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寨卡登革热基孔肯雅热三合一核酸检测试剂盒(德国Genekam原装进口）使用说明书

Lot-No.
Ref. FR342 Expiry time: 1 year

100 Tests (Ready to use PCR kit)  

 -Only for in vitro use-

-Only for research use – 

To be used by a technical person-

Principle and use:

This amplification kit has been manufactured by Genekam Biotechnology AG, Germany to detect different viruses: Chikungunya virus, Dengue virus (1-4) and Zika virus in real time PCR assay as multiplex. It is an absolute quantification. It detects viruses and indicates the load of each virus in unknown samples.  These are mosquito borne viruses, which can cause a major threat through different ways e.g. blood transfusion as well as organ transplantation to the donors along with creating normal disease profile in human population through mosquito bites. PCR is the method of choice to detect the viruses against serology methods like ELISA and Rapid tests as they cross reacts with each other.

Warning: Multiplex assays may have less sensitivity than simplex assay in some settings. Such multiplex assays have this technological disadvantage, but their advantages are economical, fast results and multiple parameters for screening, hence they should be used for fast screening e.g. blood banks to give answer whether a particular pathogen is present in unknown samples or not.

Real time PCR is based on fluorogenic dyes. In this kit, there are 4 probes / dyes, hence you have to program them in your machine, each probe indicates the presence of one specific virus:

First Probe: they are 6-Carboxy tetramethyl rhodamine (quencher) and Carboxy-fluorescein (reporter). Up to 40 Ct should be taken positive. Value between 40-45 Ct should be taken as marginal positive (doubtful). It indicates the presence of dengue virus 1 - 3.

Second probe: they are 6-Carboxy tetramethyl rhodamine (quencher) and HEX (reporter, it is available as VIC in some machines). Up to 40 Ct should be taken positive. Value between 40-45 Ct should be taken as marginal positive (doubtful). It indicates the presence of Zika virus.

Third probe: they are 6-Carboxy tetramethyl rhodamine (quencher) and ROX (reporter, it is available as ROX in some machines). Up to 40 Ct should be taken positive. Value between 40-45 Ct should be taken as marginal positive (doubtful). It indicates the presence of Chikungunya virus.

Fourth probe: they are 6-Carboxy tetramethyl rhodamine (quencher) and Cy5 (reporter, it is available as Cy5 in some machines). Up to 40 Ct should be taken positive. Value between 40-45 Ct should be taken as marginal positive (doubtful). It indicates the presence of dengue virus 2 - 4.

Hint:  Please make sure that you have realtime machine, which can detect all 4 probes in their corresponding channels. User has to select above said setting in the software of the realtime machine.

This kit needs RNA which can be isolated from  blood, urine, mosquitoes, seminal samples, liver biopsies, serum, plasma, tissue, cell cultures and any body fluid. Kindly use good methods to isolate the RNA.

Safety precautions should be taken as these viruses are infectious for human beings. Always clean your hands before the test use and clean the hands after the test. Wash your face after the test, if possible. Disinfect your working place.

IMPORTANT: we added cotton or sponge in the lid of container of the kit, to avoid damage during transportation. Please remove this cotton or sponge from the lid of each container before storage.

Composition:

It contains the following (WARNING! THAW THE TUBES SLOWLY: NEVER THAW IN HEATING BLOCK OR WITH HEAT FROM HAND):

• Tube A (2 tubes)

• Tube B (2 tubes)

• Tube Y (1 tube)

• Positive (+Ve) Control (tube D1) (1 tube). This tube must be stored at -20°C.

• Negative (-Ve) Control (tube D2) (1 tube)

Please check them before you start. Please store them at -20°C and dark.

Equipment needed:

• Laboratory centrifuge

• microtubes (0.2ml)

• Pipette-tips with and without filter (10-100µl & 1-10µl)

• Pipettes (quality pipettes)

• Paper

• Pen

• Vortexer

• 96 well microplates for PCR

• Real time machine

Procedure:

STEP A

1. Kindly thaw one tube each: A, B, Y, D1 and D2. After thaw, kindly put the tubes at 4°C (as it is better). If the kit is not in use, store them at -20 ° C.

2. Mark your microtubes with a sample number,  +ve Control and –ve Control. You can use 96 well microplate instead of tubes.

3. Add 7µl of tube A to each tube.

4. Add 10µl of B to each micro tube. Avoid to touch the wall of the microtubes.

5. Add 1µl of Y to each tube (avoid to touch the wall of the microtubes).

TIP: Add 7µlA + 10µl B + 1µl Y = 18µl per reaction. In case you want to run 10 reactions i.e. you need total 180µl, therefore you should mix 70µl of A + 100µl of B  + 10µl of Y = 180µl from which you can take 18µl and add to each tube. This way you save time and hardware.

6. Add 2µl of your RNA with sterile pipette-tip with filter to each micro tube according to your label except +Ve and -Ve (Avoid touching the wall).  Use every time a new pipette tip (for each sample) ! Mix it.

7. Use new pipette tip with filter. Add 2µl of tube D1. This is the positive control supplied with our kit. Mix it.

8. Use a new pipette tip. Add 2µl of –Ve (tube D2) to –Ve Control (don’t touch the wall).Mix it.

9. Centrifuge all tubes for 20 sec. for 8000 rpm (this is not necessary but it is better).

10. Run the program of your thermocycler as followings: Kindly check whether you have added everything correctly as the level of the volume of each micro tube must be almost the same.

You must use quencher and reporter dye to setup your software (see FAQ) asked written elsewhere and run the following program:

1. 60 minutes at 42°C
10 minutes at 70°C

2. 15 seconds at 95°C   x 45 cycles
60 seconds at 60°C

Before you start the PCR program, kindly check whether tubes are closed properly. Microtubes must be in contact with metal block (important!). There should be no air or lose contact with metal block of thermocycler.

11. After step 10 is finished take out the microtubes.

STEP B

Once the program will be finished one can see the graphics. The negative control should run along with the bottom and positive control must give a curve in the software graphics. Use your software to analyse the results. There will be Ct values in the channels, where the genotypes are positive.

Reading the results: The presence of Ct value range as shown elsewhere in each channel indicates that the samples are positive. Sometimes, internal control may fail to show the positive range inspite of fact that there are the positive results for virus specific channels. In such cases, the results are to taken as positive.

If you should find any mistakes, please let us know. Thank you.

FAQ:

1) Q: I cannot find quencher and reporter dye in my software:

A: Many software has got the words:  FAM (as reporter) and TAM (as quencher). Therefore select both in your software.

If your machines has only one word (for some machines only use the word FAM) you should select this one. For HEX, there may be an other channel available in some machines like VIC.