Real-Time Quantitative Reverse Transcription PCR for Diagnosing Viral Infections

Overview

This video demonstrates a quantitative method for detecting viral infection using real-time reverse transcription PCR. The reverse transcription step converts the RNA to cDNA, which is then amplified via PCR cycles. The presence of viral RNA in the sample is confirmed by analyzing the amplification and dissociation curves of DNA obtained from the PCR.

Protocol

1. Quantification of RNA Using a Micro-volume Spectrophotometer

1. Ensure the settings of the spectrophotometer are set to RNA.
2. Use 1-2 µL of molecular-grade water to initialize the machine and set a baseline.
3. Use 1-2 µL of each test RNA sample to assess the RNA quantity.
4. Save the readings and document.
5. Adjust the RNA to 1 µg/µL, if required.
   NOTE: RNA must be kept on ice (or in a cool block) at all times. If RNA is obtained using a column, or bead-based method the RNA is usually less than 1 µg/µL. In this situation use the RNA neat.

2. Preparation of RNA Dilution Series to Determine End Point Sensitivity

1. Make a 10-fold serial dilution of the RNA.
   1. Label tubes with the dilution series (e.g., 10^-1, 10^-2, etc.) and the RNA details.
   2. Add 45 µL of molecular-grade water to each tube.
   3. Add 5 µL of the RNA (previously diluted to 1 µg/µL) and mix well.
   4. Dispose of the pipette tip in the appropriate disinfectant and replace it with a fresh tip.
   5. Take 5 µL from the 10^-1 and add to 10^-2 tube and mix well.
   6. Repeat 2.1.3-2.1.5 with the remaining dilutions.
      NOTE: RNA must be kept on ice (or in a cool block) at all times.

3. Preparation of Real-time RT-PCR Reactions

1. Using a spreadsheet, plan the plate layout according to the number of test samples and control samples, for both the lyssavirus and ß-actin assays.
   NOTE: If 4 samples are to be tested in duplicate with positive and negative control, this equates to 10 reactions for both assays.
2. In a 'clean room' or area separate from the RNA template, wipe down the surfaces with an appropriate disinfectant prior to use or prepare a PCR workstation (if using). To prepare the workstation, wipe the cabinet surface with an appropriate disinfectant and place the items required into the workstation and close the doors. Switch on the UV light for 10 minutes.
3. Remove regents and primers from the freezer and thaw (reagents listed in Table 1 and primers in Table 2).
   NOTE: The enzyme mix is stored in glycerol so does not require thawing and must be kept on ice (or in a cool block) at all times. All other reagents can be thawed at room temperature.
4. Once thawed, mix the reagents and centrifuge briefly to collect the liquid.
   NOTE: Do not vortex the enzyme mix, just centrifuge briefly.
5. Prepare separate master mixes for lyssavirus and ß-actin. For each reaction, add 7.55 µL of molecular grade water, 10 µL of 2x Universal SYBR green reaction mix, 0.6 µL of forward primer, 0.6 µL of reverse primer, 0.25 µL of iTaq RT enzyme mix.
   1. Use a spreadsheet to calculate correct volumes to avoid errors in manual calculating. Ensure enough master-mix is prepared to compensate for pipetting errors. Therefore if 10 reactions are required (see NOTE in 3.1), prepare 12 reactions
   2. Prepare the master-mix on ice (or in a cool block) and remain on ice until placed into the real-time machine.
6. Mix the prepared master-mixes, centrifuge briefly, and dispense 19 µL into the relevant wells of strip tubes or a 96-well plate compatible with the real-time machine in use.
   NOTE: Minimize the production of bubbles in the wells whilst pipetting.
7. In a separate room, or in a UV cabinet prepared as described in 3.2, carefully add 1 µL of the RNA previously adjusted to 1 µg/µL (see step 1.5) below the surface of the appropriate master-mix well and mix gently. Discard the pipette tip into disinfectant directly after use (under the surface).
   1. Add the controls after the test samples, with the positive control added next and the no template control (NTC – molecular grade water) added last. The amount of RNA used can be altered depending on the sample type, and RNA extraction used. The amount used must be validated to ensure the reaction is optimized.
8. Seal plate using strip lids or sealer taking care to ensure all the lids are firmly closed and labeled sufficiently to orientate the samples. Label the edge of the plate/strip tubes.
9. Spin down the samples using a centrifuge to collect all the liquid at the bottom of the wells.
10. Transfer the plate to the real-time PCR machine, open the door, and place it in the holder ensuring the correct location/orientation of the samples according to the plate layout.
    NOTE: If tube strips are used, ensure the holder is in place.
11. Open up the real-time PCR machine program and choose the option for SYBR real-time experiment with dissociation curve. Program the real-time PCR machine using the thermal cycling conditions specified in Table 3, including the data collection points.
12. Select SYBR as the fluorescent dye and select unknown as the sample type and insert a name into the correct sample name box.
    NOTE: Differentiate between the replicates and also between the lyssavirus and ß-actin wells.
13. Choose a file location to save the experimental data, ensure the lamp will be switched off at the end of the run, then start the run.
    NOTE: As the first step is an RT stage, no data is collected during this time, therefore, if the lamp requires a warm-up period this can occur during the RT stage. The real-time machine and software will display the amplification curves in real-time, while the melting curve will be generated at the end of the cycle.

Representative Results

Table 1: Pan-lyssavirus real-time RT-PCR master mix reagents.

Reagent	μL/Reaction
Molecular-grade water	7.55
2x Universal RT PCR reaction mix	10
Primer Forward [20 μM]	0.6
Primer Reverse [20 μM]	0.6
RT enzyme mix	0.25
Total per reaction	19

Table 2: Pan-lyssavirus real-time RT-PCR primer details.

Assay	Primer name	Primer role	Sequence 5'-3'	Position1
Lyssavirus	JW12	RT-PCR	ATG TAA CAC CYC TAC AAT G	53-73
	N165	PCR	GCA GGG TAY TTR TAC TCA TA	165-146
ß-actin	ß-actin intronic	PCR	CGA TGA AGA TCA AGA TCA TTG	1051-1072
	ß-actin reverse	RT-PCR	AAG CAT TTG CGG TGG AC	1204-1188
Primer positions are given in relation to Pasteur virus sequence (M13215) and mouse ß-actin gene sequence (NM_007393)

Table 3: Pan-lyssavirus real-time RT-PCR cycling conditions.

Stage	Cycles	Temperature	Time	Data Collection
Reverse Transcription	1	50 °C	10 min
RT inactivation/initial denaturation	1	95 °C	5 min
Amplification	40	95 °C	10 s
		60 °C	30 s	endpoint
Dissociation curve analysis	1	9 °C	1 min
		55 °C	1 min
		55 - 95 °C	10 s	all points

Materials

Name	Company	Catalog Number	Comments
Art Barrier pipette tips (various sizes)	Thermofisher	various
Centrifuge	Beckman	Allegra 21R	Rotor capable of holding 96 well plates required. Step 3.8.
Centrifuge (micro)	Sigma
Finnpipettes (to dispense 0.5-1000 µL)	Thermofisher	various
iTaq Universal SYBR Green One-Step RT-PCR kit	Bio-Rad	172-5150	Equivalent kits can be used if validated
MX3000P or MX3005P real-time PCR system	Stratagene	N/A	Equivalent machines can be used if validated
MicroAmp reaction plate base	Any suitable		Used to hold tube strip and plates securely.
Optically clear flat clear strips (8)	ABgene	AB-0866
Perfect fit frame (if using tube strips)	Stratagene	N/A	Specific to machine
Primers: for primer details see Table 2.			Ordered at 0.05 µmole scale HPLC purified.
Thermo-Fast 96-well plates, non-skirted	ABgene	AB-600
Thermo-Fast strips (8) Thermo-tubes	ABgene	AB-0452
Vortex machine / Whirlimixer	Fisons Scientific equipment	SGP-202-010J
Unless stated, alternative equipment can be used