Mouse hepatitis virus probe method fluorescence quantitative RT-PCR kit cell line class

Fluorescence quantification of mouse hepatitis virus probe method RT-PCR kit

Name：MouseHepatitisVirus(MHV)ProbegRT-PCRKit

50 times/box

【Expected useMouse hepatitis virusMouse Hepatitis Virus (MHV) is an RNA virus that exists in the blood and internal organs, especially in the liver and kidneys, and is also found in feces and urine. It is highly contagious to young mice. Mouse hepatitis is an infectious disease caused by MHV, and infected mice are distributed, but under normal circumstances, it mostly presents as an asymptomatic infection. It can only become a lethal disease under stress factors, mainly hepatitis and encephalitis. Therefore, the rapid and accurate identification of mouse hepatitis virus plays an important role in the prevention and quarantine of this disease. This product is a specialized kit developed based on probe based fluorescence quantitative RT-PCR technology for detecting mouse hepatitis virus. It has the following characteristics:

1. Ready to use, users only need to provide sample RNA templates.

2. Primers and probes have been optimized for high sensitivity.

3. Provide a positive control to distinguish false negative samples.

4. High specificity, primers are designed based on the highly conserved region of mouse hepatitis virus and will not cross react with RNA of other viruses.

5. This product is sufficient for 50 fluorescent quantitative RT-PCR reactions using a 20 μ L probe system.

6. This product can only be used for scientific research.

[Specification and Composition]

【Transportation and storage】Low temperature transportation,-Store at 20 ℃ for a period of 12 months.

【Self provided reagents】sample RNA。

Mouse hepatitis virus probe fluorescent quantitative RT-PCR kit【 Usage 】1、 Dilute standard curve sample (Taking the 6 dilutions of 10E2-10E6 copies/μ L as an example) Due to the high concentration of the standard, the following dilution operations must be carried out in a separate area and must not contaminate the sample or other components of this kit. To increase product stability and avoid the spread of infectious pathogens, this product does not provide live samples as positive controls, only non infectious DNA fragments are provided as positive controls.

1. Mark 6 centrifuge tubes, namely 6, 5, 4, 3, 2, and 1.

2. Add 45 μ L of fluorescent PCR specific template diluent using a core gun tip (preferably using a core gun tip, the same below).

3. Add 5 μ L1 × 10E7 copies/μ L of positive control (provided by the reagent kit) to tube 6, shake thoroughly for 1 minute, and obtain 1 × 10E6 copies/μ L of standard curve sample. Put it on ice for later use.

4. Change the gun head and add 5 μ L1 × 10E6 copies/μ L of positive control (obtained from the previous dilution) to tube 5. Shake thoroughly for 1 minute to obtain a standard curve sample of 1 × 10E5 copies/μ L. Put it on ice for later use.

5. Change the gun head and add 5 μ L1 × 10E5 copies/μ L of positive control (obtained by dilution in the previous step) to tube 4. Shake thoroughly for 1 minute to obtain a standard curve sample of 1 × 10E4 copies/μ L. Put it on ice for later use.

6. Repeat the above operation until obtaining standard curve samples with 6 dilutions. Put it on ice.

2、 Sample RNApreparation

7 . If there are N samples, it is best to set N+2 extractions, with the extra one being PC (positive control for sample preparation) and one being NC (negative control for sample preparation). You can use a 10000 fold dilution of 10 μ L of positive control and a certain amount of water to make the total volume the same as the required volume for each preparation, and use it as PC. Additionally, use water as NC.

8 . Purification of RNA samples using self selected methods, this kit is compatible with most viral RNA extraction kits on the market.

threeProbeqRT PCR reaction(20 μ L system, conducted in the sample preparation room)

9 . If quantitative analysis is performed and only one repetition is made, label N+9 PCR tubes, of which N+2 are used for the N+2 samples obtained in the previous step, 1 is used for PCR negative control (using water as a template), and 6 are used for the standard curve. If qualitative analysis is performed and only one repetition is made, label N+4 PCR tubes, of which N+2 are used for the N+2 samples obtained in the previous step, 1 is used for PCR negative control (using water as a template), and 1 is used for PCR positive control (using the dilution of positive control in tube 4 as a template). Below, only quantitative analysis will be used to describe the operational steps,

10 . Add each component to the labeled tube according to the table below (this table only lists one repetition. The positive control is only set after the sample tube and negative control are set, and the positive control sample should be added after all tubes are covered and stored)

11.After closing the lid, operate the machine according to the following parametersqRT-PCR：

4： Data processing

If this reagent kit is used for quantitative detection, plot a standard curve with the log value of the positive control concentration as the horizontal axis and Ct value as the vertical axis. Calculate the log value of the RNA concentration of the sample from the standard curve based on the Ct value of the test sample, and then calculate its concentration.

13 . If this kit is used for qualitative testing and only determines positive or negative, the negative control Ct must be greater than or equal to 40. The positive control must have fluorescence logarithmic growth, typical amplification curve, and Ct value should be less than or equal to 30. For the test sample, if its Ct is greater than or equal to 40, it is negative; if it is less than or equal to 35, it is positive. If it is between 35-40, repeat once. If the Ct value of the repeated experiment is greater than or equal to 40, it is negative; if it is less than 35, it is positive.