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Human EGFR Gene 29 Mutations Detection Kit (Fluorescence PCR)

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Human EGFR Gene 29 Mutations Detection Kit (Fluorescence PCR)

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Introduction

[Product Name]Human EGFR Gene 29 Mutations Detection Kit (Fluorescence PCR) [Packaging Size] 16 tests/kit, 32 tests/kit

[Intended Use]

This kit is used to in vitro qualitatively detection of common mutations in exons 18-21 of the EGFR gene in samples from human non-small cell lung cancer patients (see Table 1 for details).
Lung cancer has become the leading cause of cancer deaths worldwide, seriously threatening human health. Non-small cell lung cancer accounts for about 80% of lung cancer patients. EGFR is currently the most important molecular target for the treatment of non-small cell lung cancer. The phosphorylation of EGFR can promote tumor cell growth, differentiation, invasion, metastasis, anti-apoptosis, and promote tumor angiogenesis. EGFR tyrosine kinase inhibitors (TKI) can block the EGFR signaling pathway by inhibiting EGFR autophosphorylation, thereby inhibiting the proliferation and differentiation of tumor cells, promoting tumor cell apoptosis, reducing tumor angiogenesis, etc., so as to achieve tumor targeted therapy [1]. A large number of studies have shown that the therapeutic efficacy of EGFR-TKI is closely related to the status of EGFR gene mutation, and can specifically inhibit the growth of tumor cells with EGFR gene mutation [2~3]. The EGFR gene is located on the short arm of chromosome 7 (7p12), with a full length of 200Kb and consists of 28 exons. The mutated region is mainly located in exons 18 to 21, codons 746 to 753 deletion mutation on exon 19 accounts for about 45% and the L858R mutation on exon 21 accounts for about 40% to 45% [4]. The NCCN Guidelines for the Diagnosis and Treatment of Non-Small Cell Lung Cancer clearly states that EGFR gene mutation testing is required before EGFR-TKI administration. This test kit is used to guide the administration of epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) drugs, and provide the basis for personalized medicine for patients with non-small cell lung cancer. This kit is only used for the detection of common mutations in the EGFR gene in patients with non-small cell lung cancer. The test results are for clinical reference only and should not be used as the sole basis for individualized treatment of patients. Clinicians should consider the patient's condition, drug indications, and treatment The reaction and other laboratory test indicators and other factors are used to comprehensively judge the test results.

[Test Principle]

This detection kit uses PCR enrichment solution, ARMS (Amplification refractory mutation system) technology combined with Taqman probe to detect common EGFR mutants such as L858R and 19del. The detection resolution and sensitivity were further improved by PCR enrichment of mutation sites.The Taqman probe is labeled with FAM that act as a reporter and also has a second dye BHQ2 which acts as a quencher. When not bound to the target sequence, the fluorescent signals of the intact probes are suppressed by the quencher dye. During the PCR amplification step, hybridization of the probes to the specific single -stranded DNA template results in cleavage of the probe by the 5' to 3' exonuclease activity of the DNA polymerase resulting in separation of the reporter and quencher dyes and the generation of a fluorescent signal.The PCR enrichment solution reduces the wild-type background sequence through enzyme digestion, and further improves the detection resolution and sensitivity. Allele- or mutation-specific amplification is achieved by ARMS (Amplification Refractory Mutation System). Taq DNA polymerase is effective at 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 ineffective amplification occurs.

The internal control selects a relatively conserved segment on exon 2 of the human EGFR gene, about 120 bp, as a quality control for reagents, DNA quality and the operation itself. Even if the DNA is degraded, the internal control can truly reflect the effective amount of DNA in the system. The detection system contains a certain proportion of dUTP, dNTP and uracil DNA glycosylase (UDG enzyme), this enzyme (also known as uracil-N-glycosylase or UNG enzyme) can interrupt PCR containing dUTP product, thus effectively preventing PCR contamination.

[Storage Condition and Shelf-life]

The kit should be stored below -18°C protected from light. The shelf life is 9 months(please use within the shelf life). After opeing, the number of repeated freezing and thawing should not be more than 4 cycles. The kit is stable for 5 days below -18°C protected from light.
See the packaging label for the production date and expiration date.

[Applicable Instruments]
Applied Biosystems 7500 Real-Time PCR Systems, Applied Biosystems 7300 Real-Time PCR Systems, QuantStudio® 5 Real-Time PCR Systems,LightCycler® 480 Real-Time PCR system, BioRad CFX96 Real-Time PCR System.

[Requirements for Samples]

Due to the heterogeneity of tumors, the collected clinical samples are often mixed with normal tissue cells. Different types of samples have different levels of normal cells, and the purity and quality of the extracted DNA also vary by sample type, especially when formalin is used. For fixed paraffin section samples, formalin will cross-link and degrade DNA, so please collect samples and extract DNA for detection in the order of the recommended sample types below. You can choose according to the actual clinical samples.

1. Recommended order of sample types: fresh tumor tissue>frozen pathological section > paraffin-embedded pathological tissue or section >plasma or serum.
2. Sampling from fresh diseased tissue should be determined to contain tumor tissue, and the recommended content is more than 30%.

3. Paraffin-embedded pathological tissue or section samples should be determined to contain tumor cells (recommended content is more than 30%), the part taken should be in the middle of the paraffin block as much as possible, the number of paraffin sections should be no less than 3, and the thickness should be 5-10 μm. The surgically removed tissue should be fixed in 4-10% formalin as soon as possible, and the fixation time should be 8-24 hours; thorough dehydration should be performed before embedding. For paraffin-embedded pathological tissue or section samples, please select samples that have not been stored for more than 3 years.

4. Take no less than 5 mL of peripheral blood samples from the patient in an EDTA anticoagulation tube. After slightly inverting and mixing, the plasma separation can be completed within 4 hours; if the peripheral blood is placed in a separation gel accelerating tube (golden tube cap), after being slightly inverted and mixed, the serum can be obtained by centrifugation after standing for coagulation.

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