Shigella nucleic acid detection kit (constant temperature fluorescence method) instruction manual

Shigella nucleic acid detection kit (constant temperature fluorescence method)

â—† Product Description

The pathogen detection series is based on a unique thermostated fluorescence detection technology that can amplify specific nucleic acid fragments of pathogenic microorganisms in food, feed and other samples. 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 Shigella. The detection limit is 10 ³ CFU/ml .

â—† Product composition (48 test)

â—† Applicable instruments

Dhelix 1610, Dhelix 3210, ESE Tube Scanner, Genie II, Deaou-308c and other constant temperature fluorescence detectors, ABI 7500, LightCycler 480, CFX 96 and other fluorescent PCR instruments.

â—† Self-supplied supplies and instruments

1 Sterilize 1.5mL or 2.0mL centrifuge tube; 2 sterilize 0.2mL PCR tube or octa tube; 3 ice box; 4 pipette (0.5-10μL, 10-100μL, 100-1000μL) and matching sterilization tips ; 5 centrifuge; 6 vortex mixer; 7 metal bath

â—† Notes

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

1) First zone: reagent preparation zone.

2) Second zone: sample preparation area.

3) The third zone: the template addition zone.

4) Zone 4: 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.

7. The detection limit is 10 ³ CFU/ml. The bacterial genomic DNA re-extracted from the cells is collected as a template by centrifugation at 1 ml 10 ³ CFU/ml.

â—† Sample processing

Refer to the 5.1 treatment sample in the GB 4789.5-2012 National Food Safety Standard for Food Microbiology Inspection Shigella Test, and pre-enrich the sample, and prepare the prepared liquid for use.

Take 25 g (ml) of the sample aseptically, add a homogenized cup containing 225 ml of Shigella bacteria broth, and use a rotary blade homogenizer at 8000 rpm/min to 10000 rpm/min. Homogenization; or into a homogenized bag containing 225 ml of Shigella bacteria broth, continuously homogenize with a tapping homogenizer for 1 min to 2 min, and mix the liquid sample with shaking. Anaerobic culture was carried out at 41.5 °C ± 1 °C for 16 h to 20 h.

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

â—† Experimental operation

The reagents were completely thawed and the components were centrifuged for 30 s.

1. Reagent preparation (reagent preparation area, placed in an ice box):

If there are N samples to be tested, refer to the table below and calculate the amount of each component according to N+2 (N samples to be tested + 1 negative control + 1 positive control), and place the reaction solution in 0.6 ml or In a 1.5 ml centrifuge tube, vortex and mix, centrifuge for 30 seconds, dispense into 0.2 ml PCR tubes, and add 1 drop of CI (about 20 μl) to each tube.

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.

3. Add a template (template add area, placed in the ice box)

2 μL of the template was added to the PCR tube containing the reaction solution in the first step, and the order was NG-I, the sample template to be tested, and PG-Shi-I. The mixture was vortexed for 30 s, centrifuged for 1 min, and the amplification reaction was immediately performed.

4. Amplification reaction (amplification and product analysis area)

1 The constant temperature instrument was reacted at 63 ° C for 30 min.

2 If a real-time PCR instrument is used, the fluorescent group is selected as FAM, the quenching group is selected as None, 63 ° C for 15 s, 63 ° C for 45 s as a cycle, and the fluorescence signal is collected at 63 ° C for 45 s, 30 cycles.

For other instruments, please refer to the instrument manual for setting.

â—† Result judgment

1 The instrument automatically determines the result. If “positive” is displayed, the sample contains Shigella; if “negative” is displayed, the sample does not contain Shigella or the content is below the detection limit.

2 On the fluorescence quantitative PCR machine, the results were determined based on the presence or absence of the "S" type amplification curve. If there is an "S" type amplification curve, the sample contains Shigella; if there is no "S" type amplification curve, the sample does not contain Shigella or the content is below the detection limit.

• The result of the NG reaction tube showed "negative", and the result of the PG reaction tube showed "positive". The test result was valid, otherwise it was invalid. If the duplicate test results are still invalid, please contact technical support.
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