Category: Application notes

Speed up your research with DCQ

Posted on by DireCtQuant Posted in Application notes, Publications

Just 3 min between your sample and the success of your qPCR, RT-qPCR and Western blot.

DireCtQuant 100ST and DireCtQuant 100W are conceptually new DNA/RNA/protein solubilisation reagents. Those reagents provide instant and complete solubilisation of the cells in the sample and permit effective extraction of proteins, genomic, plasmid, plastid, mitochondrial and viral DNA as well as messenger, structural (such as ribosomal), transport, microRNA, non-coding and viral RNA.
DireCtQuant 100ST / DireCtQuant 100W can effectively solubilise any type of sample like animal tissue, cell culture, plans and fruits, yeast, prokaryotes.

In just 3 minutes the sample is ready for analysis!

The instant disruption of the structure of the cell and the biopolymers by DireCtQuant 100ST / DireCtQuant 100W effectively blocks the endogenous enzymatic activity of the sample, preventing any degradation of macromolecules and providing exceptional long-term storage conditions for the majority of the samples referred as Stop-The-Time technology.

Fast detection of SARS-CoV-2 using DireCtQuant workflow.

Posted on by DireCtQuant Posted in Application notes, PublicationsTagged , , , , , ,

The on-going pandemic spread of Severe Acute Respiratory Syndrome Coronavirus -2 (SARS-CoV-2) requires fast and easy to implement detection methods. The virus, being the causative agent of COVID-19 disease is most frequently detected by RT-qPCR assay. This assay combines a very high sensitivity and specificity characteristic for the nucleic acid detection assays. During the assay single stranded RNA genome is converted to DNA which is detected by qPCR (also known as Real-Time PCR). Currently a lot of suppliers are offering validated assays for the detection of SARS-CoV-2 genome by RT-qPCR. In spite of the wide adoption of this technology, at present the limiting step in achieving fast and easy to implement SARS-CoV-2 detection is the sample preparation step. The detection step by RT-qPCR can take somewhere between 45 minutes and 2 hours and 96 to 384 samples in parallel can be analysed per single qPCR instrument. In contrast, the sample preparation at least doubles this time taking between 60 minutes and 4 hours to prepare this number of samples for analysis. Usually the samples are prepared by RNA extraction using column based or magnetic beads nucleic acid absorption. This is not only time and resource consuming, but also leads to the loss of nucleic acids thus reducing the sensitivity. Both, time and costs are of special interest in pandemic environment where important shortage of materials is to be expected.


Here we describe an extremely simple 5 minutes sample preparation step and its successful application for the detection of SARS-CoV-2 (Fig.1).


The samples were prepared as described in Swab DNA/ RNA isolation application note. Shortly, the sample was collected with viscose swab and extracted by submerging immediately the sampling end in 200 μl of DireCtQuant 100W. The samples were then heated at 90ºC with agitation. 1μl of the sample was subsequently used to prepare 20 μl one-step RT-qPCR reaction using the recommended protocol (1) The detection was performed by multiplex detection using CFX96Touch instrument (Bio-Rad).


The SARS-CoV-2 detection was performed by amplification of envelope (E) gene and RNA-dependent RNA Polymerase (RdRp) gene as recommended by World Health Organization (1). Internal control of human RNA sequence was used as a positive control for sample validity, this proves correct sample preparation and processing and avoids false negative results. The probes used for detection ware labelled with FAM (E gene), HEX (RdRp gene) and Cy5 (human RNA sequence).


Figure 1. Detection of SARS-CoV-2 using fast DireCtQuant workflow. A swab sample was prepared according to Swab DNA/ RNA isolation application note . The detection was performed using 1 μl of sample in 20 μl One-Step RT-qPCR reaction. Solid lines represent SARS-CoV-2 positive sample (POS), dotted lines represent SARS-CoV-2 negative sample (NEG). Note the confident detection of the internal control in both NEG and POS samples.


In conclusions,  using the DireCtQuant sample preparation the total detection time is reduced from 3 hours (1h 30min sample preparation plus 1h 30min RT-qPCR) to 1 hour 40 minutes or 45% reduction in the required time (Fig.2).


Figure 2. Time comparison between RNA extraction method and DireCtQuant method for detection of SARS-CoV-2


The simplicity and the heat extraction step reduce significantly the biohazard risk associated with testing infectious material. A huge decrease of the required work, consumables and cost are achieved. The cost per reaction (approximately 2 euros) is several times less than the majority of the column or magnetic beads-based approaches. The prepared sample can be stored for long periods of time without degradation for the confirmatory and archival purposes. This is achieved by the unique DireCtQuant formulation including Stop-the-TimeTM technology. The protocol could be extended to different types of starting material such as saliva samples, surface sample, etc. Another advantage of the method presented here is the increase in the sensitivity compared to RNA extraction method as here no extraction is performed and all the RNA present in the sample can be used for detection without any loss.



References:

  1. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Corman et al. Euro Surveill. 2020 Jan;25(3):2000045. doi: 10.2807/1560-7917.ES.2020.25.3.2000045.

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One reagent, one sample – many applications!

Posted on by DireCtQuant Posted in Application notes, Publications

From the same sample you can run PCR, RT-qPCR, Western Blot and measure DNA quantity.

The main advantage of using DireCtQuant 100ST / DireCtQuant 100W is that DNA, RNA and proteins can be analysed from a single sample without the need of prior separation and purification. Those reagents provide instant and complete solubilisation of the cells in the sample and permits effective extraction of:

  • Proteins;
  • Genomic DNA;
  • Plasmid DNA;
  • Plastid DNA;
  • mitochondrial and viral DNA;
  • messenger RNA;
  • structural (such as ribosomal) RNA;
  • transport RNA;
  • microRNA;
  • non-coding and viral RNA.

The DNA in DireCtQuant 100ST / DireCtQuant 100W solubilised sample can be directly analysed by:

  • PCR
  • quantitative (Real Time) PCR (qPCR)
  • quantified in solution using DireCtQuant DNAQ (Ref #DCQDNAQ)

The RNA in the sample can be directly analysed by reverse transcription followed by PCR or quantitative PCR (RT-qPCR)

The proteins can be directly analysed by Western blot.

DNA and RNA extraction from swabs

Posted on by DireCtQuant Posted in Application notes, PublicationsTagged , , , , ,

This protocol is designed and validated for quantitative extraction of bacteria, viruses (DNA and RNA) and epithelial cells collected with swabs in less than 5 minutes using a single tube. If the aim of the study is bacterial or virus DNA/RNA detection/quantification genomic DNA from epithelial cells within the sample can be used as positive control for correct sample collection and extraction.


Additional equipment:

  • Snap tool or scissors
  • Thermoblock (with agitation is preferred) or cup of boiling water
  • Centrifuge reaching 10.000g. (If the centrifuge is reaching only 3.000g the centrifugation time should be increased as indicated below).

The protocol is identical for DireCtQuant 100ST (#DCQ100ST) and DireCtQuant 100W (#DCQ100W).


For illustration purposes #DCQ100ST is used.


  1. Pipet 200 μl of #DCQ100ST or #DCQ100W in a 1.5 ml tube.
  2. Snip the swab applicator as high as possible, permitting the tube to be closed.
  3. Close the tube. Make sure that the swab is completely wetted by the reagent.
  4. Incubate 3 minutes at 90°C. If possible, apply agitation during the incubation.
  5. Leave the tube 1 minute on the bench to cool down.
  6. With a sterile tool reverse the swab upside down.
  7. Close the tube and centrifuge at 10.000g for 30s in order to collect all the liquid absorbed in the swab. For Minicentrifuge reaching only 3.000g increase time to 3 minutes.
  8. Discard the swab.
  9. The samples can be stored and transported at room temperature after the extraction. For more information about the lon-term storage conditions check DireCtQuant 100ST (#DCQ100ST) or DireCtQuant 100W (#DCQ100W) protocols respectively.
  10. Use 1.0 μl of the sample per 20.0 μl PCR reaction. The extracted sample should be enough for around 200 detections. For more details about the PCR reaction conditions check DireCtQuant 100ST (#DCQ100ST) or DireCtQuant 100W (#DCQ100W) protocols respectively.

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Straightforward and Precise

Posted on by DireCtQuant Posted in Application notes, Publications

Precise measurement of RNA using DireCtQuant100ST

Introduction:

The analysis of biological samples is usually performed by multistep procedures consisting of sample extraction, single or multistep purification and subsequent analysis. All this steps are associated with inevitable sample loss and introduction of technical bias. As the number of steps in given protocol grows, more technical bias is introduced and less accurate and reproducible results are obtained. Moreover multistep procedures are associated with higher cost and longer time to complete.

Objective:

To estimate the reproducibility of the RNA measurement in 4 samples prepared with DireCtQuant100ST solubilisation reagent.
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