Multiplex RT-qPCR inhibition and target dropout
A research laboratory is validating a two-target RT-qPCR assay for an enveloped RNA virus in nasopharyngeal swabs. Extraction is automated in 96-well plates with an exogenous RNase P internal control (IC) added before lysis. The run includes a no-template control (NTC), a positive control (PC), and 24 samples. PC: target A Ct 25.1, target B Ct 26.0, IC Ct 27.0. NTC: target A Ct 38.5 in one well, target B undetermined, IC undetermined. S17: target A Ct 35.2, target B Ct 36.9, IC Ct 37.8; the swab was viscous and eluted in 60 microlitres instead of 100 microlitres. S18: target A Ct 21.5, target B undetermined, IC Ct 29.1; this sample was collected 9 days after symptom onset. S19: target A Ct 33.8, target B Ct 34.1, IC Ct 29.5. The assay's limit of detection is Ct 37.0 for target A and Ct 36.5 for target B. The laboratory must decide what to report and what to repeat.
1. Classify S17, S18, S19 and the NTC. Identify inhibition, contamination, low viral load and target dropout.
Read the worked answer
NTC: target A at Ct 38.5 is a late signal beyond the stated LOD (Ct 37.0) and is therefore not a valid positive, but it is a contamination or primer-dimer warning. The run should not be accepted until the NTC is clean on repeat.
S17: IC at Ct 37.8 is more than 10 Ct later than the PC IC at 27.0. This is severe inhibition; the target A and B signals are near or beyond the LOD and cannot be interpreted. S17 is invalid, not negative.
S18: target A at Ct 21.5 is a strong positive; IC at 29.1 is acceptable (within about 2 Ct of PC). Target B is undetermined. This is target B dropout, which may reflect low target B titre, a primer/probe site variant, or a target B-negative sample. It is not proof that target B is absent.
S19: target A Ct 33.8 and target B Ct 34.1 are both below the LOD cut-offs, and IC at 29.5 is acceptable. This is a low-positive for both targets, with reportable detection but low viral load.
2. For S17, decide whether to report target A positive. Give corrective actions and acceptance criteria.
Read the worked answer
Do not report S17 as positive or negative from this run. The IC failure shows that the extraction/amplification conditions are not valid; a target Ct of 35.2 under strong inhibition could represent a higher true viral load, or could be artefact. Reporting it as a low positive would be unsafe, and reporting it as negative would also be wrong.
Corrective actions: re-extract from a new aliquot of the original sample; dilute the nucleic acid 1:5 and 1:10 in buffer to relieve inhibition; spike the IC into the diluted extract to confirm recovery; repeat target A and B in duplicate. If inhibition persists, use an alternative extraction chemistry or a column clean-up. Acceptance criteria: IC Ct within 2-3 Ct of the PC IC, target Ct below the LOD, duplicate wells concordant, and NTC clean. Only then report. If the repeat is target A positive with a valid IC, report positive; if target A remains undetected with a valid IC, report not detected.
3. For S18, design a follow-up to distinguish a target B sequence variant from low target B titre.
Read the worked answer
First repeat S18 in duplicate with the same assay and with a second target B assay that uses primers/probes against a different conserved region. Include a positive control for target B and a no-template control. If the alternate target B assay is positive, the original result is likely a primer/probe-site variant or a low-titre effect, and both targets should be reported as detected after confirmatory testing.
If both target B assays are negative while target A remains strongly positive, amplify the target B region by RT-PCR and sequence it, comparing with a reference. A mutation at the primer or probe binding site supports a variant; a negative sequence with adequate RNA quality supports genuinely low or absent target B. Also check sample timing: 9 days after symptom onset can favour target A persistence and lower target B. Report target A as detected; do not report target B as definitively negative without the alternate assay and sequence evidence.