Enterohemorrhagic Escherichia coli O157 nucleic acid detection kit (one-tube PCR-fluorescent probe method) instruction manual

Enterohemorrhagic Escherichia coli O157 nucleic acid detection kit (one-tube PCR-fluorescence probe method)

â—† Product Description

The pathogen detection series can amplify specific nucleic acid fragments of pathogenic microorganisms in food, feed and other samples, 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 enterohemorrhagic Escherichia coli O157. The detection limit is 10 ³ CFU/ml .

â—† Product composition (96 test)

â—† Applicable instruments

Real-time fluorescence PCR instrument such as ABI 7500, CFX 96, Mx 3005P, LineGene9600.

â—† Self-supplied supplies and instruments

1 ice box; 2 pipettes (0.5-10μL, 10-100μL, 100-1000μL) and matching sterilization tips; 3 centrifuges; 4 vortex mixers; 5 metal baths; 6 homogenizers, mixers or Grinding tools such as mortar; 7 electronic balance.

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) The second zone: the template addition zone.

3) Zone 3: Amplification and product analysis zone.

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

2. Work clothes and latex gloves are worn during the experiment, and tools are used independently in different areas. Gloves and lab coats need to be replaced.

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, and store the reaction solution in an appropriate volume according to the frequency of detection.

5. After the reaction is completed, the expansion tube should be placed in a sealed bag and discarded. On the same day, the lid is opened and the aerosol is easily contaminated. It is forbidden to open the lid.

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

â—† Sample processing

Refer to the 5.1 treatment sample in GB/T 4789.36-2008 Food Hygiene Microbiological Test Escherichia coli O157:H7/NM Test, and pre-enrich the sample, and prepare the prepared bacterial solution for use.

Samples should be inspected as soon as possible after collection. If it can not be tested in time, it can be stored at 2 ° C ~ 4 ° C for 18h. 25g (ml) was taken as a sterile operation into a homogenized bag containing 225ml mEC+n broth, continuously homogenized for 1min~2min on a tapping homogenizer; or placed in 225ml mEC+n The homogenized cup of the broth is homogenized for 1 min to 2 min from 8000 rpm/min to 10000 rpm/min, and cultured at 36 °C ± 1 °C for 18 h to 24 h.

For detailed steps, please follow the standard operation or check the food safety software.

â—† Experimental operation

1. Template preparation (sample preparation area)

It is recommended to use the reagents to support the bacterial DNA extraction series of products. The specific process is detailed in the product manual.

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

Cut the PCR tube containing the reaction number, and place it at room temperature to be thawed. After centrifugation for 30 seconds, uncover the sealing film. Add 5 μL of template to each reaction solution in the order of NG, sample template to be tested. , PG-O157-P. 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.

3. Amplification reaction (amplification and product analysis area)

Using a real-time PCR instrument, the fluorophore was selected for FAM and the quencher group was selected for TAMRA.

Set up the amplification reaction according to the following conditions:

4. Baseline and threshold settings

The baseline adjustment takes 3-15 cycles of fluorescence signal and the threshold line should exceed the highest point of the negative control amplification curve.

â—† Result judgment

The test sample has no Ct value or ≥40.0, the curve is straight or slightly oblique, no "S" type amplification curve, the sample can be reported negative, does not contain enterohemorrhagic Escherichia coli O157 or the content is below the detection limit;

The test sample Ct ≤ 35.0, the curve is an "S" type amplification curve, can directly report the sample positive, containing enterohemorrhagic Escherichia coli O157;

The test sample 35.0 < Ct < 40.0, a repeated experiment is required, and if the Ct value is ≥ 40.0, it is negative, otherwise it is positive.

• The NG reaction is a smooth straight line, the PG reaction is an "S" type amplification curve and the Ct value is <30. This test result is valid, otherwise it is invalid. If the duplicate test results are still invalid, please contact technical support.