downstream each 15mM, 100μl.

[0028] (2) 10×PCR buffer (containing Mg 2+ 20mM), 1.5ml.

[0029] (3) dNTPs (10 mM), 1.5 ml.

[0030] (4) Sterile ultrapure water, 3.0ml.

[0031] (5) Taq DNA polymerase (5 U / μl), 100 μl.

[0032] Wherein, the upstream primer is SEAB-F: 5'-TGATAAATAYAAAGRKAAAWAMGTAG-3' (SEQ ID No.1)

[0033] The downstream primer is SEAB-R: 5'-GTTACACCACCATACATRCAAG-3' (SEQ ID No.2).

Embodiment 2

[0034] Embodiment 2 kit operation

[0035] Take 0.5 μl of degenerate upstream primer (SEQ ID No.1), 0.5 μl of degenerate downstream primer (SEQ ID No.2), 2 μl of 10×PCR buffer, 0.5 μl of dNTPs, 0.5 μl of Taq DNA polymerase, sterile ultra Make up to 20μl with pure water, put it into a 0.2ml centrifuge tube, add 1μl of the extracted bacterial DNA to the above system, the total volume is 20μl, centrifuge and mix well, and put it on the PCR machine for automatic amplification reaction.

[0036] Methods of extracting bacterial DNA:

[0037] ① Add 1ml of absolute ethanol, 1ml of ammonia water and 1ml of petroleum ether to 5ml of dairy samples artificially contaminated with Staphylococcus aureus enterotoxin A and B, and mix well.

[0038] ② The mixture was centrifuged at 12000xg for 10 minutes. The supernatant was discarded, and the remaining pellet was dissolved with 300 μl of 10 mM / L TE (pH 7.8). Add 5 μl (10 mg / ml) of lysostaphin to the above liquid, and place in a water bath a...

experiment example 1

[0045] Taking Staphylococcus aureus, Escherichia coli, Salmonella, and Staphylococcus epidermidis as control bacteria, the above detection method was used to perform PCR detection on Staphylococcus aureus enterotoxin SEA strain, Staphylococcus aureus enterotoxin SEB strain and control strain at the same time, and then The PCR amplification products were detected by electrophoresis, and the experimental results are shown in figure 1 . It can be seen from the figure that the degenerate primer SEAB only showed positive PCR amplification reaction for Staphylococcus aureus enterotoxin SEA and SEB bacteria, and no specific fragment appeared in the control group, and the degenerate primer showed good specificity.

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Abstract

The invention discloses a PCR (Polymerase Chain Reaction) synchronization detection primer and kit as well as detection method for A and B genes of staphylococcus aureus enterotoxin. A nucleotide sequence of the synchronization detection primer provided by the invention is as shown in SEQ ID No.1 and 2. The invention also provides the PCR detection kit containing the primer. The detection kit provided by the invention can be used for accurately and sensitively detecting the A and B genes of the staphylococcus aureus enterotoxin in food; the lowest detection concentration of DNA (Deoxyribonucleic acid) is 3.58ng; the detection kit has non-cross reaction with other bacteria and has good specificity; simultaneously, the pretreatment process of the sample is simple and consumes little time; and the detection kit can be used for simultaneously detecting a large number of samples and is low in cost.

Description

technical field [0001] The invention belongs to the field of microorganism detection, in particular to a PCR synchronous detection kit for Staphylococcus aureus enterotoxin A and B genes. Background technique [0002] Staphylococcus aureus (Staphylococcus aureus, referred to as Staphylococcus aureus) is the most common pathogenic bacteria in animals and human purulent infections, ubiquitous in nature, can be found in air, water, dust and human and animal excreta, therefore There are many opportunities for food to be contaminated by it. After the food is contaminated by it, not only spoilage, but also some strains produce staphylococcal enterotoxins (Staphylococcal Enterotoxins, SEs). Enterotoxins are a class of extracellular proteins with related structures, similar virulence and different antigenicity. According to its antigenicity, it can be divided into five serotypes: SEA, SEB, SEC, SED and SEE. Most common serotype in food poisoning. All types of enterotoxins can ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12R1/445
Inventor 陈历俊刘继超姜铁民周伟明
Owner BEIJING SANYUAN FOOD
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