Standard PCR vs qPCR

Standard PCR vs qPCR

insilicoPCR decides per assay whether to apply standard PCR or qPCR/probe reporting, based on the primers defined in the primer FASTA file. Both kinds of assays can be tested in the same run (since version 0.7.0).

Standard PCR assays

An assay with only forward and reverse primers:

assayName-F
assayName-R

A positive consolidated report row is generated when the forward and reverse primers produce a valid amplicon (see Amplicon Calling).

qPCR/probe assays

An assay with forward primer, reverse primer, and probe entries:

assayName-F
assayName-R
assayName-P

A positive consolidated report row is generated only when the primer pair produces a valid amplicon and the probe is detected entirely within that amplicon. If the probe is defined in the primer file but produces no accepted hit inside the amplicon, the assay stays negative — the primer pair alone is not enough.

Because hydrolysis probes do not extend, the 3'-end mismatch rules that apply to primers are not applied to probes — a probe hit only needs to fit the total mismatch budget (see Amplicon Calling).

Mixing both in one run

Mixed primer files are fully supported. The probe requirement follows each assay:

• assays with a -P entry use qPCR reporting (probe required);
• assays without a probe use standard PCR reporting.

When at least one assay in the file defines a probe, the consolidated report includes the probe columns (Probe, ProbeLocation, ProbeSize, ProbeMismatches); those columns are left empty on rows of probe-less assays.

Versions before 0.7.0 switched the whole report to qPCR mode as soon as any -P primer was present, silently dropping all probe-less assays. If you need that behavior for some reason, run the assay types separately.