One Step RT qPCR kit

For detailed product information please refer to the IFU

Product Details

Description:

One step RT kit contains 5 X reaction buffer optimized for sensitive and specific detection of RNA species from various sources and purity backgrounds. Enzymes come separately in 20X and 200 X formulations to provide flexibility and adaptations to your substrate of interest, depending mainly on the presence of PCR inhibitors (when testing direct sample without RNA extraction or difficult samples like waste-water extracts). Optimizations on SARS COV2 templates in both swab and waste-water extracts using primer/probe sets included in WHO official protocols, in single and multiplex conditions, guarantee the reproducible and efficient qPCR reactions close to the qPCR theoretical limit of detection <5 copies per reaction for pure samples and between 5-50 copies/reaction for “tough samples” *. 

*“Tough samples”: RNA samples that are produced from either direct protocol, without column extraction and washing steps, or originating from tissues or cells that have high chemical or enzymatic PCR inhibitory potential (High phenolic substances found in plant tissues or high concentration of RNases, DNases and Proteases context found in blood samples or mammalian tissue samples or fungi cells).

Reagents supplied:

One Step RT qPCR kit, 200 rxns (Cat No: RN010S)
Cat No	Description	Tubes No.
BR030-1	5x One Step RT-qPCR buffer, 1 ml	1
PD015-0.225	20x Hot Start Taq DNA polymerase, 0.225ml	1
RN015-0.025	200x Reverse Transcriptase, 0.025ml	1
BR033- 1	Nuclease Free Water, 1 ml	2

Standard 20 μl reaction:

Component		Stock solutions	1 reaction of 20 μl	Final Concentration/Quantity (20 μl assay)
1	5x One Step RT-PCR buffer	5x	4 μl	1x
2	Fw primer	10 μΜ	0.4 μl	0.2 μM
3	Rv primer	10 μΜ	0.4 μl	0.2 μΜ
4	TaqMan Probes	10 μΜ	0.2-0.4 μl	0.1-0.2 μΜ
5	20x Hot Start Taq DNA polymerase	20x	1 μl	1x
6	200x Reverse Transcriptase	200x	0.1 μl (0.08-0.12)	1x
7	Template RNA*	0.5-107 copies of target sequence/μl	1-10 μl	10 fg-500 ng
8	Nuclease free water	N/A	Till 20 μl	N/A

Alternative suggestions:

1. After optimizing conditions and you have found the final primer/probe set you will be using, you may prepare a 10x stock and use it as such.
2. Sybr or EVA Green may be used instead of TaqMan probe in concentration of 0.2-2x. Start with 1x.
3. ROX may be added to the mix. Add according to your needs and adjust the water volume.

One Step-RT recommended conditions for single and multiplex One Step RT-qPCR reactions.

Step 1: RT reaction. This step may be set up from 40 – 62 ^oC. Increase temperature for more highly structured RNA targets.

Step 2: RT inactivation/ Hot Start Taq DNA polymerase activation.

Initial activation of Hot Start at 94^oC for 15 minutes is very important in the case of reactions with very low PCR template copies and for obtaining the maximum efficiency in qPCR setups.  DO NOT USE FOR LESS THAN 10 MIN!! 

Step 3: Cycle denaturation. It may be set 5-15 seconds. Optimize based on your target.

Step 4: Annealing/Extension step. Temperature and time depend on the primers’/probes’ Tm (melting temp) and amplicons’ length. The primers should be designed at Tm ~ 65 ^o C. Set this step at 6-10 ^oC below the lowest oligos’ tm and use 35 seconds for amplicons up to 250 bp. Annealing extension time may be lowered in 5 seconds steps (30 or 25 seconds) depending on the limit of detection and the efficiency required for the assay in question. Lowering time is recommended only if efficiency of the assay is not affected near the limit of detection.

Standard protocol for single and multiplexing

For more detailed guidelines visit the IFU manual.

Additives that improve robustness

In cases of samples with low viral content near the limit of detection (~5-10 copies/μl of wastewater sample) and/or high concentration of inhibitors one may also add extra DTT or T4gp32 protein to improve reactions’ efficiency. Final concentrations in qPCR reactions should be 1mM for DTT and 0.05 μg/μl for T4gp32. An example of the reaction setup including both additives may be found below:

Component		Concentration of Stock solutions	1 reaction of 20 μl	Final Concentration/Quantity (20 μl assay)
1	5x One Step RT-PCR buffer	5x	4 ㎕	1x
2	10x SARS COV2 Oligos mix	10x	2 ㎕	1x
3	DTT (Customer provided)	100 mM	0.2 ㎕	1 mM
4	Gp32 (Customer provided)	10 μg/μl	0.1 ㎕	0.05 μg/μl
5	20x Hot Start Taq DNA polymerase	20x	1 ㎕	1x
6	200x Reverse Transcriptase	200x	0.1 ㎕	1x
7	Template RNA*	5-105 copies of target sequence/μl	2-4 ㎕	N/A
8	Nuclease Free Water	N/A	Till 20 ㎕	N/A

Recommended suppliers:

• For DTT, any product that is compatible with molecular biology applications (example A2948/Applichem, DTT Molecular biology grade or equivalent)
• For Gp32, use any provider that is compatible with molecular biology applications. The inhouse assays have been performed with cat number M300S or L from New England Biolabs (T4 Gene 32 Protein)

DTT may reverse mildly the inhibition potential of the sample whereas both should be used when high inhibition is suspected.

Other Quality Controls: 

Certified as DNase-RNase free using fluorescently labeled RNase and DNase probes.  Efficiency between 90-110%, with R² value >0.99 of each lot is estimated by a standard curve assay produced over 5 logs of EURM019 substrate (https://crm.jrc.ec.europa.eu/p/EURM-019)

Shipping 

Shipped on blue ice or dry ice. 

Storage conditions 

Store at -20^οC ± 5^οC 

Shelf life 

1 year upon production

Example: SARS COV 2 standard EURM019

SARS COV2 standard curves produced by One Step RT qPCR kit (Cat No: RN010S). The standard curves were produced by analyzing the EURM019 SARS COV 2 standard over 5 logs. Standard dilutions were prepared, having as a given that the EURM019 stock solution contains 10⁸ copies of SARS COV2 chimeric transcripts/μl (detailed info for EURM019 used may be found at https://crm.jrc.ec.europa.eu/p/EURM-019). Since the quantification of the EURM019 standard, performed by digital droplet PCR by manufacturer, produced results between 6 – 7.6 x 10⁷ copies/μl depending on the primer set used, the actual number of copies that were introduced in each reaction may be 24-40 % less than indicated in the figure.

Reactions were set in duplicates for 10⁵-10²  copies reactions and in triplicates for 10 copies reactions.

The Baseline was manually set to 200 Relative Fluorescent Units.

The results were automatically produced by CFX Maestro software. The reactions were incubated to either CFX96 or OPUS96 qPCR machines with very similar results.

Details regarding the primer sets used may be found at: https://www.who.int/docs/default-source/coronaviruse/whoinhouseassays.pdf