Calculator Inputs
Example Data Table
| Sample | Ct | Slope | Intercept | Dilution | Elution (µL) | Input (µL) | Estimated copies/mL |
|---|---|---|---|---|---|---|---|
| A1 | 25.30 | -3.32 | 40.10 | 1 | 60 | 200 | 2,650,000 |
| B2 | 29.80 | -3.30 | 39.80 | 10 | 80 | 400 | 1,120,000 |
| C3 | 33.10 | -3.45 | 41.00 | 1 | 50 | 200 | 42,000 |
| D4 | 36.70 | -3.35 | 40.50 | 1 | 60 | 200 | 2,300 |
Formula Used
log10(copies) = (Ct − intercept) / slope
copies = 10^(log10(copies))
total copies in eluate = copies/uL × elution_uL
sample volume (mL) = sample_input_uL / 1000
copies/mL = (total copies / sample mL) × dilution_factor
How to Use This Calculator
- Enter your Ct value from the qPCR output.
- Fill slope and intercept from your assay’s standard curve.
- Provide template volume used in the reaction (µL).
- Enter elution volume and original sample input volume (µL).
- Apply any overall dilution factor from prep or re‑testing.
- Click Calculate Viral Load to view results above.
- Use CSV or PDF buttons to save your summary.
Professional Article
Quantification context and reporting units
Viral load represents the concentration of viral genomic targets in a specimen, commonly expressed as copies per milliliter. Laboratories report results to support trend monitoring, treatment response assessment, and assay performance checks. Because specimen types differ, comparable interpretation requires consistent matrices, extraction workflows, and reporting units across timepoints. Many reports also include log10(copies/mL) to simplify communication across wide ranges.
Linking Ct to target copies
Real‑time PCR generates a cycle threshold value when fluorescence crosses a defined baseline. A validated standard curve relates Ct to log10 copies using a linear model derived from serial dilutions. With a typical negative slope near −3.3, each 3.3‑cycle decrease implies roughly a tenfold increase in target copies, assuming stable efficiency. Curves are typically accepted using efficiency targets and strong linearity, and should be rechecked after reagent, instrument, or protocol changes.
Scaling for extraction and dilution
The reaction estimate becomes meaningful only after scaling back to the original sample. The calculator converts copies per reaction to copies per microliter of eluate using the template volume added to the reaction. It then multiplies by elution volume to obtain total copies recovered and divides by extracted sample volume in milliliters, finally applying any dilution factor. Enter volumes that match your workflow when concentrating, splitting eluate, or re‑testing.
Detection limits and uncertainty
Low‑level signals may fall below a laboratory’s limit of detection or limit of quantification. When estimated copies per reaction are under the selected LoD, the output is flagged to discourage over‑interpretation. Additional uncertainty arises from pipetting variation, inhibition, extraction recovery, and standard curve fit, so replicates and controls remain essential. Near cutoffs, conservative language and repeat sampling can reduce false trend conclusions.
Using logs for trend analysis
Log10 transformation compresses wide ranges and supports comparisons across orders of magnitude. Many workflows describe changes as log reductions, where a 1‑log drop equals a tenfold decrease and a 0.3‑log drop is about a twofold change. For longitudinal monitoring, stable procedures and consistent curve parameters are as important as the calculated value itself. Document dilution, volumes, and curve settings with each result for auditability. Rounding affects display only; calculations use full precision throughout internally always.
FAQs
1) What does “copies per reaction” represent?
It is the estimated number of target copies present in the PCR well, based on Ct and your standard curve. It does not yet account for extraction volumes, sample input volume, or dilution.
2) Why is the slope usually negative?
As target concentration increases, fluorescence crosses the threshold earlier, producing a lower Ct. That inverse relationship yields a negative slope when Ct is plotted against log10(copies).
3) When should I change the dilution factor?
Use it when the tested material was diluted before amplification, or when an additional dilution occurred during re‑testing. Enter 1 if no dilution was applied.
4) What does “Below LoD” mean in this tool?
It flags cases where estimated copies per reaction fall under your selected limit of detection. Such values may be unreliable for quantification and should be interpreted with your laboratory policy.
5) Can I use this for different sample types?
Yes, if you enter volumes and curve parameters that match that sample type and extraction method. Interpretation across matrices still requires consistent protocols and validated performance.
6) Why do I see both raw and log10 results?
Raw copies/mL is intuitive for reporting, while log10(copies/mL) helps compare large ranges and track fold‑changes. Many clinical and research workflows summarize trends using log differences.