qPCRBIO Probe 1-Step Virus Detect is designed for ultra-sensitive detection of viral RNA by 1-step RT-qPCR using sequence-specific probes.
The kit has been optimised with a high-concentration 4x mix, enabling greater sample input and increasing sensitivity, even when working with small volume reactions.
- Recommended kit for SARS-CoV-2 diagnostics
- Validated for qualitative detection of SARS-CoV-2 nucleic acid
- Concentrated 4x mix format, ideal for high-throughput, highly multiplexed assays
- Ultra-sensitive detection of RNA viruses
- Earlier detection of a wide range of template amounts
- Includes UltraScript RTase for thermostable reverse transcription up to 55°C
- Advanced RNase inhibitor
- Antibody-mediated hot start technology
- Compatible with all real-time PCR platforms – standard and fast cycling conditions
The kit contains proprietary components that are optimised to enable cDNA synthesis and PCR amplification to be performed in a single tube.
Developed with a high concentration 4x mix format, qPCRBIO Probe 1-Step Virus Detect offers greater sensitivity and flexibility by allowing more sample to be added to each reaction, and for smaller reaction volumes to be used with confidence. This is particularly beneficial in high throughput assays or for clinical diagnostic testing where accurate and reproducible detection of low copy viral templates is of crucial importance. This specialised kit allows accurate and sensitive detection of viral sequences over a broad range of input RNA, down to 4 copies tested per reaction (0.8 copies per μL).
Robust and reproducible RT-qPCR
Fast and efficient cDNA synthesis is provided by UltraScript Reverse Transcriptase, which can operate over a wide range of reaction temperatures. Reverse transcription can be performed in only 5 minutes and at temperatures up to 55°C, enhancing detection of GC-rich and highly structured viral RNA sequences. The PCR step is powered by PCRBIO HS Taq DNA Polymerase, which employs antibody-mediated hot start technology for specific amplification of virus-derived cDNA, with improved tolerance to common PCR inhibitors. Combined with a specially formulated buffer system developed using proprietary smart screen technology, qPCRBIO Probe 1-Step Virus Detect gives robust and reproducible RT-qPCR while minimising the potential for primer dimer and other non-specific products.
qPCRBIO Probe 1-Step Virus Detect comes ready-optimised and validated for qualitative detection of SARS-CoV-2 nucleic acid and is ideally suited to high throughput testing of COVID-19 clinical samples, either by laboratory developed assays or as a component of diagnostic testing kits. Validation has been performed using the Charité (Berlin, Germany) recommended primer-probe sequences (RdRp and E genes), and Centers for Disease Control and Prevention (Atlanta, USA) primer-probe sequences (N gene).
Universal detection kit
qPCRBIO Probe 1-Step Virus Detect contains all the required components for accurate and sensitive RT-qPCR except for primers, probe, RNA template and water. For maximum flexibility the kit is engineered for use on a wide range of probe technologies, including TaqMan®, Scorpions® and molecular beacon probes. It can be used with RNA extracted by most commercial kits, provided the amount and quality of template RNA are within an acceptable range. The kit is compatible with multiplexing assays and with all real-time PCR platforms. Use our qPCR Selection Tool to find out which ROX variant is compatible with your instrument.
This product alone does not provide any diagnostic result and is supplied for Research Use Only. It is suitable for use as a component of molecular diagnostic assays, where applicable country laws allow.
- COVID-19 / SARS-CoV-2 diagnostics and research
- RNA virus detection
- Diagnostic real-time PCR
- Detection of extremely low copy number targets
- High throughput assays
- Singleplex and multiplex
- TaqMan®, Scorpions® and molecular beacon probes
The kit should be stored between -30°C and -15°C. Avoid prolonged exposure to light. If stored correctly the kit will retain full activity for 12 months. The kit can be stored at 4°C for 1 month. The kit can go through 30 freeze/thaw cycles with no loss of activity.
Fig 1. Triplex detection of SARS-CoV-2 nucleic acid using CDC and WHO/Charité recommended sequences
Amplification of SARS-CoV-2 N, E, and RdRp genes according to CDC (N2) and WHO/Charité (E and RdRp-P2) recommended sequences, using qPCRBIO Probe 1-Step Virus Detect (purple) and SuperScript III Platinum OneStep qRT-PCR Kit (grey). Amplification curves are shown on the left and efficiency on the right. 5 serial dilutions of RNA template were used, corresponding to 40,000, 4,000, 400, 40 and 4 copies of the SARS-CoV-2 genome. Total reaction volume is 20µL. The N2 probe was labelled with Cy5,
E probe with FAM, and RdRp-P2 probe with Texas Red. Cycling conditions were RT at 55°C 10 min, denaturation at 95°C 3 min and 50 cycles of amplification at 95°C 15s, 58°C 30s.
qPCRBIO Probe 1-Step Virus Detect can reliably and sensitively detect SARS-CoV-2 in triplex reactions, giving earlier or equal Ct and increased sensitivity at lower template dilutions compared to the competitor kit.
Fig 2. Amplification of SARS-CoV-2 E gene using WHO/Charité recommended sequences after different RT times
Ct values and efficiency plots obtained with amplification of SARS-CoV-2 E gene sequences after different reverse transcription (RT) times using qPCRBIO Probe 1-Step Virus Detect (purple) and SuperScript III Platinum OneStep qRT-PCR Kit (grey). 3 dilutions of RNA template were used, corresponding to 40,000, 40 and 4 copies of SARS-CoV-2 genome per reaction. Total reaction volume is 20µL. Cycling conditions were RT at 55°C, denaturation at 95°C 3 min and 50 cycles of amplification at 95°C 15s, 58°C 30s.
qPCRBIO Probe 1-Step Virus Detect enables sensitive detection of SARS-CoV-2 nucleic acid over a range of RT times, with earlier Ct and greater efficiency compared
to the competitor kit. When using the qPCRBIO kit we recommend 5 min RT for singleplex reactions and 10 min RT for multiplex.
ROX (6-carboxy-X-rhodamine) is used as a passive reference dye in ROX-dependent real-time PCR instruments to normalize for variations of fluorescence levels that can arise mainly due to optical path variations among wells. Normalisation of the fluorescence intensity (Rn) is done in real-time PCR software by dividing the emission intensity of the specific signal by the emission intensity of ROX.
ROX does not take part in the PCR reaction and its fluorescence levels are not proportional to the quantity of DNA in each well, so the addition of this fluorophore to a mix provides a constant fluorescent signal during amplification.
Different types of real-time PCR instruments requiring a passive reference standard have different optimal concentrations of ROX, mainly due to the different optical configurations of each system (i.e. the different type of excitation source and optics used).
The addition of either too little or too much ROX would result in a very noisy signal impacting on the results of the reaction. Therefore, it is extremely important for the user to:
- Determine the correct ROX concentration to optimise real-time PCR results, and
- Check the ROX settings on the software used to set up the reaction
ROX concentrations in qPCRBIO Probe 1-Step Virus Detect Mixes:
The qPCRBIO Probe 1-Step Virus Detect Mixes come in 3 different formulations, each with a different concentration of ROX:
- qPCRBIO Probe 1-Step Go Lo-ROX (PB25.51) contains 112 nM ROX
- qPCRBIO Probe 1-Step Go Hi-ROX (PB25.52) contains 1.12 µM ROX
- qPCRBIO Probe 1-Step Go Separate ROX (PB25.54) contains no ROX and include a separate tube of 50 µM ROX additive. This enables you to choose what concentration of ROX you’d like to use.
|PB25.52-01||200 x 20μL Reactions|
|PB25.52-03||600 x 20μL Reactions|
|PB25.52-05||1’000 x 20μL Reactions|
|PB25.52-50||10’000 x 20μL Reactions|
|PB25.52-500||100’000 x 20μL Reactions|
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|4x qPCRBIO Probe 1-Step Virus Detect Hi-ROX||
1 x 1mL | 200 Reactions
3 x 1mL | 600 Reactions
1 x 5mL | 1000 Reactions
1 x 50mL | 10’000 Reactions
1 x 500mL | 100’000 Reactions
|20x UltraScript RTase (with RNase inhibitor)||
1 x 200μL | 200 Reactions
1 x 600μL | 600 Reactions
1 x 1mL | 1000 Reactions
2 x 5mL | 10’000 Reactions
1 x 100mL | 100’000 Reactions