Five diarrhea-causing Escherichia coli identification kits (PCR method) instruction manual

• product manual

The germ detection series can amplify the specific nucleic acid fragments of pathogenic microorganisms in samples such as food and feed, and the instrument monitors the fluorescence signal changes during the amplification process in real time and automatically interprets the results. This product is used for the detection of Escherichia coli. The detection limit is 10 ³ CFU/ml.

• Product composition (96 test)

â—† Required instruments

ABI, BioRad, TaKaRa and other PCR amplification equipment, electrophoresis equipment, gel imager and other electrophoresis equipment. (No need for electrophoresis equipment for qualitative interpretation using a real-time PCR machine)

â—† Self-supplied supplies and instruments

1 Sterilize 1.5mL or 2.0mL centrifuge tube; 2 ice box; 3 pipette (0.5-10μL, 10-100μL, 100-1000μL) and matching sterilization tips; 4 centrifuge; 5 vortex mixer; 6 metal bath.

â—† Sample processing

Refer to the "GB4789.6-2016 National Food Safety Standards for Food Microbiology Inspection of Escherichia Coli in Escherichia Coli" for the pre-enhancement. It is recommended to use a reagent-assisted bacterial genomic DNA extraction series.

â—† Experimental operation

1. Add a template (sample preparation area, placed in an ice box)

Take 12 pairs of reaction tubes with the number of samples to be tested +3, completely thaw the reagents, and separate the hearts for 30 s. After centrifugation, the sealing film was peeled off, and 2 μL of the template was added to each reaction solution through the solid sealing layer by puncture loading (or a small sterile sample was used to pick up a small amount of the colony to be inspected into the reaction tube).

Example of loading: (There are 2 samples to be tested)

Take 5 rows of 12 reaction tubes, add NG-DW in the first row, add NG-Ecoli in the second row, add sample 1 in the third row, add sample 2 in the fourth row, add PG in the fifth row. -Ecoli.

After the matching PCR tube cap was covered, the mixture was vortexed for 30 s, centrifuged for 1 min, and the PCR amplification reaction was immediately performed.

2. Amplification reaction (amplification and product analysis area)

Set up the amplification reaction according to the following conditions: (If you need to use fluorescence method for detection, please select FAM channel)

3. Product electrophoresis (amplification and product analysis area)

Weigh 4. 0 g of agarose powder, add to 200 mL of 1 × TAE running buffer, and mix well. Use a microwave oven to repeatedly heat to boiling until the agarose powder is completely melted to form a clear, transparent solution. When the agarose solution is cooled to about 60 ° C, add ethidium bromide ( EB ) to a final concentration of 0.5 μg / mL, mix thoroughly, and gently pour into the mold where the comb has been placed, the gel length is greater than 10cm, the thickness should be 3mm~5mm. Check for bubbles under the comb or between the combs, and carefully remove the air bubbles from the agarose gel with a disposable tip. After the agarose gel has completely set and hardened, gently pull out the comb, carefully place the block and the glue bed into the electrophoresis tank, and place the sample well at the cathode end. Add 1×TAE electrophoresis buffer to the electrophoresis tank, and the liquid level is 1mm~2mm above the rubber surface. After mixing 5 μL of the PCR product with 1 μL of 6× loading buffer, the mixture was pipetted into the subsurface gel with a micropipette and carefully loaded into the well; the PCR reaction product of the positive control was added to the last one. Lane; 2 μL molecular weight Marker was added to the first lane. Turn on the electrophoresis power supply, calculate and set the electrophoresis voltage value according to the formula: voltage = distance between the positive and negative electrodes of the electrophoresis tank (cm) × 5V / cm; start the voltage switch, the electrophoresis starts with bubbles appearing in the positive and negative platinum wires quasi. After electrophoresis for 30 min to 45 min, the power is turned off. The gel was removed and placed in a gel imager to observe the results, photograph and record the data.

• Equivalent nucleic acid fluorescent dyes such as Gold view, Gal-Rad, etc. can be used instead of EB.
• It is recommended to use DNA Marker: DL2000 or 100bp ladder, etc. Marker that can indicate a band of 100bp~1500bp.

• Result analysis

• Yin positive interpretation:

When performing electrophoresis detection, it is necessary to compare with Marker to judge whether the target is positive when a single significant band appears at the corresponding position of the target target; otherwise, the target is negative. (When detecting with a real-time PCR machine, the hole is judged to be positive when the detection hole Ct≤30; when the detection hole Ct>30, the detection hole is negative)

Example electropherogram

• Validity interpretation:

It should be satisfied at the same time: 1. All lanes in the blank control are negative; 2. In the negative control, the uidA lane is positive, and the other lanes are negative; 2. All lanes in the positive control are positive, and the experiment can be judged to be effective. If the above conditions cannot be met, the experiment is invalid and the experiment needs to be repeated. If the result is still invalid after repeated, please contact our technical support.

• Result interpretation:

If the experiment is valid, it can be interpreted according to the following table:

• More than 97% of Escherichia coli is positive for uidA , and the amplification result can be judged by referring to the detection result of the gene in the negative control;
• When the result is judged to be EPEC positive, if the bfpB target is positive, the sample contains a typical EPEC; if the bfpB target is negative, the sample is atypical EPEC;
• When the result is judged to be STEC/EHEC positive, if the eae target is positive, the sample contains typical EHEC; if the eae target is negative, the sample is atypical EHEC.

â—† Notes

1. This reagent has high detection sensitivity. In order to prevent pollution, the experiment is to be partitioned.

1) First zone: sample preparation zone.

2) Second zone: amplification and product analysis zone.

★ It is best to physically isolate the partitions to avoid contamination caused by human factors.

2. Wear work clothes and latex gloves during the experiment, use pipettes, and treat all waste generated in the test in time.

3. Strictly follow the operation steps, reagent preparation and sample loading steps, please operate in strict accordance with the instructions on the ice box.

4. The components in the reaction solution are sensitive to light and should be stored away from light . The reagent should be completely thawed before use, but repeated freezing and thawing should be avoided. It is recommended to centrifuge for 30 seconds before use.

5. Do not mix different batches of reagents used within the validity period.

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