Description:

MaxDirect Plus RT-PCR Master Mix is capable to detect SARS-CoV2 directly from samples. The RNA Extraction Kit is NOT needed anymore. MaxDirect Plus RT-PCRs works directly from RNA templates.

Storage:

Componentes
MaxDirect Plus Plus RT-PCR 2x Probe Master Mix	-25 °C and -15 °C
10x S-CoV2 Duplex Assay	-25 °C and -15 °C
S-CoV2 positive control	-25 °C and -15 °C
Direct Booster solution	4 and 12 °C

 

Shipping temperature:

Although this Master Mix is stable at +20°C for up to 9 days (if not opened).  We strongly recommend using 2kg. of ice packs to keep the Master Mix in optimal conditions (no heavy dry ice needed).

MaxDirect Plus RT-PCR Probe 2x Master Mix will not lose activity within 10 freeze-thaw-cycles. Additional freeze-thaw cycles could affect the Master Mix’s performance. Keep the kit out from lighting environments.

 

Kit Components:

Component	Content	Volume
MaxDirect Plus Plus RT-PCR 2x Probe Master Mix	DNA Polimerase, dNTP, buffer, PCR additives	1,5 ml
10x S-CoV2 Duplex Assay	Primer/Probe Mix for SARS-CoV-2 N-gen and RNaseP	300 µl
S-CoV2 positive control	DNA Plasmid with target region for SARS-CoV-2 and target region for RNaseP	70 µl
Direct Booster solution	Additives that improve the sensitivity of PCR Direct	100 µl

 

PCR Set Up For ALL Sample Collection Methods:

Step	Temperature	Duration	Cycles
Reverse Transcriptase	70 °C	10 min.	1
Denaturation	95 °C	5 seg.	45
Annealing	65 °C	20 seg.*

• Fluorescence measurement in FAM and Cy5-channel

Thaw kit components on ice, then mix and centrifuge all components.

For the preparation of the PCR Master Mix, the number of total reactions, including positive and negative controls, is calculated. It is recommended to prepare an additional reaction to compensate for the loss of pipetting. Mix and centrifuge the components.

 

1st. OPTION (DRY SWAB based sample collection)

MaxDirect Plus RT-PCR Amplification                    Protocol

Put the dry swab stick thoroughly in 350 µl RNase-free water. Remove debris from the swab by short centrifugation. Then heat the samples for 5 min at 95 °C. The assay detects SARS-CoV2 N1-gene (FAM- channel) and human RNaseP-gene (Cy5-channel).

 

Assay setup

Reagente	Volumen
MaxDirect Plus Plus RT-PCR 2x Master Mix	15 µl
10x Duplex Assay	3 µl
PCR-Clean wáter	5.1 µl
Direct Booster solution	0.9 µl
Dry swab RNA sample/positive control/negative control	6 µl
Total	30 µl

The positive control can be used directly or diluted 1 to 10, which will give a solid signal.

 

2nd.OPTION(GARGLE based sample collection)MaxDirect Plus RT-PCR amplification protocol.

Gargle with 5 ml of Clean Water for PCR or 0.9% saline solution for at least 20 seconds and then spit into a PCR tube.

Gargle specimens can be inactivated by heating the tube for 3 min at 95 ° C, the heat inactivated gargle solution can be stored for a maximum of 3 hours at 0-12 ° C before use. But application is recommended immediately after heat inactivation. Take proper precautions against aerosol formation during the gargling process.

Note.- Make sure the patient does not eat, drink, chew gum, or smoke for at least 30 minutes before collecting the sample.

 

Assay setup for Gargle based collection samples:

Content	Volumen
MaxDirect Plus Plus RT-PCR 2x Master Mix	15 µl
10X Duplex Assay	3 µl
PCR-Clean Water (Agua de PCR)	5.1 µl
Direct Booster Solution	0.9 µl
Gargling solution/positive control/negative control	6 µl
Total	30 µl

 

3rd.OPTION (RNA Extraction based method)

MaxDirect Plus RT-PCR Master Mix Protocol for extracted/purified RNA samples

Follow the instruction of your RNA extraction kit to process the samples.

The assay detects SARS-CoV2 N1-gene (FAM-channel) and human RNaseP-gene (Cy5-channel).

 

    Assay setup 

Reagent	Volume
MaxDirect Plus Plus RT-PCR 2x Master Mix	10 µl
10X Duplex Assay	2 µl
PCR-Clean water	3 µl
RNA extracted sample	5 µl
Total	20 µl

 

Interpretation of data

FAM – N1	Cy5 – RNaseP	Interpretation
+	–	Unclear, repeat experiment
+	+	Valid – SARS-CoV2 positive
–	+	Valid – SARS-CoV2 negative
–	–	Invalid, repeat experiment

 

General considerations

• Use sterile, DNA/DNase/RNase-clean consumables to avoid contaminations.
• Clean all surfaces and equipment in direct environment before starting the experiment to lower the risk of nucleic acid-, DNase/RNase- and protease contaminations.
• Do not use this product after its expiry date.
• Include positive and negative controls in your experiment to validate your results.