APIS ESR1 Mutations Kit (RUO*)
The APIS ESR1 Mutations Kit is an advanced qPCR assay for the sensitive and precise detection of mutations within the oestrogen receptor gene. The APIS ESR1 Mutations Kit is a qualitative test, detecting eleven ESR1 mutations across three exons: exon 5 (E380Q), exon 7 (S463P) and exon 8 (P535H, L536R, L536Q, L536H, L536P, Y537C, Y537S, Y537N and D538G).
The APIS ESR1 Mutations Kit includes mutation-specific probes to enable highly sensitive detection of the target mutations. Additionally, it uses PCR clamp and blocker technology, which ensures specific amplification of the mutant sequence, even in the presence of a high wild-type background.
*APIS ESR1 Mutations Kit is distributed by Biocartis for Research Use Only (RUO), not for use in diagnostic procedures. Data and conclusions have not been validated by Biocartis NV.
Benefits of Using APIS ESR1 Mutations Kit
- Wide target coverage • the kit has been designed to cover 11 ESR1 mutations
- High specificity • includes clamp and blocking technology to ensure no wild-type detection
- High sensitivity • all mutations are detected at ≤ 1% MAF (Mutant Allele Frequency) and ≤ 0.5% MAF in high WT background
- Easy to use • our assays are designed to be user-friendly, with a simple protocol. The reagents have been optimised for precise and sensitive detection in human DNA.
SPECIMEN REQUIREMENTS
- 30 µl of DNA, e.g., extracted cell-free DNA (cfDNA).
- It is recommended to extract samples using a cell-free-specific kit and to use the sample without dilution.
Assay Mutations and Analytical Sensitivity in Contrived Samples
The APIS ESR1 Mutations Kit detects 11 ESR1 mutations across three exons. Contrived samples of 0.008–1.6% mutant allele frequency (MAF) were prepared with a total copy of 5000 per reaction. A total of 24 replicates were generated per target, across two instruments and two kit lots. The Limit of Detection (LoD) is based on the highest mutant allele frequency with 95% correct calls observed for each target in the 24 replicates. The claimed LoD for the targets in % MAF is ≤ 1% for all targets (Table 1).
Table 1. 11 ESR1 mutations detected in the APIS ESR1 Mutations Kit. LoD in % MAF and copies (cp). The mutant copies at LoD and WT copies at LoD describe the copies of each DNA fragment in the contrived LoD samples.
Sensitivity in High WT background
To assess kit performance in high WT background, samples were contrived with WT and mutant DNA at 0.5% MAF, with a total DNA copy number of 10,000 or 50,000 per reaction. Table 2 shows the mutations for which the APIS ESR1 Mutations Kit achieves sensitivity of ≤0.5% MAF in a high WT background.
Table 2. ESR1 Mutations detected at ≤0.5% MAF in high WT background. The Mutant DNA and WT copies in the contrived 0.5% MAF samples are shown.
* Targets detected with 0.5% MAF sensitivity in samples with both 10,000 and 50,000 total copies
Supporting Data for the APIS ESR1 Mutations Kit Linearity
An 8-level dilution series of DNA fragments specific to each mutation were used to evaluate PCR efficiency and R-sq values (Table 2 and Figure 1).
SPECIFICATIONS
The kit has undergone testing as part of APIS' unique assay development pipeline, which includes in silico and wet laboratory testing. As a result, researchers can have confidence in the accuracy, sensitivity, specificity, and reliability of the kit. The qPCR assay’s efficiency and linearity were assessed using contrived synthetic DNA samples.
