What Is the Limit of Detection?
The limit of detection (LOD) is the lowest amount or concentration of analyte that can be reliably detected by an analytical procedure, although it may not be quantified with acceptable accuracy or precision (typically 3 × standard deviation).
During molecular assay development and validation, establishing LOD is essential for determining analytical sensitivity. Published recommendations suggest generating approximately 20 measurements distributed above, below, and around the estimated LOD (identified through preliminary dilution studies), to support analytical sensitivity assessment 1,2.
LOD Determination Using Mimix Reference Standards
Mimix™ reference standards provide a reliable and convenient approach for evaluating the analytical sensitivity of molecular assays across a broad range of technologies, including Next Generation Sequencing (NGS), Quantitative PCR (qPCR), Digital PCR (dPCR), Digital Droplet PCR (ddPCR), and Sanger Sequencing.
Mimix mutant and matched wild-type reference standards can be combined at defined ratios to generate samples with known variant allelic frequencies (VAF). These samples can then be used to establish dilution series spanning the expected detection limits of the assay.
By analyzing samples with progressively decreasing VAFs, laboratories can generate an LOD dilution curve and determine the lowest VAF that can be detected reliably and reproducibly.
Material required to generate LOD dilution curve
| Material Required | Specification |
|---|---|
| Mutant Mimix Reference Standard | 50% VAF |
| Matched Wild-Type Mimix Reference Standard | 100% Wild Type |
| Total DNA Required | 1µg mutant DNA and 5µg wild type DNA |
| DNA Concentration | 50ng/µL |
Notes: Refer to the batch-specific Certificate of Analysis for the VAF and concentration of the Mimix Reference Standard.
**Mimix Reference Standards are provided at 50 ng/µL. Where a different input concentration is required, use the concentration dilution table
below to prepare the appropriate dilution.
Dilution Series for Molecular Applications
Dilution schemes can be used for various molecular workflows: NGS, dPCR, qPCR, Sanger Sequencing or a custom LOD study design.
Below is an example of a generic LOD dilution series suitable for generating LOD curves over a range of VAF. The dilution scheme presented is for example purposes. Users should optimize dilution points and replicate numbers according to their study design and intended use of the assay.
| Target VAF | 50% Mutant DNA | Wild-Type DNA |
|---|---|---|
| 50% (control)* | 10 µL | 0 µL |
| 20% | 4 µL | 6 µL |
| 10% | 2 µL | 8 µL |
| 5% | 1 µL | 9 µL |
| 2% | 2µL of prepared 10% dilution | 8 µL |
| 1% | 1 µL of prepared 10% dilution | 9 µL |
| 0.5% | 1 µL of prepared 5% dilution | 9 µL |
| 0.1% | 1 µL of prepared 1% dilution | 9 µL |
| 0.01% | 1 µL of prepared 0.1% dilution | 9 µL |
| 0% | 0 µL | 10 µL |
*We recommend that a test on the undiluted reference standard is performed to accurately determine the actual allelic frequency observed with the method of interest.
DNA Concentration Dilution Guide
For Sanger Sequencing, dPCR, qPCR and NGS applications, a DNA concentration of between 5-50ng/µL is commonly required. The provided 50ng/µL DNA can be diluted with Tris-EDTA (TE) buffer (Tris-HCl 10mM, 1mM EDTA, pH 8.0). Refer to table below for DNA concentration dilution guide.
| Target DNA Concentration | Reference standard (50ng/µL Mutant or Wild Type DNA) | TE Buffer | Total Volume |
|---|---|---|---|
| 5 ng/µL | 2 µL | 18 µL | 20 µL |
| 10 ng/µL | 4 µL | 16 µL | 20 µL |
| 25 ng/µL | 10 µL | 10 µL | 20 µL |
For concentrations or volumes different from the above examples, dilution volumes can be calculated using the equation C1V1=C2V2 where:
C1 = Stock concentration
V1 = Volume of stock to add
C2 = Desired final concentration
V2 = Final volume
Solve for V1=(C2V2)/C1
Example (to make 100 µL of 10 ng/µL):
50 ng/µL * V1 = (10 ng/µL) (100 µL)
V1 = 1000/50 = 20 µL of stock will be brought up to final volume of 100 uL using 80 µL of stock will be brought up to a final volume of 100 µL using 80 µL
of TE buffer.
References
- Westgard, J. O. 2008. Basic method validation, 3rd ed. Westgard QC, Inc., Madison, WI.
- CLSI/NCCLS. 2004. Protocols for determination for limits of detection and limits of quantitation. Approved guideline. CLSI document EP17-A. Clinical and Laboratory Standards Institute, Wayne, PA.
Written by Ezgi Gokhan, PhD, Senior Scientist, R&D
Ezgi is a Senior Scientist in the Mimix R&D team. She obtained her PhD from Brunel University, where she focused on mitotic exit molecules and their roles in cancer, as well as in the reformation and maintenance of the nuclear envelope after mitosis. She has been part of the team for almost 2 years, during which she helped launch multiple products and developed custom reference standards for major companies in the industry. Previously, she worked in clinical affairs, where she was involved in obtaining FDA and IVDR approvals for FISH probes that are used to diagnose patients with blood cancers in the clinic.
For Research Use Only. Not for diagnostic procedures.