Hifair^TM V Multiplex One Step RT-qPCR Probe Kit (UDG Plus)

Mfr.No.

13650ES92, 13650ES60, 13650ES80, 13650ES50

Description

Hifair^TM V Multiplex One Step RT-qPCR Probe Kit (UDG Plus) is a multiplex quantitative PCR reagent. In the process of the experiment, reverse transcription and quantitative PCR were carried out in the same tube, which simplified the experimental operation and reduced the risk of contamination. In this kit, the first strand cDNA was efficiently synthesized by heat-resistant Hifair^TM V Reverse Transcriptase and quantitatively amplified by Unicon^TM HotStart Taq DNA Polymerase. The kit mainly contains optimized MP buffer, enzymes mix, etc. The buffer solution already contains Mg2+ and dNTP. In addition, the factors that can effectively inhibit the non-specific PCR amplification and improve the amplification efficiency of multiple qPCR reactions are added, which can ensure the amplification efficiency and carry out up to multiple amplification reactions. The dUTP/UDG system was added to effectively prevent the risk of aerosol contamination.

Features

High sensitivity：The carefully optimized buffer system improves the detection sensitivity of low-concentration templates up to 250 copies/mL；
Super stability: The product has passed real-time stability testing for 18 months..
dUP/UDG anti-pollution system：dUTP/ UDG anti-pollution system is introduced to degrade aerosol pollution of products efficiently, reduce false positive, and ensure authentic results；
Support fast program: compatible with fast program, 40min can produce results;

Product Components

Components No.	Name	13650ES50	13650ES60	13650ES80	13650ES92
		(50 T)	(100 T)	(1,000 T)	(10,000 T)
13650-A	2×HifairTM V MP Buffer	750 μL	1.5 mL	15 mL	150 mL
13650-B	HifairTM V Enzyme Mix	60 μL	120 μL	1.2 mL	12 mL

Note:
1) 2×HifairTM V MP Buffer is the abbreviation of HifairTM V Multiplex One Step RT-qPCR Probe Buffer, which includes dNTPs, dUTP, Mg2+, stabilizers, enhancers, and more.
2) HifairTM V Enzyme Mix mainly contains heat-resistant HifairTM V reverse transcriptase, UNICONTM HotStart Taq DNA polymeras and UDGase.

Product Details

Reaction Composition

Components	Volume (μL)	Final Concentration
2×HifairTM V MP Buffer	15	1×
HifairTM V Enzyme Mix	1.2	-
Primer / Probe Mix (2.5 μM)	3	0.25 μM
Template RNA	10-Jan	-
RNase Free H2O	to 30	-

Note：Be sure to mix well before use, avoid excessive bubbles caused by violent vibration.

a) Primer/Probe concentration: Primer mix including multiplex primer, depending on the situation optimal primer concentration may be between 0.1 and 1.0 μM.Probe mix including multiplex probe labeling difference fluorescent group, depending on the situation optimal probe concentration may be between 0.05 and 0.5 μM.

c) Template dilution: qPCR is highly sensitive and it is recommended to dilute the template. The control Ct value is suitable between 20 and 35.

d) System preparation: unhead with filter element. Avoid cross contamination and aerosol contamination.

Optimized Cycling Protocol

	Reaction stage	Temperature	Time	Cycle
1	Reverse transcription	50°Ca	10 min	1
2	Initial denaturation	95°C	5 min	1
3	Amplification reaction	95°C	15 sec	45 cycles
3	Amplification reaction	60°Cb	30 secc	45 cycles

Note：a) Reverse transcription: The temperature can select 42°C or 50°C for 10-15 minutes.

b) Amplification reaction: The temperature is adjusted according to the Tm value of the designed primers.

c) Fluorescence signal acquisition: Please set the experimental procedure according to the requirements of the instrument manual.

3. Application equipment

Equipment with Rox：ABI 5700, 7000, 7300, 7700, 7900HT Fast, StepOne™, StepOne Plus™

Equipment with Low Rox：ABI 7500, 7500 Fast, ViiA™7, QuantStudio™ 3 and 5, QuantStudio™ 6,7,12k Flex

Stratagene MX3000P™, MX3005P™, MX4000P™

Equipment without Rox：

Bio-Rad CFX96™, CFX384™, iCycler iQ™, iQ™5, MyiQ™, MiniOpticon™, Opticon®, Opticon® 2, Chromo4™
Eppendorf Mastercycler® ep realplex, realplex 2 s; Qiagen Corbett Rotor-Gene® Q, Rotor-Gene® 3000, Rotor-Gene® 6000
Roche Applied Science LightCycler® 480, LightCycler® 2.0, Lightcycler® 96
Thermo Scientific PikoReal Cycler; Cepheid SmartCycler®; Illumina Eco qPCR

Notes

Please wear the necessary PPE, such lab coat and gloves, to ensure your health and safety！
Please use RNasefree consumables for the experiment.

Sensitivity test
The 13650 stored at -20°C for 18 months (experimental group) and those within the shelf life (control group) were simultaneously tested for the gradient dilution (500 copies/ml, 250 copies/ml) of the pseudovirus SARS-CoV-2, targeting the ORF gene and N gene. Eight replicate wells were tested for each concentration, with a detection rate of 100%. Moreover, there were no significant differences in the amplification curve peak shapes and Ct values.

Lot	FAM (number of positives)		VIC(number of positives)
Lot	250copies/ml	500copies/ml	250copies/ml	500copies/ml
experimental group	8-Aug	8-Aug	8-Aug	8-Aug
control group	8-Aug	8-Aug	8-Aug	8-Aug

Real-time stability test
The 13650 stored at -20°C for 18 months and those within their shelf life. Both groups were tested simultaneously for the SARS-CoV-2 pseudovirus ORF gene and N gene at a concentration of 1000 copies/ml with 20 replicate wells. The detection rate for both was 100%, and there were no significant differences in the shape of the amplification curves or the Ct values.

Red: 13650 within their shelf life；blue：13650 stored at -20°C for 18 months。

Stable performance after repeated freeze-thaw cycles

Subjecting the 13650 reagent to 30 and 50 freeze-thaw cycles using dry ice, alongside reagents stored normally at -20°C, and simultaneously testing for the SARS-CoV-2 pseudovirus ORF gene, the results indicated that the performance of the 13650 reagent was unaffected after 50 freeze-thaw cycles.

Red: 13650 stored normally at -20°C; Purple：13650 to 30 freeze-thaw cycles；Green：13650 to 50 freeze-thaw cycles.

Compatible with Standard and Rapid Protocols

Using 13650ES, the ORF gene and N gene of SARS-CoV-2 pseudovirus were detected under the same reaction system with both standard and rapid protocols. The results showed that under a 4-plex system, there was no difference in the amplification curve peak shapes and Ct values between the two protocols.

Applications

RT-qPCR, Multiplex PCR, Probe-Based qPCR

Storage	The product should be stored at -25°C~-15°C for 18 months.

Manual	Manual

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Combination SKU	Supplier No.	Size	Your price
BHR20800037-1	13650ES92	10000T	$6265	Add to Cart
BHR20800037-2	13650ES60	100 T	$105	Add to Cart
BHR20800037-3	13650ES80	1000T	$895	Add to Cart
BHR20800037-4	13650ES50	50 T	$55	Add to Cart

Hifair^TM V Multiplex One Step RT-qPCR Probe Kit (UDG Plus)

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