How to Interpret qPCR Results Near the Detection Limit
A sample can be clearly positive for a selected target even when there is too little DNA for a reliable numerical concentration. This is why detection and quantification have different limits.
“Detected below LOQ” is a valid result category
A valid + means the target met the assay’s positive-call criteria, but its amount is below the validated limit of quantification (LOQ). The analytical ability to establish presence can extend below the quantitative range. A non-detect is not zero, and an invalid sample must not be reported as a negative.
How can qPCR detect DNA below its quantification limit?
The LOD describes reliable detection at a stated probability and with a defined positive-call rule. The LOQ is where numerical results start meeting the method’s stated precision and other quantitative performance criteria. For the same validated method, there can be a zone where amplification is convincingly target-positive but the copy estimate is too variable to report as a trustworthy concentration. These limits should be determined experimentally.
Illustrative example, not a MICBUSTERS assay specification: If a sample-level LOQ were 1.0E+02 target-gene copies/mL, a valid amplification consistent with a smaller amount could be reported +. It would be incorrect to print “25 copies/mL” as a precise routine result simply because a calculation produced that number. A control failure, suspicious curve or contaminated blank would require further review before any positive call.
Detection is probabilistic near the LOD: splitting a very low-copy sample between reaction wells can give one positive and one negative through random allocation. The LOD is therefore not an absolute biological threshold below which the target cannot exist. Each sample’s effective limits depend on processed volume, extract fraction, recovery, dilution and inhibition. See the qPCR accuracy guide and inhibition troubleshooting.
What do the MICBUSTERS certificate symbols mean?
| Display | Meaning | Safe reading |
|---|---|---|
1.0E+03 (or another number) | Quantified result in the unit shown on the certificate. | Compare with similar valid samples and the same denominator. |
+ | Target detected but not quantifiable under the method’s rule. | Positive evidence of target DNA; no defensible numerical concentration. |
<# | Target not detected; # stands for the detection limit printed with this sample. | For example, <1.0E+02 means a valid negative with the displayed limit, not zero target copies. |
ND | Negative for this target in a non-quantitative assay. | No quantitative concentration or numeric target trend follows from ND. |
! | Rejected because of a problem with the sample matrix or associated sample-quality check. | Resolve inhibition or recovery, repeat or send to a suitable laboratory. Do not call it negative. |
The number following < is a sample-specific displayed detection limit. It may differ between samples if volume, extraction recovery or dilution differs. Always read the target name and reporting unit alongside the symbol. The internal process standard and other controls must pass the applicable acceptance rules before interpreting a positive or negative. See field qPCR controls.
How to read scientific notation on a qPCR certificate
The letter E means “times ten to the power of.” The reported unit, such as target-gene copies/mL or copies/swab, still applies to every number. Certificates may use this notation for results from 100 upwards; the form 1.0E+01 may also be shown.
| Certificate | Ordinary number | Equivalent |
|---|---|---|
1.0E+01 | 10 | 1.0 × 101 |
1.0E+02 | 100 | 1.0 × 102 |
1.0E+03 | 1,000 | 1.0 × 103 |
1.0E+04 | 10,000 | 1.0 × 104 |
1.0E+05 | 100,000 | 1.0 × 105 |
1.0E+06 | 1,000,000 | 1.0 × 106 |
1.0E+07 | 10,000,000 | 1.0 × 107 |
1.0E+08 | 100,000,000 | 1.0 × 108 |
These are gene copies (or the certificate’s validated gene-unit label), not automatically living cells. Read gene copies versus cells for the 16S copy-number example.
How should near-limit results be trended?
- Keep the status visible. Plot quantified values as numbers, and mark
+,<#,NDand!as distinct categories. Do not routinely replace+or a non-detect by zero or half a limit. - Compare like with like. Use the same target, sample point, matrix, method, sampled volume or area and reporting unit. Track changing limits as well as changing concentrations.
- Confirm a boundary crossing. A shift from
<#to+can justify resampling or closer surveillance. Two+results cannot establish a quantitative increase; a result rising well above LOQ can. - Act on the operational context. A positive MicC or MicH target near the LOD may merit a surface sample or review of corrosion evidence, but cannot on its own establish active MIC.
If samples were collected just after a biocide, DNA may persist after inactivation; interpret the time series with treatment contact, sampling location and independent activity or corrosion evidence. Read qPCR and dead-cell DNA after biocide treatment.
Turn certificate symbols into a monitoring decision
MICBUSTERS can help you set detection, repeat-sampling and escalation rules for your target panel before a field campaign.
Frequently asked questions
Does a + mean the sample is truly positive?
It means the selected target met the method’s validated positive-call rule and the relevant controls passed, but its concentration cannot be quantified reliably.
Can a non-detect rule out MIC?
No. It refers only to the selected target, tested sample and its displayed limit. A local biofilm, another organism or another corrosion mechanism may still be present.
Scientific sources and further reading
- Forootan A et al. Methods to determine limit of detection and limit of quantification in quantitative real-time PCR. Biomolecular Detection and Quantification. 2017;12:1–6.
- Bustin SA et al. MIQE 2.0: Revision of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments Guidelines. Clinical Chemistry. 2025;71:634–651.
- MICBUSTERS. How to Sample and Interpret MicC and MicH qPCR Results (sample-specific reporting and trends).
Interpretation note: DNA qPCR measures selected amplifiable target sequences in the processed sample. Sampling, assay coverage, matrix effects and controls limit the conclusions. MIC diagnosis also requires relevant surface, chemistry and corrosion evidence.