Key Features

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Ultrasensitive

Limit of detection down to 0.1% VAF

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Comprehensive

Wide range of mutations covered

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Quantitative

Able to quantitate mutation VAF in original sample

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Sample flexibility

Applicable to a variety of sample types

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Benefits

  • Detects 400 EGFR mutations and quantitates 43 mutations
  • Enables distinction of cis/trans mutations in C797S/T790M
  • Compatible with plasma samples, tumor biopsies, paraffin-embedded sections (FFPE), and fresh frozen tumors
  • Accepts 10-100 ng cfDNA from blood plasma, or 10-1000 ng DNA from tissue biopsy
  • Accepts 2-10 ml of blood, 1-5ml of blood plasma, as little as 1mm*1mm (5 μm) tissue sections (fresh frozen or FFPE)
  • Delivers results in 8 hours, with 2.5 hours of hands-on time

List of Detected and Quantitated Mutations

EGFR Mutation Testing

Sample Data

Quantitative PCR
  • Variant verification by Sanger sequencing
  • Because NuProbe’s BDA technology enriches the variant allele frequency (VAF) of mutations by over 200-fold, Sanger’s limit of detection of 20% VAF is effectively improved to 0.1% VAF. Sanger sequencing can be applied to confirm the identity of quantities detected and quantitated by qPCR using standard sequencing protocols.
Quantitative PCR
  • Variant verification by Sanger sequencing
  • Because NuProbe’s BDA technology enriches the variant allele frequency (VAF) of mutations by over 200-fold, Sanger’s limit of detection of 20% VAF is effectively improved to 0.1% VAF. Sanger sequencing can be applied to confirm the identity of quantities detected and quantitated by qPCR using standard sequencing protocols.

Workflow

Workflow Step 1

Step 1: DNA Extraction

DNA is extracted from clinical samples with commercial extraction kits such as cobas® cfDNA Sample Preparation Kit, QIAamp® DNA Blood Mini Kit, GeneRead® DNA FFPE Kit.
40 min (40 min hands-on)

Sanger Sequencing Workflow

Step 2: qPCR BDA Reaction

NuProbe’s qPCR/Sanger kit reagents and clinical samples are prepared for qPCR reactions. After running the reaction, Ct results and BDA products are obtained.
1.5 hrs (30 min hands-on)

2.5 hrs total time for qPCR reactions (STEP1 + STEP2)

Workflow Step 3

Step 3: Sanger Sequencing (Optional)

BDA products are prepared for Sanger sequencing, including PCR product purification, cycle sequencing and sequencing clean-up. Purified Sanger products are analyzed by capillary electrophoresis.
5.5 hrs (1.5 hrs hands-on)

8 hrs total time including Sanger Sequencing (STEP1+STEP2+STEP3)

Compatible Instruments

qPCR

Quantitative PCR BioRad CFX96

BioRad CFX96


Quantitative PCR ABI 7500

ABI 7500


Quantitative PCR Cobas z480

Cobas z480

Sanger sequencing

Sanger Sequencing ABI 3500 Dx Genetic Analyzer

ABI 3500 Dx Genetic Analyzer

Background

For research use only. Not for use in diagnostic procedures.

EGFR mutations are present in 10-30% non-small cell lung cancer (NSCLC) patients. Specific mutations can guide treatment with targeted therapies such as TARCEVA® (erlotinib), TAGRISSO® (osimertinib), IRESSA® (gefitinib).

The VarTrace EGFR Assay enables highly sensitive detection and quantification of EGFR mutations for patients with advanced or metastatic NSCLC. The kit uses NuProbe’s PCR-based Blocker Displacement Amplification (BDA) technology to enable the selective amplification of low abundant sequence variants (SNV and indels) in a background of wildtype DNA. Following PCR enrichment, Sanger sequencing is applied to reveal the identity of the variants.

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