Qiagen, 870121, therascreen EGFR RGQ PCR Kit (24)

For 24 reactions: 1 Control Assay, 7 Mutation Assays, Positive Control, Water, Taq DNA Polymerase

Features

- FDA-approved for in vitro diagnostic use with GILOTRIF or IRESSA
- Ready-to-use system with simple workflow
- Specific and sensitive due to ARMS and Scorpions PCR technologies

Principle

The therascreen EGFR RGQ PCR Kit is a real-time PCR test for the qualitative detection of exon 19 deletions and exon 21 (L858R) substitution mutations of the epidermal growth factor receptor (EGFR) gene in DNA derived from formalin-fixed paraffin-embedded (FFPE) non-small cell lung cancer (NSCLC) tumor tissue. The test is intended to be used to select patients with NSCLC for whom GILOTRIF (afatinib) or IRESSA (gefitinib) EGFR tyrosine kinase inhibitors (TKIs) are indicated. Safety and efficacy of GILOTRIF (afatinib) has not been established in patients whose tumors have exon 20 insertions and T790M mutations. Safety and efficacy of IRESSA (gefitinib) has not been established in patients whose tumors have L861Q, G719X, S768I, exon 20 insertions and T790M mutations. These EGFR mutations are also detected by the therascreen EGFR RGQ PCR Kit.

ARMS

Allele- or mutation-specific amplification is achieved by ARMS (Amplification Refractory Mutation System). ARMS primers preferentially anneal with DNA containing the mutation and allow Taq DNA polymerase to initiate PCR, effectively distinguishing between a match and a mismatch at the 3' end of a PCR primer. Specific mutated sequences are selectively amplified, even in samples where the majority of the sequences do not carry the mutation. When the primer is fully matched, the amplification proceeds with full efficiency. When the 3' base is mismatched, only low-level background amplification occurs.

Scorpions

Detection of amplification is performed using Scorpions. Scorpions are bi-functional molecules containing a PCR primer covalently linked to a probe. The technology uses a fluorescence-based method to indicate the presence of the mutation. The Scorpion primer hybridizes with a DNA sequence upstream of the target region. The primer is then extended by Taq DNA polymerase and the target region is copied. The newly copied region is complementary to the probe region of the Scorpion. Following a temperature increase within the real-time PCR cycler, the extended Scorpions primer denatures. When the solution cools, the Scorpions probe self hybridizes. The fluorophore is separated from the quencher and a fluorescence signal is generated.

Procedure

Extract DNA samples from FFPE NSCLC tumor tissue collected from NSCLC patients using the QIAamp DSP DNA FFPE Tissue Kit, which has been validated for use with the therascreen EGFR RGQ PCR Kit. The therascreen EGFR RGQ PCR Kit uses a two-step procedure. The first step is performance of the control assay to assess the total amplifiable DNA in a sample. The second step is to test with the mutation assays in order to detect the presence or absence of EGFR mutations.

Applications

Mutation: Exon
Deletions: 19
2235_2249del15: 6223
2236_2250del15: 6225
2239_2253del15: 6254
2239_2256del18: 6255
2240_2254del15: 12369
2240_2257del18: 12370
2239_2248TTAAGAGAAG>C: 12382
2239_2251>C: 12383
2237_2255>T: 12384
2239_2258>CA: 12387
2238_2252>GCA: 12419
2238_2248>GC: 12422
2235_2252>AAT: 13551
L858R: 21
T790M**: 20
L861Q*: 21
G719A*: 18
S768I*: 20
Insertions**: 20
2310_2311insGGT: 12378

The therascreen EGFR RGQ PCR Kit enables the detection of 21 mutations of the human EGFR gene using DNA extracted from FFPE NSCLC tumor tissue samples. The therascreen EGFR RGQ PCR Kit is only intended to discriminate between EGFR mutation-negative (wild-type) and EGFR mutant tumors. EGFR mutations detected by the therascreen EGFR RGQ PCR Kit include:

*Safety and efficacy of IRESSA (gefitinib) has not been established for these mutations.

**Safety and efficacy of GILOTRIF (afatinib) and IRESSA (gefitinib) has not been established for these mutations.