Detection of RNA virus infections, e.g. of Flaviviridae (Flavivirus, Pestivirus und Hepacivirus).

With the new HotScriptase RT 2X Cell master mix from Genaxxon RNA viruses (e.g. Zika virus, dengue virus, yellow fever virus) can be detected directly from body fluids as blood, salvia or semen even without isolating or purifying RNA prior to RT-PCR. The normally difficult detection of RNA from Flaviriridae will be carried out easily and fast using the new enzyme HotScriptase RT without any isothermal reverse transcription step.

By avoiding expensive, time-consuming cell lysis or RNA purification, Genaxxon HotScriptase RT Cell Mastermix is ideal for high-throughput applications, RNAi knockdown, gene expression experiments or realtime PCR.

The HotScriptase RT Cell 2-time mix enables you to quantify and detect RNA directly from cell suspension in PCR, without a need of separate lysis or purification steps. Simply add cells or cell suspension (100 to <10,000 cells) directly to our HotScriptase RT Cell master mix, place the reaction mixture in your PCR instrument, and start the run.

Human pathogenic Flaviviruses are, e.g. the yellow fever virus, dengue virus (dengue fever), the FSME virus or the das Zika virus (ZIKV).

More information regarding Flaviviridae or the Zika virus can be found on Wikipedia: Flaviviridae - Zika virus.

Excerpt vom the HotScriptase RT Cell master mix Protocol

  1. Prepare the PCR reaction mixture according to Table 1.
    The master mix typically contains all of the components needed for RT-PCR except primers and template.
  2. Program the thermal cycler according to the manufacturer’s instructions.
    A typical RT-PCR cycling program is outlined in Table 2. For maximum yield and specificity, temperatures and cycling times should be optimized for each new target or primer pair.
  3. Place PCR tubes in the thermal cycler and start program

Table 1: Recommendations for PCR / Reaction Setup (50µL PCR reaction)

Components

Volume

Final concentration

Primer forward (10µM)

Primer reverse (10µM)

HotScriptase RT Cell Mastermix

Template/Sample extract*

Nuclease-free water

1µL

1µL


12.5µL

2µL - 10.5µL

up to 25µL total reaction volume

0.5µM (0.05-1µM)

0.5µM (0.05-1µM)


1X

10 to <10000 cells

 

 *Recommended final template concentration is between 5ng/µL to 500ng/µL.


Table 2:
Typical “Zero-Step” RT-PCR protocol (an isothermal reverse transcription step is not needed)

Step

Temperature

Time

Initial denaturation

Denaturation

Annealing/Extension*

Hold

95°C

95°C

>57°C – 75°C

<10°C

2 min.

15 sec.

min. 60 sec. (25 – 40 cycles)

hold

 NOTE:      HotScriptase RT does not have an activity optimum at 68°C! For this reason you can use much higher temperatures for elongation if needed (if the primer sequence does allow higher temperatures)

NOTE:      A “Two-Step” as well as “Three-Step” PCR protocol can be used.
If primer needs  annealing temperatures below 68°C a three step protocol is recommended.

NOTE:      A new RT-PCR is ideally established by running a temperature gradient in order to find the best annealing/extension temperature for each new primer pair.

NOTE:      Optimal PCR protocol times and temperatures may vary depending on the used cycler, the nature of template and the amplicon length.

RT-PCR HotScriptase

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