PROTOCOL
· Composition of duplex RTqPCR mastermixes (final volume of RTqPCR reaction of 20 µl)
Beta RTqPCR
2x One Step: 10 µl
Forward primer 10µM: 0.8 µl (400 nm)
Reverse primer 10µM: 0.8 µl (400 nm)
Probe_Beta 10µM: 0.4 µl (200 nm)
Probe_NoBeta 10µM: 0.4 µl (200 nm)
Takara Ex Taq: 0.4 µl
Prime Script Enzyme: 0.4 µl
H2O PCR: 1.8 µl
Extracted RNA: 5 µl
Gamma RTqPCR
2x One Step: 10 µl
Forward primer 10µM: 0.8 µl (400 nm)
Reverse primer 10µM: 0.8 µl (400 nm)
Probe_Gamma 10µM: 0.4 µl (200 nm)
Probe_NoGamma1 10µM: 0.2 µl (100 nm)
Probe_NoGamma2 10µM: 0.2 µl (100 nm)
Takara Ex Taq: 0.4 µl
Prime Script Enzyme: 0.4 µl
H2O PCR: 1.8 µl
Extracted RNA: 5 µl
Delta RTqPCR
2x One Step: 10 µl
Forward primer 10µM: 0.8 µl (400 nm)
Reverse primer 10µM: 0.8 µl (400 nm)
Probe_Delta 10µM: 0.4 µl (200 nm)
Probe_NoDelta 10µM: 0.4 µl (200 nm)
Takara Ex Taq: 0.4 µl
Prime Script Enzyme: 0.4 µl
H2O PCR: 1.8 µl
Extracted RNA: 5 µl
· Controls to be included in each RTqPCR assay:
- 2 wells with 5 µl of nuclease-free water (RTqPCR negative controls)
- 2 wells with 5 µl of RNA extraction negative control (RNA extraction negative controls)
- 1 well with 5 µl of each corresponding synthetic SARS-CoV-2 RNA controls at 1000 copies/µL (RTqPCR positive controls)
· For each sample, 2 wells of undiluted RNA and 2 wells of 1/10 dilution are analyzed.
· For quantification of each target, standard curves are constructed, using a minimum of 5 10-fold dilutions and 3 wells for each dilution, using 2 synthetic SARS-CoV-2 RNA controls as reference materials (one corresponding to the variant containing the specific mutation and another corresponding to a variant without the specific mutation).
· Thermocycler conditions (common for all duplex RTqPCR assays)
- 10 min at 50ºC (x1)
- 3 min at 95ºC (x1)
- 3 sec at 95ºC and 30 sec at 60ºC (x45)
INTERPRETATION OF RESULTS
a) RTqPCR controls (see Tables 2, 3, 4)
b) Samples
· The calculation of each specific SARS-CoV-2 target concentration in genome copies per reaction (gc/rxn) in each well is performed using the standard curve.
· Occurrence of inhibition and calculation of mean viral titers are estimated by comparing concentrations obtained from duplicate wells tested for the two RNA dilutions (undiluted RNA and 1/10 dilution), as described in Carcereny et al., 2021 (1). Mean concentration of samples and standard error are calculated using as many data as possible, taking into consideration the following steps:
a. Calculate mean concentration as gc/rxn for each RNA dilution:
ai. Data from RNA dilutions containing “No Cq” or Cq≥40 in both wells are not be used for calculation.
aii. When in the analysis of the 2 wells of any RNA dilution, one of the wells has a Cq value <40 and the other has "No Cq" or Cq≥40, this last well is assigned a concentration equal to the theoretical limit of detection (LoD) of 1 gc/rxn.
b. Calculate mean concentration as gc/rxn for each sample:
bi. When the difference between the concentration estimated from undiluted RNA and 1/10 dilution is < 0.5 log10, mean concentration of sample is calculated using data from the 4 wells.
bii. When the difference between the concentration estimated from undiluted RNA and 1/10 dilution is ≥ 0.5 log10, inhibition is considered and mean concentration of sample is calculated using data from the 1/10 dilution.
· The proportion of SARS-CoV-2 genomes corresponding to variants containing the specific signature mutation is calculated using the formula:
Beta Duplex RTqPCR:
% = gc/rxn (Probe_Beta) / [gc/rxn (Probe_Beta) + cg/rxn (Probe_NoBeta)] x 100
Gamma Duplex RTqPCR:
% = gc/rxn (Probe_Gamma) / [gc/rxn (Probe_Gamma) + cg/rxn (Probes_NoGamma (1+2)] x 100
Delta Duplex RTqPCR:
% = gc/rxn (Probe_Delta) / [gc/rxn (Probe_Delta) + cg/rxn (Probe_NoDelta)] x 100
· Cases in which any of the concentrations fall below the limit of quantification (LoQ), the percentage is calculated using the corresponding LoQ. When both concentrations are <LoQ, percentage cannot be estimated.