Accutest LyoMorph-Q ESR1 Mutation Detection Kit

ESR1 mutations are studied in the context of acquired resistance to aromatase inhibitors and endocrine therapy. This kit is intended for molecular research and is not a tool for diagnosis or treatment selection.

Not in every case. Several variants share a fluorescence channel within a reaction, so a positive result may identify a mutation group rather than one unique variant. The codon groups 380, 422, 463 and 534–538 can be distinguished.

Treat each primer/probe mix as a separate reaction identity and maintain a plate map linking the sample, assay mix, controls and fluorescence channels. A complete 15-variant profile requires all four mixes, although some research workflows may screen mixes containing higher-frequency variants first.

Compatible LyoMorph-Q mutation panels share standardized chemistry and cycling conditions, allowing multiple panels to run together on the same 96-well qPCR plate. Instrument optics, fluorescence channels and each assay’s specific requirements must still be verified.

The reported 95% LoD is three mutant copies for eight variants, five copies for six variants and twenty copies for S463P. The protocol defines the 95% LoD criterion as at least 19 clear positive results in 20 replicates without indeterminate results.

VAF is the proportion of mutant alleles relative to total target alleles, but detection also depends on the absolute number of mutant molecules in the reaction. In the reported study, three mutant copies at 0.01% VAF were demonstrated against approximately 30,000 wild-type target copies. By comparison, 30 ng of human DNA contains approximately 9,000 ESR1 allele copies, so 0.01% VAF represents an average of about 0.9 mutant copies per reaction. Under random molecular sampling, approximately 40% of reactions would contain no mutant molecules, making 95% detection mathematically impossible at that input and VAF.

The protocol specifies a minimum input of 1 ng and recommends at least 5 ng where available. Listed maximum inputs are 30 ng cfDNA, 25 ng FFPE-derived gDNA and 100 ng gDNA. Input beyond these limits may reduce specificity.

The kit is designed primarily for sensitive mutation screening, not absolute mutant copy-number quantification. Ct values should not be interpreted directly as mutant molecule counts.

A missing RNase P signal may indicate insufficient amplifiable human DNA, PCR inhibition, sample degradation, pipetting error or incorrect instrument settings. Review sample input, the assigned fluorescence channel, amplification curves and run controls before interpreting a mutation-negative result. Follow the current protocol’s internal-control acceptance criteria.

The assay has been verified on the Agilent AriaDx. Other equivalent 96-well qPCR systems require local verification of thermal performance, optical compatibility and analysis settings.

The complete kit can ship at ambient temperature. The lyophilized reagent mixes are stable for up to 18 months at 15–25°C, with demonstrated stability for 30 days at 37°C. After receipt and reconstitution, follow the component-specific storage instructions in the current protocol.