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TOROIVD® Probe 1-step RT-qPCR 5G kit
    Publish time 2020-08-07 12:52    

TOROIVD® Probe 1-step RT-qPCR 5G kit provides for sensitive, reproducible detection up to five RNA/DNA targets in a single multiplex reaction .Particularly useful for virus detection with TaqMan®probe assays , the kit includes UNG, thermostable MMLV reverse transcriptase, RNase inhibitor ,TOROIVD® 5G DNA polymerase and reaction buffer.  The improved enzymes also enables a high resistance to PCR inhibitors and high stability in room temperature. The 1-step system is suitable for high-throughput analysis because of its simple reaction setup. In addition, this system can reduce the risk of cross-contamination with uracil-N-glycoslyaseUNG) . The kit is suitable for high-speed RT-qPCR and enables accurate detection and quantification of targets, making it possible to obtain highly reproducible and reliable realtime PCR results over a wide dynamic range.



DESCRIPTION

TOROIVD® Probe 1-step RT-qPCR 5G kit provides for sensitive, reproducible detection up to five RNA/DNA targets in a single multiplex reaction .Particularly useful for virus detection with TaqMan®probe assays , the kit includes UNG, thermostable MMLV reverse transcriptase, RNase inhibitor ,TOROIVD® 5G DNA polymerase and reaction buffer.  The improved enzymes also enables a high resistance to PCR inhibitors and high stability in room temperature. The 1-step system is suitable for high-throughput analysis because of its simple reaction setup. In addition, this system can reduce the risk of cross-contamination with uracil-N-glycoslyaseUNG. The kit is suitable for high-speed RT-qPCR and enables accurate detection and quantification of targets, making it possible to obtain highly reproducible and reliable realtime PCR results over a wide dynamic range.


Features

-Rapid and highly sensitive
 This kit can achieve the rapid and highly sensitive quantification of a low-copy targets  by a 1-step RT-qPCR method with probes and be suitable for the quantification of RNA/DNA viruses or mRNA expressed at a low level. 

-Optimized for multiplexing
This kit has been validated for multiplexing up to five targets simultaneously, allowing for additional targets and/or controls to be run simultaneously for efficiency or quality control purposes. 

-Inhibitor tolerant
The unique proprietary formulation of this kit allows robust performance even in the presence of substances that can normally inhibit PCR, such as heparin, hematin, or EDTA, increasing your confidence when working with a variety of complex clinical samples. 

-Wide dynamic range compatible with RNA and DNA
This kit has been optimized to provide high specificity and dynamic range for use with both RNA and DNA targets. This input flexibility can help streamline the number of different workflows in your lab to improve efficiency. 

-Broad instrument compatibilityThis kit can be run in either fast or standard cycling conditions with equivalent performance across a wide variety of real-time cyclers. The 50×ROX Reference dyenot suppliedis added can be applied to the real-time cyclers that require a passive reference dye. 

-Utilization of dUTP 

This kit contains dUTP in the reaction buffer and UNG in the enzyme mix . Therefore, the rate of false-positive detection can be reduced by adding UNG .

Primer/probe design 

 -Design of primers 

Primer length: 18–25bpGC content of primer: 40–60%

Tm of primer: 60–65°CPurification grade: OPC or HPLC grade 

Target length: 70–200 bp Larger targets (>200 bp) tend to reduce the efficiency and specificity of amplification. 

 - Design of  probes
  -Probe length: 20–30bpGC content of probe: 40–60%

Tm of probe: 65–70°CPurification grade : HPLC grade.  

- Checking the performance of primers and probes

-Prepare a dilution series with five or more dilutions of template RNA/DNA. Perform RT-qPCR assay using the diluted RNA/DNA with the newly designed primers and probe, and draw a standard curve. 

- Confirm that the PCR efficiency is between 90% and 110% and R2 is equal to or greater than 0.99. if the PCR efficiency or R2 are outside of these ranges, the reaction conditions should be optimized. If this does not improve the result, the primers and/or probe should be redesigned. 


Components

The kit includes the following  reagents, and QPR-303U can be used for 400 reactions for a total 25μl reaction volume. All reagents should be stored at −20°C. 

RT-qPCR Enzyme Mix           260μl/tube×2    

2×5G qPCR Buffer GB            1ml/tube×5 

  Notes:  RT-qPCR Enzyme Mix  contains  TOROIVD®III reverse transcriptase, UNG,TOROIVD®5G DNA polymerase and RNase inhibitor.2×5G qPCR Buffer GB  contains 0.4mM dA/C/G/T/UTP, 5mM Mg2+reaction buffer and stabilizeretc.).

Protocol 

1. This kit should be fully thawed before use. Gently vortexed and briefly centrifuged. 

2. Purified or crude template RNA can be may be used directly or after dilution

3. Prepare the following reaction mixture in a thin-walled qPCR tube or plate.

Components              25μl reaction

2×5G qPCR Buffer GB                12.5μl

 

 

Premix A

 

 

 

 

Premix C

RT-qPCR Enzyme Mix                   1.3μl

50× ROX                                 0/0.05/0.5μl

DNase/Rnase Free Water                    Yul

10μM Forward primer                          1.25μl

 

 

Premix B

10μM Reverse primer                          1.25μl

10μM TaqMan® probe                          0.5μl

 DNase/Rnase Free Water                   Zul

  Template RNA solution                       5μl                   

4. Gently mix the  reaction solutions  and spin down in microcentrifuge.

Notes 

- 2.5mM MgCl2 final concentration have been added in this reaction mixture. But for the direct RT-qPCR to crude template RNA the MgCl2 concentration may need to be optimized between 2.5-8mM. 
 -The primer concentration should be optimized between 0.2-0.8 μM and  TaqMan® probe optimized between 0.1-0.4 μM  with 10-50copies templates /reaction . So the best primers-probe concentration sets was selected by orthogonal design of experiments.

-The 50× ROX is not supplied with this kit. It is recommended that you use 50×ROX (ROX-050) optionally. 50× ROX reference dyes are used for analyses with instruments that correct for cross-talk between wells, such as the real- time PCR instruments by Applied Biosystems and Agilent Technologies. 0.5ul 50×ROX Reference Dye was added for a total 25ul reaction volume in when using the following instruments,Applied Biosystems 7300/ 7700/7900HT, StepOnePlus, etc. And 0.05ul was added for using the following instruments,Applied Biosystems 7500/7500Fast StepOnePlus ,Agilent Technologies AriaMx,etc.No ROX Reference Dye is required when using other brand instruments,such as LightCycler 96/LightCycler 480 system (Roche), CFX96 Real-Time PCR Detection System (Bio-Rad), Smart Cycler System (Cepheid) ,etc.

Cycling conditions 

The recommended 2-step PCR protocol is described below. Use this protocol first and optimize PCR conditions when necessary. Perform 3-step PCR when using primers with low Tm values or when 2-step PCR is not feasible.

 

UNG treatment:                     37°C            2min           1cycle  

Reverse transcription           52°C            5min            1cycle

Pre-denaturation                  95°C            1min           1cycle

 Denaturation                         95°C           10sec           40cycles      

Extension /Annealing            60°C            30sec          40cycles

Data collection should be performed at the extension step. 


Note
 

-The indicated RT temperature can be optimized between 50-60°C and time between  2-10min

-The indicated Pre-denaturation temperature can be optimized 95-98°Cand time between 10sec-5min

-The indicated denaturation temperature can be optimized 95-98°Cand time between 3sec-10sec

-The indicated Extension /Annealing temperature can be optimized 60-65°Cand time between 5sec-30sec

Application data  

Template RNA:

SARS-CoV-2 reference RNAGBWE091111China);

N Gene2.4×105copies/ul; E Gene1.8×105copies/ul;ORF1ab Gene0.70×105copies/ulDilution factor is 103104105106.

Primer and ProbeFrom China CDC

ORF1ab-F5`- CCCTGTGGGTTTTACACTTAA-3`

ORF1ab-R 5`- ACGATTGTGCATCAGCTGA-3`

ORF1ab-P:  5`-FAM-CCGTCTGCGGTATGTGGAAAGGTTATGG-3`BHQ1

N-F: 5`- GGGGAACTTCTCCTGCTAGAAT-3`

N-R: 5`- CAGACATTTTGCTCTCAAGCTG-3`

N-P: 5`-FAM-TTGCTGCTGCTTGACAGATT-3`TAMRA

Instrument

Line Gene 9600 PlusBioer.

 

STORAGE

This reagent can be stored at 4°C for 1 months. For longer storage,  this reagent should be kept at -20°C for 2 years.





 


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