# 13-May-2020 COVID-19: Calculating the detection limit for a SARS-CoV-2 RT-PCR assay using Analyse-it

Recent improvements to the Analyse-it Method Validation edition, in version 5.50 and later, include the addition of probit regression. Probit regression is useful when establishing the detection limit (LoD) for an RT-qPCR assay.

The CLSI EP17-A2: Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures protocol provides guidance for estimating LoD and is recognized by the FDA. In this blog post, we will look at how to perform the relevant part of the CLSI EP17-A2 protocol using Analyse-it.

For details on experimental design, see section 5.5 in the CLSI EP17-A2 guideline. In Analyse-it, you should arrange the data in 2 columns: the first should be the concentration, and the second should be the result, positive or negative. You should have a minimum of 20 replicates at each concentration. We have put together a hypothetical example in the workbook COVID-19 Detection Limit.xlsx which you can use the follow the steps below:

To Analyse-it:

- Select a cell in the dataset.
- On the
**Analyse-it**ribbon tab, in the**Statistical Analyses**group, click**Fit Model**, and then click**Probit**.The analysis task pane opens.

- In the
**Y**drop-down list, select the Result variable, and then in the**Event**drop-down list, select the Positive state (1). - In the
**X**drop-down list, select the RNA variable, and then click the drop-down menu icon next to the variable drop-down, and in the**Transform**drop-down list, select**Log 10**. - In the
**Plot**drop-down list, select**Scaled - X against Y**to show the X axis as a logarithmic scale. - Clear the
**Individual**check-box, and select the**Aggregate**check-box. - On the
**Analyse**-**it**ribbon tab, in the**Fit Model**group, click**Predict**, and then click**X given Probability**. - On the analysis task pane, on the
**Fit**panel, next to the**Predict X given Probability**, click**More**…, and then on the**Predict X given Probability**panel, click**Add**and select**LoD=95%**.NOTE: If using Analyse-it pre-version 5.65, on the

**Fit**panel, in the**Predict X given Probability**edit box, type 0.95. - Click
**Calculate**.

The analysis report shows the probit regression fit:

The Predict X for given Probability section shows the limit of detection (LoD) as the point where the probability of a positive result is 95%.

For more information, see the online documentation:

Estimating the detection limit using a probit fit

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