PCR (polymerase chain reaction) synchronous detection kit for staphylococcus aureus enterotoxin A and B genes

A staphylococcal intestinal and synchronous detection technology, applied in the field of microbial detection, can solve the problems of long detection time of two-way agar diffusion method, unfavorable promotion and use of grassroots units, long detection time, etc., to achieve low cost of sample detection, long storage time, no Effects of radioactive contamination

Inactive Publication Date: 2011-09-14
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) synchronous detection kit for staphylococcus aureus enterotoxin A and B genes
  • PCR (polymerase chain reaction) synchronous detection kit for staphylococcus aureus enterotoxin A and B genes
  • PCR (polymerase chain reaction) synchronous detection kit for staphylococcus aureus enterotoxin A and B genes

Examples

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Embodiment 1

[0024] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Example 1 Construction of the PCR kit for detecting Staphylococcus aureus enterotoxin A and B with degenerate primers

[0025] (1) Degenerate primer SEAB upstream and downstream each 15mM, 100μl.

[0026] (2) 10×PCR buffer (containing Mg 2+ 20mM), 1.5ml.

[0027] (3) dNTPs (10 mM), 1.5 ml.

[0028] (4) Sterile ultrapure water, 3.0ml.

[0029] (5) Taq DNA polymerase (5 U / μl), 100 μl.

Embodiment 2

[0030] Embodiment 2 kit operation

[0031] Take 0.5 μl of degenerate upstream primers, 0.5 μl of degenerate downstream primers, 2 μl of 10×PCR buffer, 0.5 μl of dNTPs, 0.5 μl of Taq DNA polymerase, make up to 20 μl with sterile ultrapure water, and put them into a 0.2ml centrifuge tube , Add 1 μl of the extracted bacterial DNA into the above system, the total volume is 20 μl, centrifuge and mix well, and then put it on a PCR machine for automatic amplification reaction.

[0032] Methods of extracting bacterial DNA:

[0033] 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.

[0034] 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 at 37° C. for...

experiment example 1

[0041] 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) synchronous detection primer for staphylococcus aureus enterotoxin A and B genes, a detection kit thereof and a detection method. The nucleotide sequence of the synchronous detection primer is as shown in SEQ (sequence) ID (identity) No. 1 and 2. The invention further provides the PCR detection kit containing the primer. By adopting the detection kit, the staphylococcus aureus enterotoxin A and B in a food can be accurately and sensitively detected, and the lowest detection concentration of DNA (deoxyribonucleic acid) is 3.58ng; furthermore, the detection kit has no cross reaction with other bacteria, and the specificity is good; simultaneously, the pretreatment process of samples is simple, the consumed time is short, a large number of the samples can be detected simultaneously, and the cost is low.

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): C12Q1/68C12Q1/14C12N15/11
Inventor 陈历俊刘继超姜铁民周伟明
Owner BEIJING SANYUAN FOOD
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