Reverse Transcriptase Assay (PERT)-SkyGen

Description

WHO stipulates that biological products cannot contain retrovirus contamination. Since there are many kinds of retroviruses, the presence or absence of retroviruses is usually detected indirectly by detecting the activity of reverse transcriptase.The method for detecting reverse transcriptase activity prescribed by pharmacopoeia is PERT (Product-Enhanced Reverse Transcription).Its principle is to replace reverse transcriptase with samples to be tested for RT-PCR, and to calculate whether reverse transcriptase exists in samples and its concentration by using the standard curve of standard Product preparation.And then calculate whether the sample contains retrovirus and its concentration. The PERT method, based on enzyme activity, is widely used to detect several broad categories of known and unknown retroviruses, including oncovirus, lentivirus and spumavirus. However, the conventional PERT method is a two-step RT-PCR and need electrophoresis detection, which is cumbersome and has large errors, and is not suitable for high-throughput operation. In order to overcome this shortcoming, we developed a one-tube real-time PERT product. It has the following features:

1. All-in-one kit, the users don’t need to optimize every components.

2.Probe qPCR-based one-tube reaction, carry-over contaminations minimized.

3.Small coefficient of variation.

4.High sensitivity, a few dozen of reverse transcriptase molecules can be detected. 

5.The linear range of quantitation is at least 6 orders of magnitude.

6.Low background because of the low residual reverse transcriptase activity in the DNA polymerase used in the kit.

7.Applicable to samples in various forms, including cell culture supernatant, cell lysate and purified reverse transcriptase.

8.Supplies positive control for making standard curve and for ruling out invalid experiment.

9.Suitable for high throughput screening.

10.This product is sufficient for 50 PERT reactions of the 20 mL system.

11.This product can only be used for scientific research, including biological products and cell bank and other quality inspection links.

Kit Component

Storage and Handling

Transport at low temperature, store at -20℃, the storage period is 12 months

Materials Required but not Supplied in the Kit

Sample, ultra-pure water, Total Protein Assay Kit

Procedures

Section 1: Prepare samples for PERT in a biosafety cabinet

1.Cell culture supernatant: Collect the supernatant, centrifuge at 11000×g for 10 min,filter with 0.45 mm membrane,and centrifuge 100,000×g for 60 min to obtain virus particles. Resuspend the virus particles pellet with PERT Sample Diluent and then placed on ice for later use. 1-5 mL of the sample will be used for each PERT test.

2.Cell lysate:After washing,1E6 cells were precipitated and re-suspended in 200 mL PERT Sample Diluent, and the cell lysate were placed on ice for 20 minutes.The protein concentration is determined with Total Protein Assay Kit,and 0.1-1 mg protein sample is needed for each PERT experiment.

Section 2:prepare a 10-fold serial dilutions of MMLV reverse transcriptase.

3.Mark 10 centrifuge tubes No. 1, No. 2,..... No. 9, No. 10，respectively.

4.Add 45 mL PERT Sample Diluent to each of the 10 tubes.

5.Add 5 mL MMLV Reverse Transcriptase (200 U/mL, provided by the kit) into tube No. 1, gently mix for 1 minute and then put it on ice. This is Diluent 1 and the MMLV Reverse Transcriptase concentration is 20 U/mL.

6.Transfer 5 mL Diluent 1 tube No. 2, mix for 1 minute ant put in ice. This is Diluent 2 and the MMLV Reverse Transcriptase concentration is 20 U/mL.

7.Transfer 5 mL Diluent 2 tube No. 3, mix for 1 minute ant put in ice. This is Diluent 2 and the MMLV Reverse Transcriptase concentration is 2 U/mL.

8.Repeat the above procedure until Dilution 10 is obtained. Generally, Dilution 3 –Dilution 8 are used for the experiment. The diluted enzymes can be stored at -20℃ for at least 3 months.

Section 3: Set PERT reaction

9. For the first time using the kit, add 150 mL ultra-pure water into the PERT Prime-Probe Mixture tube, vortex for 30 seconds, then put it on ice for use. The Mixture should be stored at -20℃ .

10.For the first time using the kit, add 100 mL ultra-pure water into the PERT RNA Template tube, vortex for 30 seconds and then put on ice for later use and at -80℃ for long-term storage. 

11.For N samples to be tested, label N+8 0.2 mL PCR tubes, of which N tubes are used for N samples, one tube is used for No Template Control (NTC), one tube is used for No Sample Control (NSC), and 6 tubes are used for the six dilutions (Dilution 3-Dilution 8) to make the standard curve. Add each component according to the following table:

12.Note:If the sample contains DNA polymerase,some DNA polymerases also have residual RT activity,so the sample without reverse transcriptase may also have a positive result.Users may add 2 μL of DNA polymerase RT activity inhibitor to each tube to shield it (and reduce by 2 μL of ultrapure water).This product must be purchased separately.

Section 4: Data Processing

13. Experimental validity judgment: If Ct values appear in NTC or NSC and the fluorescence signal has a standard inverted Samplification curve,it indicates that the reagent or the environment is polluted, then the whole experiment is invalid.If there is no amplification of all positive controls (standard curved tube), it indicates that there is a problem with the reagent, instrument or operation, and the whole experiment is invalid. If the experiment is invalid, it is necessary to find the cause before continuing the experiment. If the experiment is valid, further analysis can be performed.

14.Make the standard curve: take the log value of the concentration of MMLV reverse transcriptase dilution in pU/μL as the horizontal axis and the Ct value of FAM channel as the vertical axis to draw a standard curve.

15.Use the Ct value of N samples to calculate the log value of its corresponding reverse transcriptase concentration in from the standard curve,and then use the log value to calculate its corresponding reverse transcriptase concentration,and then calculate the activity unit used according to the volume.

16.If the weight of the measured reverse transcriptase is known, its specific activity (activity units /mg) can be calculated.The specific activity of the MLV reverse transcriptase provided in this product is 280,000U/mg.

17.If you know the molecular weight of the reverse transcriptase measured, you can also calculate how many active units each reverse transcriptase molecule has.Suppose that the specific activity of an  AMV reverse transcriptase sample is 36500U/mg.Since the molecular weight of AMV reverse transcriptase is 158kDa, the molar number of 1 mg AMV reverse transcriptase is 1/1.58E8,and the corresponding molecular number is 6.02E23×(1/1.58E8) =3.81E15. Since the activity of 1mg is 35600U,each AMV reverse transcriptase molecule quals to 36500U/3.81E15=0.96E- 11U=9.6pU.The molecular weight of MMLV reverse transcriptase is 78kDa and HIV- 1 reverse transcriptase is 117kDa.

18.If it is known how many reverse transcriptase molecules each AMV virus particle contains,it can be calculate how many reverse transcriptase units each virus particle has. According to the literature, each avian retrovirus (including AMV virus) particle contains about 70 reverse transcriptase molecules,and each mouse retrovirus particle and human HIV virus particle has about 70-80 reverse transcriptase molecules.Therefore, each AMV virus particle has 9.6×70=673 pU of AMV reverse transcrptase.

Documents

Reverse Transcriptase Assay (PERT)_Flyer