4X一步法 RT-qPCR 预混型探针法试剂盒(含 UNG，可兼容引物探针预混)

4X One-Step RT-qPCR Pre-Mixed Probe Assay Kit (with UNG, compatible with primer probe pre-mix)

Product Code: CM0273

- +

¥2,200

Product Description

This is a specialized kit for one-step RT-qPCR based on the probe method. Reverse transcription and qPCR reactions are completed in the same tube, which makes the operation simple and fast.
This product uses a new modified Evo Super M-MLV reverse transcriptase with optimized Accurate Taq HS polymerase can synthesize cDNA and perform stable qPCR amplification in a short period of time, and supports pre-mixing of primer probes. The product is widely used, with high impurity tolerance, and can amplify genes of different complexity, while it can be used for single or multiplexed detection. It is suitable for the detection of DNA/RNA and other trace viruses.
The dUTP/UNG anti-contamination system is introduced in this product. dUTP replaces dTTP in the PCR reaction, and the UNG enzyme selectively hydrolyzes dU-containing DNA strands without affecting non-dU-containing DNA strands, thus removing dU-containing contaminating templates introduced in the preparation of the PCR reaction system, and thus preventing false-positive results of the PCR effectively; At the same time, monoclonal antibody that can inhibit the activity of DNA Polymerase at room temperature is also added to this product, which can effectively inhibit non-specific amplification, improve the sensitivity of the reaction and enhance the accuracy of the results.

Product Advantages

1. This is a one-step RT-qPCR reaction kit, which can complete the reverse transcription and qPCR reaction in a single tube, simplifying the operation, improving the efficiency, and effectively reducing the possibility of contamination caused by multiple operations.
2. This product adopts the new modified Evo Super M-MLV reverse transcriptase with optimized Accurate Taq HS polymerase with carefully optimized reaction system for high specificity and amplification efficiency.
3. This product supports premixing of primer probes.
4. The product is highly sensitive and can stably detect single-digit copy templates, and can perform single or multiple detection.
5. This product can perform DNA/RNA co-detection.
6. dUTP/UNG is a contamination prevention system that removes contamination of dU-containing PCR products, thereby preventing false-positive PCR results and improving the accuracy of the results.

Product Composition
individual parts making up a compound norm
4X One Step Probe Mixture II (UNG Plus)* 1.25 ml

*: contains Evo Super M-MLV RTase Enzyme,Accurate Taq HS DNA Polymerase, UNG Enzyme and RNase Inhibitor, and reaction buffer.

Preservation and transportation
Storage temperature: -20℃ storage
Transportation temperature:Dry ice transportation or -20°C ice pack transportation.

I. Advance premixed primer probe stability experiments

Example 1

Total RNA from 293T cells was used as a template for the 37℃ accelerated stability assay. Accelerated samples and untreated samples were pre-mixed with primer probes and placed at 37℃ for 3 days and 5 days, respectively, and assayed simultaneously. The amount of template added to the reaction system was 10 ng, 1 ng and 100 pg. The quantification instrument used was ABI QuantStudio.TM 5 Real-Time PCR Systems. the results of the experiments were as follows:

[FAM channel]

The results are shown above:
1. The performance of accelerated samples (red and blue curves) and untreated samples (green curve) after 3 and 5 days at 37°C are comparable.
2、This product can be single weight testing.

[VIC Channel]

The results are shown above:
1, different concentrations of templates can get good amplification within 45 cycles;
2. Negative controls were not detected within 45 cycles;
3, the amplification efficiency was 93.41 TP3T, R2= 0.9998.

Example 2

Porcine blue-ear virus PRRSV JXA1 standard [ GBW(E)090929 ], pig blood RNA as a template, RNase free water instead of the template as a negative control, pre-mixed primer probe will be placed at 37 ℃ 3 days accelerated samples and untreated samples at the same time for the dual detection, 4 ℃ 30 days accelerated samples and untreated samples at the same time for the dual detection. The test was performed on both accelerated and untreated samples placed at 4℃ for 30 days, respectively.
The ORF gene (GC 56%, FAM channel) and CYTB gene (GC 40%, HEX channel) were added in 10 copies and the pig blood RNA template was added in 100 pg. The amount of PRRSV JXA1 template was 10 copies, and the amount of porcine blood RNA template was 100 pg. The quantification instrument used was ABI QuantStudio.TM 5 Real-Time PCR Systems. the results of the experiments were as follows:

[FAM channel]

[HEX Channel]

The results are shown above:
1. The performance of accelerated samples placed at 37°C for 3 days, accelerated samples placed at 4°C for 30 days (rose color curve) and untreated samples (light green color curve) are comparable.
2. Negative controls were not detected within 45 cycles.
3. This product can be dually tested.

 

II. Support for expedited procedures

Example 1

Human nasal C RNA virus, human glandular 7 DNA virus standard and 293T cell Total RNA were used as templates for triple detection. 10 copies of human nasal C RNA virus template, 25 copies of human glandular 7 DNA virus template and 100 pg of 293T cell Total RNA template were added to the reaction system, and 14 replicate wells were tested for all three templates. All three templates were tested in 14 replicate wells, and RNase free water was used as a negative control instead of the template. The standard and rapid procedures were performed after simultaneous preparation, and the quantification instrument used was ABI QuantStudio.TM 5 Real-Time PCR Systems.

The program is as follows:

The results of the experiment are as follows:

[FAM channel]

[ROX channel]

[Cy5 channel]

The results are shown above:
1, the product is extremely sensitive, for low copy and trace amounts of the template can be well detected.
2, the product supports rapid detection, standard mode and rapid mode can be well detected, and 14 duplicate holes are detected.
3、This product can be used for multiple testing, and can be used for DNA/RNA co-testing.
4. Negative controls were not detected within 45 cycles.

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