CN102747139B - Detection kit for Bacillus cereus, Enterobacter sakazakii and staphylococcus aureus, and detection method thereof - Google Patents

Abstract

The invention is applicable to the technical field of microorganisms, and provides a detection kit and a detection method for Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus. The kit includes DNA extraction solution and PCR reaction solution. The detection kit and detection method of Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus of the present invention can detect Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus at the same time, and compared with the traditional culture method, it has rapid and sensitive , safety and other advantages, can be applied to the detection of Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus.

Description

Detection kits and detection methods for Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus

technical field

The invention belongs to the technical field of microorganisms, and in particular relates to a detection kit and a detection method for Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus.

Background technique

Bacillus cereus is a rod-shaped, spore-producing Gram-positive bacterium. Because Bacillus cereus is widely distributed in nature, it often exists in soil, fly ash, rotten grass and air, and is very easy to be used in food processing and transportation. , storage, sales process, through flies, cockroaches and other insects and unhygienic utensils and hand pollution. It can lead to abscess, meningitis, osteomyelitis, endocarditis, etc. in clinical reports, but most commonly it causes different types of food poisoning: diarrhea type and vomiting type. Enterobacter sakazakii (also known as Enterobacter sakazakii) is a type of Enterobacteriaceae, which was renamed Enterobacter sakazakii from Enterobacter cloacae in 1980. Enterobacter sakazakii can cause severe neonatal meningitis, enterocolitis and bacteremia, and the mortality rate is as high as 50%. Staphylococcus aureus is an important pathogenic bacterium of humans, which belongs to the genus Staphylococcus, has another name of "fleshophilus", and is a representative of Gram-positive bacteria. The pathogenicity of Staphylococcus aureus mainly depends on the invasive enzymes and toxins it produces, which can cause local suppurative infection, pneumonia, pseudomembranous enteritis, pericarditis, etc., and even systemic infection such as sepsis and sepsis , The symptoms of poisoning are severe, mainly manifested as vomiting, fever, and diarrhea.

The internationally accepted Staphylococcus aureus is cultured and detected as follows:

(1) Sample treatment: Aseptically take 25g or 25mL food samples, put them into 225mL sterilized normal saline for homogenization, and make a 10-1 dilution.

(2) Bacterial enrichment culture: Add the 10-1 dilution into 7.5% NaCl broth or tryptone broth, and incubate at 37°C for 24 hours.

(3) Separation and culture: Streak the blood plate and Baird-Parker plate with the above dilution or culture solution, respectively, and culture at 37°C for 24-48 hours. Staphylococcus aureus is a golden or white colony on the blood plate, which is large and raised, with a smooth surface and a hemolytic ring around it. On the Baird-Parker plate, the colony is round, 2-3mm in diameter, gray or black in color, surrounded by a turbid zone.

(4) Staining observation: Pick suspicious colonies from the plate for Gram staining. Staphylococcus aureus is Gram-positive, arranged in a grape shape under the microscope without spores and capsules, with a diameter of 0.5-1 μm.

(5) Plasma coagulase test: Take 0.5mL of rabbit plasma and 0.5mL of Staphylococcus aureus meat infusion broth for 24 hours and mix well, culture at 36±1°C, observe once every half hour, and observe continuously for 6 Hours, coagulation occurs, that is, when the small test tube is tilted or inverted, the content does not flow, and it is judged positive. At the same time do negative and positive controls.

(6) Thermostable nuclease test

Treat the 24-hour broth culture in a boiling water bath for 15 minutes, inoculate it on a toluidine blue-DNA plate with an inoculation loop, and incubate at 36±1°C for 24 hours. If a light pink color appears around the inoculation line, it is positive. Staphylococcus aureus was positive in this test.

Enterobacter sakazakii was detected by culture method as follows:

(1) Pre-enrichment and enrichment

Take 100g (mL) of the test sample and add it to a Erlenmeyer flask filled with 900mL buffered peptone water preheated to 44°C, shake slowly by hand until fully dissolved, and incubate at 36°C±1°C for 18h±2h. Transfer 1mL to 10mL ST-Vm broth and incubate at 44°C±0.5°C for 24h±2h.

(2) Separation

Gently mix the mLST-Vm broth culture, take 1 ring of the enrichment culture, streak inoculate on two Enterobacter sakazakii chromogenic medium plates, and incubate at 36°C±1°C for 24h±2h. Pick 1 to 5 suspicious colonies and inoculate them on TSA plates. Incubate at 25°C±1°C for 48h±4h.

(3) Identification

Pick yellow suspicious colonies directly from the TSA plate for biochemical identification. You can choose biochemical identification kit or automatic microbial biochemical identification system.

(4) Results and reports

Based on the colony morphology and biochemical characteristics, it is reported that Enterobacter sakazakii was detected or not detected in every 100g (mL) sample.

According to the above general method, the simultaneous detection of the three bacteria Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus in the sample has a long period (at least 48 hours), cumbersome operation, low sensitivity, easy contamination, complicated procedures and required The shortcomings of various reagents (biochemical test reagents and serum) are far from being able to meet the requirements of rapid detection of Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus.

Contents of the invention

In view of this, the present invention provides a detection kit for Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus, which solves the problem of simultaneous detection of Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus in the prior art. Low, long cycle and other technical problems, as well as detection methods for Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus.

The present invention is achieved like this,

A detection kit for Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus, comprising a DNA extraction solution and a PCR reaction solution, the PCR reaction solution including a first reaction solution, a second reaction solution and paraffin, the first One reaction solution includes Bacillus cereus forward primer, Bacillus cereus reverse primer, Bacillus cereus probe, Enterobacter sakazakii first primer, Enterobacter sakazakii second primer, Enterobacter sakazakii probe , Staphylococcus aureus first primer, Staphylococcus aureus second primer, Staphylococcus aureus probe, described Bacillus cereus forward primer sequence as shown in SEQ ID NO: 1, Bacillus cereus reverse primer The sequence is shown in SEQ ID NO: 2, the probe sequence of Bacillus cereus is shown in SEQ ID NO: 3, the sequence of the first primer of Enterobacter sakazakii is shown in SEQ ID NO: 4, the second primer of Enterobacter sakazakii The sequence is shown in SEQ ID NO: 5, the probe sequence of Enterobacter sakazakii is shown in SEQ ID NO: 6, the first primer sequence of Staphylococcus aureus is shown in SEQ ID NO: 7, the second primer of Staphylococcus aureus The sequence is shown in SEQ ID NO: 8, and the probe sequence of Staphylococcus aureus is shown in SEQ ID NO: 9.

The present invention further provides a detection method for Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus, comprising the following steps:

Provide DNA samples of Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus;

Fluorescent quantitative PCR detection is performed, and the aforementioned detection kits for Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus are used in the detection step of fluorescent quantitative PCR.

The detection kit and detection method for Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus of the present invention can detect Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus at the same time, without long-term enrichment of the samples to be tested Cultivation, adding detection primers and probes at the same time, has the advantages of rapidity, sensitivity and safety compared with traditional culture methods, and can be applied to the detection of Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus.

Description of drawings

Fig. 1 is the positive control PCR amplification figure in the detection kit of Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus in the embodiment of the present invention;

Fig. 2 is the negative control PCR amplification figure of the Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus detection kits of the embodiment of the present invention;

Fig. 3 is the positive sample PCR amplification figure in the detection method of Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus in the embodiment of the present invention;

Fig. 4 is the negative sample PCR amplification figure in the detection method of Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus in the embodiment of the present invention;

Fig. 5 is a flow chart of the detection method of Bacillus cereus culture.

Detailed ways

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

Real-time fluorescent quantitative PCR is widely used in basic scientific research, clinical diagnosis, disease research and drug development and other fields. The basic principle is: a fluorescent chemical substance is introduced into the PCR reaction system, and as the PCR reaction progresses, the PCR reaction product accumulates continuously, and the fluorescence signal intensity also increases proportionally. After each cycle, a fluorescence signal intensity is collected, so that the change of the product amount can be monitored through the change of the fluorescence intensity, and a fluorescence amplification curve is obtained. There is a linear relationship between the logarithmic value of the PCR product amount and the amount of the initial template, which can be Perform quantitative analysis.

An embodiment of the present invention provides a detection kit for Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus, comprising a DNA extraction solution, a PCR reaction solution, and the PCR reaction solution includes a first reaction solution, a second reaction solution and Paraffin, the first reaction solution includes Bacillus cereus forward primer, Bacillus cereus reverse primer, Bacillus cereus probe, Enterobacter sakazakii first primer, Enterobacter sakazakii second primer, Sakazakii Enterobacter aureus probe, Staphylococcus aureus first primer, Staphylococcus aureus second primer, Staphylococcus aureus probe, the Bacillus cereus forward primer sequence is shown in SEQ ID NO: 1, wax sample The reverse primer sequence for Bacillus is shown in SEQ ID NO: 2, the probe sequence for Bacillus cereus is shown in SEQ ID NO: 3, the first primer sequence for Enterobacter sakazakii is shown in SEQ ID NO: 4, Sakazaki The sequence of the second primer for Enterobacter sakazakii is shown in SEQ ID NO: 5, the sequence of the probe for Enterobacter sakazakii is shown in SEQ ID NO: 6, and the sequence of the first primer for Staphylococcus aureus is shown in SEQ ID NO: 7. The coccus second primer sequence is shown in SEQ ID NO: 8, and the Staphylococcus aureus probe sequence is shown in SEQ ID NO: 9.

Specifically, the detection kits for Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus according to the embodiment of the present invention include DNA extraction solution and PCR reaction solution, which can simultaneously detect Bacillus cereus, Enterobacter sakazakii and aureus Staphylococcus detection; the kit also includes Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus negative control, positive control. Specifications of the kit:

The specification of the negative control is 1 tube, 40μl/tube, the specification of the positive control is 1 tube, 40μl/tube, the DNA extraction solution is 5ml/tube (1 tube in total), and the PCR reaction solution is 8 tubes/strip x 6 strips, 20μl /Tube.

The negative control is Tris-EDTA buffer (0.01M pH8.0) that does not contain Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus genes.

The positive control is a positive template control prepared by using Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus and DNA extraction, which is diluted with Tris-EDTA buffer (0.01MpH8.0) and stored frozen. The negative and positive controls are used for self-inspection during detection to prevent detection errors. Please refer to Figure 1 and Figure 2, Figure 1 shows the PCR amplification pattern of the positive control in the kit, and Figure 2 shows the PCR amplification pattern of the negative control in the kit.

Specifically, the specific components of the DNA extraction solution (for extracting DNA) are: Tris-EDTA buffer solution (0.01M pH8.0), containing 0.01% NP-40.

Specifically, the PCR reaction solution includes the first reaction solution, the second reaction solution and paraffin, and the first reaction solution and the second reaction solution are separated by the paraffin wax. During the PCR reaction, since the paraffin wax melts at a temperature of 95° C., the second The first reaction liquid and the second reaction liquid are mixed, and the PCR reaction can be carried out. For the specific content of the paraffin, please refer to the patent (200910190112.7). The PCR reaction reagent is stored in a common centrifuge tube, the first reaction solution is located at the lower end, the paraffin is located in the middle, and the second reaction solution is located on the paraffin.

Specifically, the first reaction solution includes buffer, magnesium chloride, dNTPs, Bacillus cereus forward primer, Bacillus cereus reverse primer, Bacillus cereus probe, Enterobacter sakazakii first primer, Enterobacter sakazakii Bacillus second primer, Enterobacter sakazakii probe, Staphylococcus aureus first primer, Staphylococcus aureus second primer, Staphylococcus aureus probe, and, sterilized ultrapure water, the total volume of the first reaction solution 18 μl/tube.

The buffer is 10×Buffer, and the buffer (Bao Bioengineering (Dalian) Co., Ltd.) does not contain magnesium ions. The volume of the buffer is 2-4 microliters/tube;

The concentration of the magnesium chloride is 25mM, and the volume of the magnesium chloride is 2-4 microliters/tube;

The concentration of the dNTPs is 2.5mM, and the volume is 2-4 microliters/tube;

The concentration of the Bacillus cereus forward primer is 50 μM, and the volume is 0.1-0.2 μl/tube, such as 0.15 μl/tube. The nucleotide sequence of the Bacillus cereus forward primer is shown in SEQ ID NO: 1 , specifically: 5'-AAATCAA ACAAATTGTT GCGC-3'-;

The concentration of the Bacillus cereus reverse primer is 50 μM, and the volume is 0.1-0.2 μl/tube, such as 0.15 μl/tube. The nucleotide sequence of the Bacillus cereus reverse primer is shown in SEQ ID NO: 2 ,Specifically:

5'-CCATATTAGTGAAAGCCGCACT-3';

The concentration of the Bacillus cereus probe is 50 μM, and the volume is 0.1-0.2 μl/tube, such as 0.15 μl/tube. The nucleotide sequence of the Bacillus cereus probe is shown in SEQ ID NO: 3, Specifically: 5'-(FAM)TAGTT TGTTTAACAA CGCGCG(DABCYL)-3';

The concentration of the first primer of Enterobacter sakazakii is 50 μM, and the volume is 0.1-0.2 microliter/tube, such as 0.15 microliter/tube. The nucleotide sequence of the first primer of Enterobacter sakazakii is shown in SEQ ID NO: 4 , specifically: 5'-AGCTTCCG CAGTGAAACG TCACG-3';

The concentration of the second primer of Enterobacter sakazakii is 50 μM, and the volume is 0.1-0.2 microliter/tube, such as 0.15 microliter/tube. The nucleotide sequence of the second primer of Enterobacter sakazakii is shown in SEQ ID NO: 5 , specifically: 5'-GCACTGCAAAGTGACGCCTTCG-3';

The concentration of the Enterobacter sakazakii probe is 50 μM, and the volume is 0.1-0.2 microliter/tube, such as 0.15 microliter/tube. The nucleotide sequence of the Enterobacter sakazakii probe is shown in SEQ ID NO: 6, Specifically:

5'-(HEX)CTGGCTC GCGAAAACGC ACGC(DABCYL)-3';

The concentration of the first primer for Staphylococcus aureus is 50 μM, and the volume is 0.1-0.2 microliters/tube, such as 0.15 microliters/tube. The nucleotide sequence of the first primer for Staphylococcus aureus is shown in SEQ ID NO: 7. Specifically, it is: 5'-ATACACCT GAAACAAAGCATCC-3'-;

The concentration of the second primer for Staphylococcus aureus is 50 μM, and the volume is 0.1-0.2 microliter/tube, such as 0.15 microliter/tube. The nucleotide sequence of the second primer for Staphylococcus aureus is shown in SEQ ID NO: 8. show, specifically:

5'-AAGTCCTGGTATAAAGAGATGTGG-3';

The concentration of the Staphylococcus aureus probe is 50 μM, and the volume is 0.1-0.2 microliters/tube, such as 0.15 microliters/tube. The nucleotide sequence of the Staphylococcus aureus probe is shown in SEQ ID NO: 9, specifically For: 5'-(ROX)GGTGTAGAG AAATATGGTC CTGA(DABCYL)-3';

The amount of the sterilized ultrapure water is 4.2-11.1 microliters/tube, and the volume of the first reaction solution is 18 microliters/tube.

Specifically, the second reaction solution includes Taq enzyme, chromogen and sterilized ultrapure water, and the total volume of the second reaction solution is 2 microliters/tube.

The volume of the Taq enzyme is 0.15-0.3 microliter/tube, the volume of the chromogenic reagent is 0.01-0.02 microliter/tube, and the chromogenic reagent is, for example, bromophenol blue. The amount of the sterilized ultrapure water is 1.68-1.84 microliters/tube, so that the volume of the second reaction solution satisfies 2 microliters/tube.

Specifically, the volume of the paraffin is 20 microliters/tube.

The detection kit for Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus in the embodiment of the present invention can detect Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus at the same time. Safety and other advantages, can be applied to the detection of Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus.

The embodiment of the present invention further provides a detection method for Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus, comprising the following steps:

Step S01, enrichment culture and specimen processing:

Provide DNA samples of Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus;

Step S02, real-time fluorescent PCR:

Perform real-time fluorescent quantitative PCR detection, and use the above-mentioned Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus detection kits in the fluorescent quantitative PCR detection step.

Specifically, step S01 is performed under sterile conditions.

Specifically, in step S01, a sample is collected, and the sample may or may not contain Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus, which needs to be determined by the detection method of the embodiment of the present invention. Therefore, in this step S01, the DNA samples of Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus may or may not contain the DNA of Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus.

Place the collected specimens in a constant temperature room at 37°C for 3 hours of enrichment culture; then perform the following DNA lysis treatment:

Take 1ml of the enrichment solution and add 50μL of the DNA extraction solution, and repeatedly aspirate 3 times to fully mix the specimen.

Specifically, in step S02, the supernatant obtained in step S01 is taken for fluorescent quantitative PCR analysis. The fluorescent quantitative PCR equipment used in this step is not limited, ABI series, Bio-Rad series (ICycler/MJ Opticon 2 ), Stratagene MX series, Roche LightCycler, Cepheid SmartCycler, Corbett Rotor-Gene, Hangzhou Bioer series.

The conditions of the PCR reaction in step S02 are:

50-55℃: 2-5min;

94-95°C: 2-3min;

94-95°C: 5-10S, 55-60°C: 40-80S; (40 cycles).

In the detection method of Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus in the embodiment of the present invention, a positive control and a negative control in the kit are provided for each detection to prevent contamination during the detection process.

The above detection methods for Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus will be described in detail below in conjunction with specific examples.

Embodiment one

The detection method of Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus in the embodiment of the present invention comprises the following steps:

1. Collect samples of Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus, the number of samples is 20, some samples contain Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus, some samples do not Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus. Place the collected specimens in a constant temperature room at 37°C for 3 hours of enrichment culture; then perform the following DNA lysis treatment:

Take 1ml of the enrichment solution and add 50μL of the DNA extraction solution, pump repeatedly 3 times to fully mix the specimen, and obtain the DNA samples of Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus;

2. Take the above PCR reaction solution, each component in the PCR reaction solution is:

The fluorescent quantitative PCR reaction was carried out and detected under the following conditions:

50-55℃: 2-5min;

94-95°C: 2-3min;

94-95°C: 5-10S, 55-60°C: 40-80S; (40 cycles).

Embodiment two

The detection method of Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus in the embodiment of the present invention comprises the following steps:

1. Collect samples of Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus, the number of samples is 20, some samples contain Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus, some samples do not Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus. Place the collected specimens in a constant temperature room at 37°C for 3 hours of enrichment culture; then perform the following DNA lysis treatment:

Take 1ml of the enrichment solution and add 50μL of the DNA extraction solution, pump repeatedly 3 times to fully mix the specimen, and obtain the DNA samples of Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus;

2. Take the above PCR reaction solution, each component in the PCR reaction solution is:

The fluorescent quantitative PCR reaction was carried out and detected under the following conditions:

50-55℃: 2-5min;

94-95°C: 2-3min;

94-95°C: 5-10S, 55-60°C: 40-80S; (40 cycles).

Embodiment three

The detection method of Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus in the embodiment of the present invention comprises the following steps:

1. Collect samples of Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus, the number of samples is 20, some samples contain Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus, some samples do not Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus. Place the collected specimens in a constant temperature room at 37°C for 3 hours of enrichment culture; then perform the following DNA lysis treatment:

Take 1ml of the enrichment solution and add 50μL of the DNA extraction solution, pump repeatedly 3 times to fully mix the specimen, and obtain the DNA samples of Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus;

2. Take the above PCR reaction solution, each component in the PCR reaction solution is:

The fluorescent quantitative PCR reaction was carried out and detected under the following conditions:

50-55℃: 2-5min;

94-95°C: 2-3min;

94-95°C: 5-10S, 55-60°C: 40-80S; (40 cycles).

comparative example

The detection method of Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus in this comparative example comprises the following steps:

1. Collect samples containing Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus. The samples in this comparative example are consistent with the samples in Example 1. Place the collected samples in a constant temperature room at 37°C for 3 hours of enrichment culture ; Then carry out the following DNA cleavage process:

Take 1ml of the enrichment solution and add 50μL of the DNA extraction solution, pump repeatedly 3 times to fully mix the specimen, and obtain the DNA samples of Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus;

2. Perform detection according to the international common method mentioned in the previous background technology.

Please refer to the following table, the following table shows the results of the method of application embodiment one and comparative example to detect 20 examples of samples:

It can be seen from the table that the method of the first embodiment of the present invention and the method of the comparative example have consistent results.

Please refer to Fig. 3, Fig. 4, Fig. 3 is the positive sample PCR amplification figure in the detection method of Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus in the embodiment of the present invention; After comparing Fig. 3 with Fig. 1, Fig. 4 and Fig. 2, it can be known that Bacillus cereus, Bacillus cereus, The detection method of Enterobacter sakazakii and Staphylococcus aureus can detect positive samples and negative samples in the samples, and the detection results are correct.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention should be included in the protection of the present invention. within range.

Claims (5)

1. a detection kit for Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus, comprising DNA extraction liquid, PCR reaction liquid, it is characterized in that, described PCR reaction liquid comprises first reaction liquid, second reaction liquid and paraffin, the first reaction liquid includes Bacillus cereus forward primer, Bacillus cereus reverse primer, Bacillus cereus probe, Enterobacter sakazakii first primer, Enterobacter sakazakii second primer, Enterobacter sakazakii probe, Staphylococcus aureus first primer, Staphylococcus aureus second primer, Staphylococcus aureus probe, described Bacillus cereus forward primer sequence is shown in SEQ ID NO: 1, wax The reverse primer sequence of Bacillus sakazakii is shown in SEQ ID NO: 2, the probe sequence of Bacillus cereus is shown in SEQ ID NO: 3, and the first primer sequence of Enterobacter sakazakii is shown in SEQ ID NO: 4. The sequence of the second primer for Enterobacter sakizakii is shown in SEQ ID NO: 5, the sequence of the probe for Enterobacter sakazakii is shown in SEQ ID NO: 6, and the sequence of the first primer for Staphylococcus aureus is shown in SEQ ID NO: 7, golden yellow The sequence of the second primer for Staphylococcus is shown in SEQ ID NO: 8, the sequence of the probe for Staphylococcus aureus is shown in SEQ ID NO: 9, and the first reaction solution and the second reaction solution are separated by the paraffin. 2. Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus detection kit as claimed in claim 1, is characterized in that, described first reaction solution also comprises sterilized ultrapure water, damping fluid, magnesium chloride, dNTPs. 3. Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus detection kit as claimed in claim 1, is characterized in that, described second reaction solution comprises Taq enzyme, sterilized ultrapure water and chromogen . 4. Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus detection kit as claimed in claim 2 or 3, is characterized in that, each component volume of described first reaction solution is: 5. Bacillus cereus, Enterobacter sakazakii and Staphylococcus aureus detection kit as claimed in claim 2 or 3, is characterized in that, the volume of each component of described second reaction solution is: Taq enzyme 0.15～0.3 μl/tube Chromogen 0.01～0.02 microliters/tube Sterilized ultrapure water 1.68-1.84 microliters/tube.