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Reagent	Storage Temperature	Storage Time
cobas® EGFR Mutation Test v2*	2°C to 8°C	Once opened, stable for 4 uses over 90 days or until the expiration date indicated, whichever comes first

Note: With the exception of the PK reagent, do not freeze reagents
*EGFR MMX-1, EGFR MMX-2, EGFR MMX-3 v2, and working MMX (prepared by the addition of MGAC to EGFR MMX-1 or EGFR MMX-2 or EGFR MMX-3 v2) should be protected from prolonged exposure to light. Working MMX must be stored at 2ºC to 8ºC in the dark. The prepared samples and controls must be added within 1 hour of preparation of the working MMX. Amplification must be started within 1 hour from the time that the processed samples and controls are added to the working MMX.", "Country": "XG", "Code": "Storage Conditions (Product)", "Name": "Storage Conditions (Product)" }, { "Language": "en", "Value": "Tissue: The sample extraction process takes about 3 hours. This may vary according to the number of samples being extracted.
Plasma: The sample extraction process takes about 2 hours. This may vary according to the number of samples being extracted.

Tissue and Plasma: The Amplification and Detection step takes 90 minutes.", "Country": "XG", "Code": "Assay Time", "Name": "Assay Time" }, { "Language": "en", "Value": "The cobas^® EGFR Mutation Test v2 (cobas^® EGFR Test) is a research use only real-time PCR test for the qualitative detection and semi-quantitative measurement of defined mutations of the epidermal growth factor receptor (EGFR) gene.
Defined EGFR mutations are detected using DNA isolated from circulating cell-free DNA (cfDNA) from plasma derived from EDTA anti-coagulated peripheral whole blood.

The cobas^® EGFR Test is based on two major processes: (1) manual sample preparation to obtain DNA from plasma; and (2) PCR amplification and detection of target DNA using complementary primer pairs and oligonucleotide probes labeled with fluorescent dyes. The cobas^® EGFR Test is designed to detect the following mutations:

• Exon 18: G719X (G719A, G719C, and G719S)
• Exon 19: deletions and complex mutations (defined as the combination of a deletion and an insertion)
• Exon 20: S768I, T790M, and insertions
• Exon 21: L858R and L861Q

Mutation detection is achieved through PCR analysis with the cobas z 480 analyzer. A mutant control and negative control are included in each run to confirm the validity of the run.


Sample preparation

Plasma samples are processed and circulating cell free DNA (cfDNA) isolated using the cobas^® cfDNA Sample Preparation Kit, a generic manual sample preparation based on nucleic acid binding to glass fibers. Two milliliters (mL) of plasma are processed with a protease and chaotropic binding buffer that protects the cfDNA from DNases. Subsequently, isopropanol is added to the binding mixture that is then centrifuged through a column with a glass fiber filter insert. During centrifugation, the cfDNA is bound to the surface of the glass fiber filter. Unbound substances, such as salts, proteins and other impurities, are removed by centrifugation. The adsorbed nucleic acids are washed and then eluted with an aqueous solution. The target DNA is then amplified and detected on the cobas z 480 analyzer using the amplification and detection reagents provided in the cobas^® EGFR Test kit.

PCR amplification

Target selection

The cobas^® EGFR Test uses primers that define specific base-pair sequences for each of the targeted mutations. For the exon 19 deletion mutations, sequences ranging from 125 to 141 base pairs are targeted; for the L858R substitution mutation in exon 21, a 138 base pair sequence is targeted; for the T790M substitution mutation in exon 20, a 118 base pair sequence is targeted; for the G719X substitution mutation in exon 18, sequences ranging from 104-106 base pairs are targeted; for the S768I substitution mutation in exon 20, a 133 base pair sequence is targeted; for the exon 20 insertion mutations, sequences ranging from 125 to 143 base pairs are targeted; for the L861Q substitution mutation in exon 21, a 129 base pair sequence is targeted. Amplification occurs only in the regions of the EGFR gene between the primers; the entire EGFR gene is not amplified.

Target amplification

A derivative of Thermus species Z05-AS1 DNA polymerase is utilized for target amplification. First, the PCR mixture is heated to denature the DNA and expose the primer target sequences. As the mixture cools, the upstream and downstream primers anneal to the target DNA sequences. The Z05 DNA polymerase, in the presence of divalent metal cation and excess dNTP, extends each annealed primer, thus synthesizing a second DNA strand. This completes the first cycle of PCR, yielding a double-stranded DNA copy which includes the targeted base-pair regions of the EGFR gene. This process is repeated for a number of cycles, with each cycle effectively doubling the amount of amplicon DNA.

Automated real-time mutation detection

The cobas^® EGFR Test utilizes real-time PCR technology. Each target-specific, oligonucleotide probe in the reaction is labeled with a fluorescent dye that serves as a reporter, and with a quencher molecule that absorbs (quenches) fluorescent emissions from the reporter dye within an intact probe. During each cycle of amplification, the probe complementary to the single-stranded DNA sequence in the amplicon binds and is subsequently cleaved by the 5’ to 3’ nuclease activity of the Z05-AS1 DNA Polymerase. Once the reporter dye is separated from the quencher by this nuclease activity, fluorescence of a characteristic wavelength can be measured when the reporter dye is excited by the appropriate spectrum of light. Four different reporter dyes are used to label the mutations targeted by the test. Amplification of the seven targeted EGFR sequences are detected independently across three reactions by measuring fluorescence at the four characteristic wavelengths in dedicated optical channels.

Selective amplification

Selective amplification of target nucleic acid from the sample is achieved in the cobas^® EGFR Test by the use of AmpErase (uracil-N-glycosylase) enzyme and deoxyuridine triphosphate (dUTP).¹ The AmpErase enzyme recognizes and catalyzes the destruction of DNA strands containing deoxyuridine but not DNA containing thymidine. Deoxyuridine is not present in naturally occurring DNA but is always present in amplicon due to the use of dUTP in place of deoxythymidine triphosphate as one of the nucleotide triphosphates in the Master Mix reagents; therefore, only amplicon contains deoxyuridine. Deoxyuridine renders contaminating amplicon susceptible to destruction by AmpErase enzyme prior to amplification of the target DNA. The AmpErase enzyme, which is included in the Master Mix reagents, catalyzes the cleavage of deoxyuridine-containing DNA at the deoxyuridine residues by opening the deoxyribose chain at the C1-position. When heated in the first thermal cycling step at alkaline pH, the amplicon DNA chain breaks at the position of the deoxyuridine, thereby rendering the DNA non-amplifiable. The AmpErase enzyme is inactive at temperatures above 55ºC, i.e., throughout the thermal cycling steps, and therefore does not destroy target amplicon. The cobas^® EGFR Test has been demonstrated to inactivate deoxyuridine-containing EGFR mutant amplicon.", "Country": "XG", "Code": "Principle", "Name": "Principle" }, { "Language": "en", "Value": "

Kit/Cassette	Components and Reagent Ingredients	Quantity per Test	Safety Symbol and Warninga
cobas® EGFR Mutation Test v2 Kit 24 Tests (P/N: 07248555190)	EGFR MMX-1 (EGFR Master Mix 1) (P/N: 06496466001) Tris buffer Potassium chloride Glycerol EDTA Tween 20 3.13% Dimethyl sulfoxide 0.09% Sodium azide < 0.10% dNTPs < 0.01% Z05-AS1 DNA polymerase (microbial) <0.01% AmpErase (uracil-N-glycosylase) enzyme (microbial) < 0.01% Aptamer < 0.01% Upstream and downstream EGFR primers < 0.01% Fluorescent labeled EGFR probes	2 x 0.48 mL	N/A
EGFR MMX-2 (EGFR Master Mix 2) (P/N: 06496474001) Tris buffer Potassium chloride Glycerol EDTA Tween 20 3.13% Dimethyl sulfoxide 0.09% Sodium azide <0.10% dNTPs < 0.01% Z05-AS1 DNA polymerase (microbial) < 0.01% AmpErase (uracil-N-glycosylase) enzyme (microbial) < 0.01% Aptamer < 0.01% Upstream and downstream EGFR primers < 0.01% Fluorescent labeled EGFR probes	2 x 0.48 mL	N/A
EGFR MMX-3 v2 (EGFR Master Mix 3) (P/N: 07248610001) Tris buffer Potassium chloride Glycerol EDTA Tween 20 3.13% Dimethyl sulfoxide 0.09% Sodium azide < 0.10% dNTPs < 0.01% Z05-AS1 DNA polymerase (microbial) < 0.01% AmpErase (uracil-N-glycosylase) enzyme (microbial) < 0.01% Aptamer < 0.01% Upstream and downstream EGFR primers < 0.01% Fluorescent labeled EGFR probes	2 x 0.48 mL	N/A
MGAC (Magnesium acetate) (P/N: 06496539001) Magnesium acetate 0.09% Sodium azide	6 x 0.2 mL	N/A
EGFR MC (EGFR Mutant Control) (P/N: 06496504001) Tris buffer EDTA Poly-rA RNA (synthetic) 0.05% Sodium azide < 0.1% Plasmid DNA containing EGFR exon 18, 19, 20 and 21 sequences (microbial) < 0.1% EGFR wild-type DNA (cell culture)	6 x 0.1 mL	N/A
DNA SD (DNA Specimen Diluent) (P/N: 06496512001) Tris-HCl buffer 0.09% Sodium azide	2 x 3.5 mL	N/A

^aProduct safety labeling primarily follows EU GHS guidance.
^bParaffin Binding Buffer is used for plasma samples.", "Country": "XG", "Code": "Content", "Name": "Content" } ] } } ] }