Lyo-Ready Probe 1-Step Kit

Lyo-Ready Probe 1-Step Kit is a lyophilisable RT-qPCR kit designed for ultra-sensitive detection of viral RNA sequences.

This specially formulated kit is ideal for the development of diagnostic assays. It contains a glycerol-free 4x qPCR mix comprising hot start Taq polymerase, dNTPs, MgCl2 and a blend of excipients to provide optimal functionality without loss of activity post lyophilisation.

Features

  • Ultra-sensitive detection of RNA viruses
  • Offers the same high performance post-lyophilisation as our standard kits
  • Includes a glycerol-free 4x qPCR mix
  • Pre-blended with excipients for reliable lyophilisation into beads or cakes
  • Provided with separate, high-concentration UltraScript RTase (with RNase inhibitor)
  • Validated for qualitative detection of SARS-CoV-2 nucleic acid
  • Compatible with high-throughput, highly-multiplexed assays
CAT No.
Product
Size
Price
QTY
PB90.01-03
Lyo-Ready Probe Mix
600 x 20 μL Reactions
PB90.01-10
Lyo-Ready Probe Mix
2000 x 20 μL Reactions (2 x 5 mL)
PB90.01-50
Lyo-Ready Probe Mix
10 000 x 20 μL Reactions (1 x 50 mL)
PB90.01-500
Lyo-Ready Probe Mix
100 000 x 20 μL Reactions (1 x 500 mL)
PB90.11-03
Pre-Lyo Probe 1-Step Evaluation Kit
600 x 20 μL Reactions
PB90.12-10
Lyo-Ready Probe 1-Step Kit
2000 x 20 μL Reactions (2 x 5 mL)
PB90.12-50
Lyo-Ready Probe 1-Step Kit
10 000 x 20 μL Reactions (1 x 50 mL)
PB90.12-500
Lyo-Ready Probe 1-Step Kit
100 000 x 20 μL Reactions (1 x 500 mL)

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More Information

Lyo-Ready Probe 1-Step Kit is specially formulated for ultra-sensitive detection of viral RNA using probe-based RT-qPCR and is ideal for the development of lyophilised diagnostic tests.

Optimised for lyophilisation

Lyophilisation offers many advantages including ambient storage, extended shelf-life and increased versatility in sample volume. The kit has been optimised to offer the same high performance post-lyophilisation as our standard 1-step RT-qPCR kits.

Robust and reliable RT-qPCR

Lyo-Ready Probe 1-Step Kit includes a glycerol-free 4x qPCR mix containing hot start Taq polymerase, dNTPs, MgCl2 and a blend of excipients to ensure reliable lyophilisation, without loss of activity. Antibody-mediated hot start technology ensures specific amplification of virus-derived cDNA, with improved tolerance to the common PCR inhibitors found in clinical samples.

UltraScript Reverse Transcriptase is provided separately in a highly concentrated 3200x format. This robust and thermostable modified MMLV reverse transcriptase is engineered to enhance cDNA synthesis speed and yield with accurate transcript representation and enables fast and efficient reverse transcription up to 55 °C.

SARS-CoV-2 detection

The kit has been validated for SARS-CoV-2 detection in singleplex (see Figure 1) and multiplex (see Figure 2) and offers reliable performance with no loss of efficiency or sensitivity after lyophilisation.

Customisable diagnostic assay development

At PCR Biosystems we understand that every molecular diagnostic assay is different and the process to lyophilise them can be challenging. To help with initial verification work, we offer a Pre-Lyo Probe 1-Step Evaluation Kit for testing purposes.

ISO 13485

Lyo-Ready Probe 1-Step Kit has been manufactured under an ISO 13485 certified Quality Management System and is suitable for further manufacturing use as a component, reagent or reagent assembly for molecular biology diagnostics.

Applications

  • Development of lyophilised molecular diagnostic tests
  • Detection of extremely low copy number targets
  • Compatible with TaqMan®, Scorpions® and molecular beacon probes

Specifications

Lyo-Ready Probe 1-Step Kit

Component

2000 Reactions

10 000 Reactions

100 000 Reactions

4x Lyo-Ready Probe Mix

2 x 5 mL

1 x 50 mL

1 x 500 mL

3200x UltraScript RTase (with RNase inhibitor)

1 x 12.5 μL

1 x 62.5 μL

1 x 625 μL

Pre-Lyo Probe 1-Step Evaluation Kit

Component

600 Reactions

4x Lyo-Ready Probe Mix

3 x 1 mL

20x UltraScript RTase (with RNase inhibitor)

1 x 600 µL

Lyo-Ready Probe Mix

Component

600 Reactions

2000 Reactions

10 000 Reactions

100 000 Reactions

4x Lyo-Ready Probe Mix

3 x 1 mL

2 x 5 mL

1 x 50mL

1 x 500mL

Lyo-Ready Probe 1-Step Kit

Component

4x Lyo-Ready Probe Mix

3200x UltraScript RTase (with RNase inhibitor)



2000 Reactions

2 x 5 mL

1 x 12.5 μL

10 000 Reactions

1 x 50 mL

1 x 62.5 μL

100 000 Reactions

1 x 500 mL

1 x 625 μL

Pre-Lyo Probe 1-Step Evaluation Kit

Component

4x Lyo-Ready Probe Mix

20x UltraScript RTase (with RNase inhibitor)



600 Reactions

3 x 1 mL

1 x 600 µL

Lyo-Ready Probe Mix

Component

4x Lyo-Ready Probe Mix



600 Reactions

3 x 1 mL

2000 Reactions

2 x 5 mL

10 000 Reactions

1 x 50mL

100 000 Reactions

1 x 500mL

Reaction Volume

Storage

20μL

On arrival, products should be stored between -30 °C and -15 °C. If stored correctly the kit will retain full activity for 12 months.

Reaction Volume

20μL

Storage

On arrival, products should be stored between -30 °C and -15 °C. If stored correctly the kit will retain full activity for 12 months.

FAQs

Beyond what Ct value are results unreliable?

Ct values can vary between template concentrations, reaction optimisation, instruments and laboratories, so care must be taken when selecting a cut-off Ct value. Generally, Ct values over 35-40 would begin to be considered unreliable. However, late Cts could be observed for inefficient reactions using low template copy number. It is always good practice to normalise cut-offs with relative or absolute quantification methods. It is also recommended to run and analyse melt curves or gels of the products to determine products of any late amplifications.

Can other components be added to the Lyo-Ready Probe 1-Step Mix before lyophilisation?

We have not screened for any other components to add to the Lyo-Ready Probe 1-Step Mix apart from primers, probes and ROX. If you would like to add extra lyo-compatible components, you will need to test their activity in both the wet mix and the lyophilised material, as well as assess their stability over the time. PCR Biosystems is not responsible for any loss of performance caused by the added component.

Can ROX be added to the Lyo-Ready Probe 1-Step Mix and can it have a negative impact on the reaction?

Yes, ROX (6-carboxy-X-rhodamine), available separately, can be added to the kit and it would not interfere with the lyophilisation process.

ROX 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 template 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:

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

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

Can the activation time for the HS Taq DNA Polymerase be altered?

We recommend using a minimum of 2 minutes for activating the polymerase. Longer times of up to 15 minutes can also be used with no detrimental effects to the enzyme.

Do I need to use an RNase inhibitor in my RT reaction?

No, the 3200x UltraScript RTase provided in the kit already contains an RNase inhibitor to prevent any degradation and increase sensitivity.

General troubleshooting for low product or late Ct values

If you are observing unusually late Ct values, try diluting the template RNA. By doing this, you are diluting any inhibitors which may be present to a concentration where they do not inhibit the reaction. Additionally, try increasing the reverse transcription step to 55 °C and increasing the annealing/extension temperatures. This may help resolve difficulties caused by secondary structures present in the RNA template and/or primers.

In cases where reaction inhibition may be involved, try reducing the amount of template1 or add 0.4 – 4.4 mg/ml BSA to the reaction2.

For more specific problems contact technical@pcrbio.com with the following information:

  • Amplicon size
  • Reaction setup
  • Cycling conditions
  • Screen grabs of amplification traces and melting profile

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

2  Plante et al. The use of bovine serum albumin to improve the RT‐qPCR detection of foodborne viruses rinsed from vegetable surfaces. Applied Microbiology. 52:3 (2010) doi: https://doi.org/10.1111/j.1472-765X.2010.02989.x

How do I handle the Lyo-Ready Probe 1-Step Kit?

The Lyo-Ready Probe 1-Step Kit contains 2 components: 4x Lyo-Ready Probe Mix and 3200x UltraScript Reverse Transcriptase (RTase). 1.25 μL of 3200x UltraScript RTase should be added for each mL of 4x Lyo-Ready Probe Mix used. Due to the high concentration of the RTase, the volume supplied is very small, so we recommend spinning the tube every time before pipetting the desired volume of enzyme.

Whilst the 2 components of the kit are stable and have a longer shelf life (see expiry date of each component), the 4x Lyo-Ready Probe 1-Step Mix (obtained by adding the 3200x UltraScript RTase to the 4x Lyo-Ready Probe Mix) should be used immediately or can be stored for up to 3 days at 4 °C. We therefore recommend preparing only the amount of 4x Lyo-Ready Probe 1-Step Mix required and to return the remaining separate components to storage at between -30 °C and -15 °C.

Is it normal if the fluorescence of 4x Lyo-Ready Probe Mix differs from the one obtained with competitors’ products?

Different products could give a different plateau of fluorescence. However, this has no impact on quantification accuracy and Ct values will not differ among products.

What is the MgCl2 concentration in the 4x Lyo-Ready Probe Mix?

4x Lyo-Ready Probe Mix contains MgCl2 at a concentration of 18 mM. This means the final concentration in the reaction is 4.5 mM.

What priming method can I use?

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

What shall I do in case of problems with lyophilisation?

In the product manual we are providing a general lyophilisation protocol, tested for 2 mL glass vials filled with 500 μL of the Lyo-Ready Probe 1-Step Mix. We observed that lyophilisation is facilitated (i.e., the program is shorter, and cakes have a more uniform aspect with no signs of shrinkage) when the Lyo-Ready Probe 1-Step Mix is diluted to 1x or 2x (with oligonucleotides and water), so we recommend doing this dilution before starting the run.

Depending on the volume lyophilised, the material of the vials (i.e., glass vs plastic) and the lyophiliser used, the cycle may be optimised.

If the cakes are not properly formed after the cycle, we recommend repeating with a more conservative cycle, in which the primary drying is carried at -50 °C and time is extended to guarantee the correct cake formation. Secondary drying phase could also be extended if required.

What should be considered when doing multiplex amplification with the Lyo-Ready Probe 1-Step Kit?

The primers should be designed to ensure that they have similar annealing temperatures, are specific to the target, and do not form primer dimers. The length of the reverse transcription phase can be extended to 10 minutes to ensure there is enough template for priming and amplification.

When performing a multiplex, what is the recommended concentration of each primer?

We recommend using 400-1000 nM of each primer. There is a degree of flexibility around this recommended concentration, however the primer concentration should not be increased beyond this range as this may significantly affect the activity of the enzyme. Normally the concentration of the probe is half of that of the amplifying primers, but optimisation and validation are required for each primer set.

More Information

Lyo-Ready Probe 1-Step Kit is specially formulated for ultra-sensitive detection of viral RNA using probe-based RT-qPCR and is ideal for the development of lyophilised diagnostic tests.

Optimised for lyophilisation

Lyophilisation offers many advantages including ambient storage, extended shelf-life and increased versatility in sample volume. The kit has been optimised to offer the same high performance post-lyophilisation as our standard 1-step RT-qPCR kits.

Robust and reliable RT-qPCR

Lyo-Ready Probe 1-Step Kit includes a glycerol-free 4x qPCR mix containing hot start Taq polymerase, dNTPs, MgCl2 and a blend of excipients to ensure reliable lyophilisation, without loss of activity. Antibody-mediated hot start technology ensures specific amplification of virus-derived cDNA, with improved tolerance to the common PCR inhibitors found in clinical samples.

UltraScript Reverse Transcriptase is provided separately in a highly concentrated 3200x format. This robust and thermostable modified MMLV reverse transcriptase is engineered to enhance cDNA synthesis speed and yield with accurate transcript representation and enables fast and efficient reverse transcription up to 55 °C.

SARS-CoV-2 detection

The kit has been validated for SARS-CoV-2 detection in singleplex (see Figure 1) and multiplex (see Figure 2) and offers reliable performance with no loss of efficiency or sensitivity after lyophilisation.

Customisable diagnostic assay development

At PCR Biosystems we understand that every molecular diagnostic assay is different and the process to lyophilise them can be challenging. To help with initial verification work, we offer a Pre-Lyo Probe 1-Step Evaluation Kit for testing purposes.

ISO 13485

Lyo-Ready Probe 1-Step Kit has been manufactured under an ISO 13485 certified Quality Management System and is suitable for further manufacturing use as a component, reagent or reagent assembly for molecular biology diagnostics.

Applications

  • Development of lyophilised molecular diagnostic tests
  • Detection of extremely low copy number targets
  • Compatible with TaqMan®, Scorpions® and molecular beacon probes

Specifications

Lyo-Ready Probe 1-Step Kit

Component

2000 Reactions

10 000 Reactions

100 000 Reactions

4x Lyo-Ready Probe Mix

2 x 5 mL

1 x 50 mL

1 x 500 mL

3200x UltraScript RTase (with RNase inhibitor)

1 x 12.5 μL

1 x 62.5 μL

1 x 625 μL

Pre-Lyo Probe 1-Step Evaluation Kit

Component

600 Reactions

4x Lyo-Ready Probe Mix

3 x 1 mL

20x UltraScript RTase (with RNase inhibitor)

1 x 600 µL

Lyo-Ready Probe Mix

Component

600 Reactions

2000 Reactions

10 000 Reactions

100 000 Reactions

4x Lyo-Ready Probe Mix

3 x 1 mL

2 x 5 mL

1 x 50mL

1 x 500mL

Lyo-Ready Probe 1-Step Kit

Component

4x Lyo-Ready Probe Mix

3200x UltraScript RTase (with RNase inhibitor)



2000 Reactions

2 x 5 mL

1 x 12.5 μL

10 000 Reactions

1 x 50 mL

1 x 62.5 μL

100 000 Reactions

1 x 500 mL

1 x 625 μL

Pre-Lyo Probe 1-Step Evaluation Kit

Component

4x Lyo-Ready Probe Mix

20x UltraScript RTase (with RNase inhibitor)



600 Reactions

3 x 1 mL

1 x 600 µL

Lyo-Ready Probe Mix

Component

4x Lyo-Ready Probe Mix



600 Reactions

3 x 1 mL

2000 Reactions

2 x 5 mL

10 000 Reactions

1 x 50mL

100 000 Reactions

1 x 500mL

Reaction Volume

Storage

20μL

On arrival, products should be stored between -30 °C and -15 °C. If stored correctly the kit will retain full activity for 12 months.

Reaction Volume

20μL

Storage

On arrival, products should be stored between -30 °C and -15 °C. If stored correctly the kit will retain full activity for 12 months.

FAQs

Beyond what Ct value are results unreliable?

Ct values can vary between template concentrations, reaction optimisation, instruments and laboratories, so care must be taken when selecting a cut-off Ct value. Generally, Ct values over 35-40 would begin to be considered unreliable. However, late Cts could be observed for inefficient reactions using low template copy number. It is always good practice to normalise cut-offs with relative or absolute quantification methods. It is also recommended to run and analyse melt curves or gels of the products to determine products of any late amplifications.

Can other components be added to the Lyo-Ready Probe 1-Step Mix before lyophilisation?

We have not screened for any other components to add to the Lyo-Ready Probe 1-Step Mix apart from primers, probes and ROX. If you would like to add extra lyo-compatible components, you will need to test their activity in both the wet mix and the lyophilised material, as well as assess their stability over the time. PCR Biosystems is not responsible for any loss of performance caused by the added component.

Can ROX be added to the Lyo-Ready Probe 1-Step Mix and can it have a negative impact on the reaction?

Yes, ROX (6-carboxy-X-rhodamine), available separately, can be added to the kit and it would not interfere with the lyophilisation process.

ROX 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 template 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:

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

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

Can the activation time for the HS Taq DNA Polymerase be altered?

We recommend using a minimum of 2 minutes for activating the polymerase. Longer times of up to 15 minutes can also be used with no detrimental effects to the enzyme.

Do I need to use an RNase inhibitor in my RT reaction?

No, the 3200x UltraScript RTase provided in the kit already contains an RNase inhibitor to prevent any degradation and increase sensitivity.

General troubleshooting for low product or late Ct values

If you are observing unusually late Ct values, try diluting the template RNA. By doing this, you are diluting any inhibitors which may be present to a concentration where they do not inhibit the reaction. Additionally, try increasing the reverse transcription step to 55 °C and increasing the annealing/extension temperatures. This may help resolve difficulties caused by secondary structures present in the RNA template and/or primers.

In cases where reaction inhibition may be involved, try reducing the amount of template1 or add 0.4 – 4.4 mg/ml BSA to the reaction2.

For more specific problems contact technical@pcrbio.com with the following information:

  • Amplicon size
  • Reaction setup
  • Cycling conditions
  • Screen grabs of amplification traces and melting profile

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

2  Plante et al. The use of bovine serum albumin to improve the RT‐qPCR detection of foodborne viruses rinsed from vegetable surfaces. Applied Microbiology. 52:3 (2010) doi: https://doi.org/10.1111/j.1472-765X.2010.02989.x

How do I handle the Lyo-Ready Probe 1-Step Kit?

The Lyo-Ready Probe 1-Step Kit contains 2 components: 4x Lyo-Ready Probe Mix and 3200x UltraScript Reverse Transcriptase (RTase). 1.25 μL of 3200x UltraScript RTase should be added for each mL of 4x Lyo-Ready Probe Mix used. Due to the high concentration of the RTase, the volume supplied is very small, so we recommend spinning the tube every time before pipetting the desired volume of enzyme.

Whilst the 2 components of the kit are stable and have a longer shelf life (see expiry date of each component), the 4x Lyo-Ready Probe 1-Step Mix (obtained by adding the 3200x UltraScript RTase to the 4x Lyo-Ready Probe Mix) should be used immediately or can be stored for up to 3 days at 4 °C. We therefore recommend preparing only the amount of 4x Lyo-Ready Probe 1-Step Mix required and to return the remaining separate components to storage at between -30 °C and -15 °C.

Is it normal if the fluorescence of 4x Lyo-Ready Probe Mix differs from the one obtained with competitors’ products?

Different products could give a different plateau of fluorescence. However, this has no impact on quantification accuracy and Ct values will not differ among products.

What is the MgCl2 concentration in the 4x Lyo-Ready Probe Mix?

4x Lyo-Ready Probe Mix contains MgCl2 at a concentration of 18 mM. This means the final concentration in the reaction is 4.5 mM.

What priming method can I use?

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

What shall I do in case of problems with lyophilisation?

In the product manual we are providing a general lyophilisation protocol, tested for 2 mL glass vials filled with 500 μL of the Lyo-Ready Probe 1-Step Mix. We observed that lyophilisation is facilitated (i.e., the program is shorter, and cakes have a more uniform aspect with no signs of shrinkage) when the Lyo-Ready Probe 1-Step Mix is diluted to 1x or 2x (with oligonucleotides and water), so we recommend doing this dilution before starting the run.

Depending on the volume lyophilised, the material of the vials (i.e., glass vs plastic) and the lyophiliser used, the cycle may be optimised.

If the cakes are not properly formed after the cycle, we recommend repeating with a more conservative cycle, in which the primary drying is carried at -50 °C and time is extended to guarantee the correct cake formation. Secondary drying phase could also be extended if required.

What should be considered when doing multiplex amplification with the Lyo-Ready Probe 1-Step Kit?

The primers should be designed to ensure that they have similar annealing temperatures, are specific to the target, and do not form primer dimers. The length of the reverse transcription phase can be extended to 10 minutes to ensure there is enough template for priming and amplification.

When performing a multiplex, what is the recommended concentration of each primer?

We recommend using 400-1000 nM of each primer. There is a degree of flexibility around this recommended concentration, however the primer concentration should not be increased beyond this range as this may significantly affect the activity of the enzyme. Normally the concentration of the probe is half of that of the amplifying primers, but optimisation and validation are required for each primer set.