Brucella melitensis nucleic acid detection kit

Brucella melitensis nucleic acid detection kit

50T/box

Real time fluorescencePCR method

Brucella melitensis nucleic acid detection kitMain components

RT-PCR reaction solution, mixed enzyme solution, positive control, negative control.

Storage conditions and expiration date

light-proof-Store below 20 ℃ to avoid repeated freezing and thawing, with a shelf life of 12 months.

Multi channel real-time fluorescence quantitative PCR instruments including ABI series, Bio Rad series, Agilent Stratagene MX series, Roche LightCycler R480, Cepheid SmartCycler, Rotor Gene series, Hangzhou Borui series, etc.

Reagent Preparation (Reagent Preparation Area)

（1) Take out the reagent kit from the refrigerator, extract the necessary reagents for the experiment from the kit, thoroughly melt and mix, and centrifuge instantly to remove any liquid adhering to the tube wall.

（2) Calculate the required number of reactions (n) for the current experiment, and determine the amount of various reagents needed for the experiment based on the reaction system shown in the table below.

N=number of negative controls (1T)+number of positive controls (1T)+margin of error (1T)+number of samples

（3) Add the above reagents into a sterile centrifuge tube, mix thoroughly, and centrifuge immediately. Divide the reagent into PCR reaction tubes at a rate of 20 μ L per tube.

（4) Cover the PCR reaction tube tightly and transfer it to the sample processing area. Put the remaining reagents back into the refrigerator below -20 ℃ for freezing storage.

2. Sample preparation (sample processing area)

useQIAGEN and Roche DNA extraction kits are used to extract quDNA. The specific operation should follow the instructions of their kits.

3. Sample addition

In the above preparedAdd 5 μ l of processed DNA and a final volume of 25 μ l/tube to the PCR reaction sample tube, cover the tube with the PCR reaction cap and mix well. Immediately centrifuge at low speed and transfer to the PCR instrument. For the negative control and positive control tubes, add 5 μ L each of the negative control or positive control samples provided in the kit, with a final volume of 25 μ l/tube. Cover the PCR reaction cap and mix well. Immediately centrifuge at low speed and transfer to the PCR instrument.

PCR (PCR amplification region)

（1) Take the prepared PCR reaction tubes from the sample processing area, place them in the corresponding position of the real-time fluorescence quantitative PCR instrument sample slot, and record the placement order.

（2) Set the instrument's nucleic acid amplification parameters according to the table below for PCR amplification.

Note:When setting up the ABI series fluorescence PCR instrument, do not select ROX calibration, and select None for quenching groups.

Reference value (reference range)

1. Validity assessment of reagent kit:

（1) Positive control: There is a typical S-shaped amplification curve or Ct value ≤ 35.

（2) Negative control: Ct value>38 or no Ct value, linear or slightly diagonal, no exponential growth period.

2. Determination of specimen results:

（1) Positive: The Ct value of the specimen test result is ≤ 35 or there is a significant exponential growth period.

（2) Suspicious: The Ct value of the specimen test result is within the range of 35-38. At this point, the specimen should be subjected to repeated testing. If the Ct value of the repeated experiment is still within the range of 35-38 and there is a significant exponential growth period, it is judged as positive. Otherwise, it is judged as negative.

（3) Negative: The Ct value of the specimen test result is greater than 38 or there is no Ct value.

Limitations of Inspection Methods

When the concentration of the tested nucleic acid in the test sample is lower than the maximum limit of this kitThe detection limit of DI may result in false negative results.

This kit is only for preliminary screening of specimens. For positive results detected by this kit, further confirmation should be made using culture methods.

【 Precautions 】

Laboratories using this reagent kit should strictly follow the management regulations for gene amplification testing laboratories issued by relevant national departments;

to avoidRNA degradation, the sample processing process should be operated under 0-4 ℃ conditions, and immediately tested on the machine after completing the experiment; The instruments and consumables used in the sample processing process should be treated with ribonucleases;

All items in each area are for exclusive use and must not be used interchangeably to avoid contamination; After the inspection is completed, the workbench should be cleaned immediately;

When absorbing the reaction solution, try to avoid generating bubbles as much as possible; upBefore using the PCR instrument, it is important to check if all reaction tubes are tightly sealed to prevent inaccurate results caused by liquid evaporation;

All reagents in the reagent kit should be thoroughly melted and mixed at room temperature before use, and should be centrifuged immediately;

The negative and positive controls provided in the reagent kit should be transferred to the specimen preparation area and stored separately before the first use;

To prevent fluorescence interference, direct contact with bare hands should be avoidedPCR reaction tube, and any labeling on the PCR reaction tube should be avoided;

The suction head used during the testing process should be directly hit onto the containerIn the waste tank containing 10% hypochlorous acid, PCR reaction tubes that have been tested should not be opened and should be sterilized together with other waste items before being discarded; Workbenches and various experimental supplies should be regularly disinfected with 10% hypochlorous acid, 75% alcohol, or ultraviolet lamps;

9. The parameters related to instrument amplification should be set according to the relevant requirements of this manual; Different batch numbers of reagents cannot be mixed; And use it within its validity period.