ddPLEX ESR1 Mutation Detection Kit

The ESR1 gene encodes estrogen receptor (ER) subtype alpha. The ddPLEX ESR1 Mutation Detection Kit quantitatively interrogates seven key ESR1 variants across three exons: exon 5 (E380Q), exon 7 (S463P) and exon 8 (L536R, Y537C, Y537S, Y537N and D538G), and their wild-type counterparts.

The kit consists of sufficient reagents for 120 reactions (including 60 reactions of each for the Mutant Assay and Wild-Type Assay), a synthetic DNA based ESR1 Positive Control, multiplex supermix, and DTT.

The kit is used with the Bio-Rad QX600™ Droplet Digital™ PCR System or QX600™ AutoDG Droplet Digital™ PCR System and is compatible with QX Manager Standard Edition and Premium Edition Software for data analysis.

Features & Benefits

• Highly sensitive (0.01%–0.025% VAF*) single-well discrimination of seven key ESR1 gene mutations
• Same-day results with a streamlined workflow
• Flexible sample input of DNA from plasma or FFPE
• Kit includes positive and internal controls
• Sensitivity increased with patented wild-type suppression chemistry
• ddPCR-powered reproducibility and specificity using the QX600 ddPCR system and QX Manager Software

* Variant Allele Frequency

Performance

Analytical Sensitivity

The limit of blank (LoB) was established for the ddPLEX ESR1 Mutant assay using two reagent lots. Fifty-four replicates of wild-type DNA were tested at an input amount of 40,000 copies. The positive droplet counts were rank ordered for each variant and the 95th percentile was determined to be the LoB. Between the two reagent lots, the higher LoB estimate was used.

To establish the limit of detection (LoD) for the ddPLEX ESR1 mutant assay, a titration of synthetic variant DNA in a background of wild-type cell line DNA was tested at a total input amount of 40,000 copies into the ddPLEX ESR1 mutant assay. Variant allele frequencies were 0.5, 0.2, 0.1, 0.05, 0.025, and 0.01%. Eight replicates of each of the samples for each ESR1 variant were tested. The LoD was determined as the lowest variant allele frequency at which all replicates were above the LoB. A summary of the LoB and LoD results is provided in Table 1.

Table 1. Summary of LoB and LoD results for the ESR1 Mutant Assay

Analytical Specificity

Samples with each of the ESR1 variants at 0.1% in a background of wild-type cell line DNA were tested at an input amount of 40,000 copies into the ddPLEX ESR1 mutant assay. Values below the LoB were considered as negative. The concentration of positive targets was plotted (see Figure 1). Specific detection of the spiked variant is observed with low non-specific detection.

Figure 1. Analytical specificity of the ddPLEX ESR1 mutant assay

Reproducibility

The reproducibility of the ddPLEX ESR1 Kit was assessed by testing samples with 1% and 5% allele frequency at a total input amount of 10,000 copies into the mutant assay and 4,000 copies into the wild-type assay. Samples were prepared with synthetic ESR1 mutant DNA mixed with cell line DNA. Two operators tested each sample in quadruplicate with two different reagent lots across two QX600 instruments. For each sample and target, the mean sample concentration (back calculated from the reported reaction concentration), standard deviation, and coefficient of variation (CV) were calculated over all variables. The CV for wild-type targets was less than 12% and for mutant targets was less than 19% for all samples tested (see Table 2).

Table 2. Reproducibility of the ddPLEX ESR1 Assay

Applications & Uses

• For the detection, discrimination, and quantification of key ESR1 mutations for breast cancer research, from cfDNA in plasma and/or DNA in formalin-fixed paraffin embedded (FFPE) tissue samples.

Included Components

The ddPLEX ESR1 Mutation Detection Kit (17009501) includes the following components for 120 reactions and can accommodate up to 42 samples and controls:

The four components above can also be purchased individually.

Configuration Options (or Instrument Compatibility)

The ddPLEX ESR1 Mutation Detection Kit should be used with the QX600 ddPCR System and either the Manual Droplet Generator or Automated Droplet Generator. Equipment, key consumables, and reagents necessary for the QX600 system are listed below (additional items may be needed, please see the User Guide for more details).

Key Droplet Generation & Reader Equipment, Consumables & Reagents

Reagents & Consumables for Automated Droplet Generation Only

Reagents & Consumables for Manual Droplet Generation Only

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Related Category

• Digital PCR Assays

For Research Use Only. Not for use in diagnostic procedures.