qPCRBIO Probe 1-Step Virus Detect

Ct values can vary depending on sample concentration, reaction optimization, equipment, and laboratory, so caution is advised when selecting a cutoff Ct value. Typically, Ct values above 35 may be considered unreliable. However, late Ct values can be observed for inefficient reactions when using low sample quantities. Good practice is to standardize cutoff values using relative or absolute quantification methods. Additionally, run and analyze melting curves or gels of products to identify any late amplification products.

ROX (6-carboxy-X-rhodamine) is used as a passive reference dye in ROX-dependent real-time PCR instruments to normalize fluorescence fluctuations that may occur due to optical variations between wells. Rn (normalized fluorescence intensity) is achieved in real-time PCR software by dividing the emission intensity of the specific signal by that of ROX.

ROX is not involved in the PCR reaction, and its fluorescence intensity does not change in proportion to the amount of DNA in each well, providing a constant fluorescence signal during amplification.

Different real-time PCR instruments require an optimal concentration of ROX for reference, usually due to the distinct optical configurations of each system (e.g., different types of excitation sources and optics used).

Adding too little or too much ROX will result in noisy signals, affecting the reaction outcomes. Therefore, it is crucial for users to:

1. Determine the correct ROX concentration to optimize real-time PCR results, and
2. Verify the ROX settings on the software used to set up the reaction.

A useful selection tool for commonly used systems can be found here.

We recommend a minimum of 2 minutes to activate the polymerase. Longer times up to 15 minutes can also be used without adversely affecting the enzyme.

1-step

Both cDNA synthesis and PCR occur in the same mix. This option is suitable for high-throughput applications due to speed and ease of setup. Additionally, there is less risk of contamination. However, it is not ideal for low-quality RNA samples or if cDNA needs to be stored or analyzed separately.

2-step

cDNA synthesis and PCR occur separately. This option is preferable if cDNA product needs to be retained for analysis. It also allows for higher optimization of reaction levels. It allows control over enzyme type and concentration, RNA input, and cDNA concentration, leading to higher sensitivity than the 1-step format.

No, UltraScript® RTase 20x contains an RNase inhibitor to prevent degradation and enhance sensitivity.

If you observe unusually late Ct values, try diluting the sample RNA. By doing this, you are diluting any inhibitors that may be present at a concentration that does not inhibit the reaction. Additionally, try increasing the reverse transcription reaction temperature to 55°C and increase the annealing/extension temperature. This may help overcome difficulties due to secondary structure within the sample RNA and/or primers.

In the event of potential reaction inhibition, try reducing the sample amount¹ or adding 0.4 – 4.4 mg/ml BSA to the reaction².

For more specific issues, contact technical@pcrbio.com providing the following information:

• Amplicon size
• Reaction setup
• Cycling conditions
• Screenshots of amplification plots and melt profiles

1 Scipioni et al. A SYBR Green RT-PCR assay in one tube for detection of human and bovine noroviruses and control of inhibition. Virology Journal.5:94 (2008). doi: 1186/1743-422X-5-94

2 Plante et al. Use of bovine serum albumin to improve the detection of enteric viruses from washing of vegetable produce. Applied Microbiology. 52:3 (2010) doi: https://doi.org/10.1111/j.1472-765X.2010.02989.x

Different products are likely to fluoresce at different levels. However, this does not impact the quantitative accuracy and Ct values will not differ between products.

Probe-based kits like qPCRBIO Probe 1-Step Virus Detect provide higher sensitivity and are less likely to amplify non-targets. Multiplexes can be measured by using amplicons with different fluorophores to target probes, which cannot be achieved with dyes.

Dye-based systems like qPCRBIO SyGreen 1-Step Detect and 1-Step Go detect any intact dsDNA, so they will show primer-dimer or non-target amplification. These can be separated from product peaks by melt curve analysis.

ROX is a passive reference dye, meaning it does not participate in the PCR. It is used to normalize fluorescence fluctuations not related to PCR. You can use our qPCR Selection Tool in the Resources section to determine which of our qPCR mixes are best suited for your qPCR machine.

All qPCRBIO Probe 1-Step Virus Detect 4x mixes contain MgCl₂ at a concentration of 18 mM. This means the final concentration in the reaction is 4.5 mM.

Gene-specific primers can be used in the 1-step reaction.

Primers should be designed to ensure they have similar attachment temperatures, are target-specific, and do not form primer-dimers. The reverse transcription step time can be extended to 10 minutes to ensure sufficient material for attachment and amplification.

We recommend 400-1000 nM for each primer. There is some flexibility around this recommended concentration, however, primer concentrations should not exceed this range as it may significantly affect enzyme activity.