For： ADx-EG01
AmoyDxTM EGFR 29 Mutations Detection Kit
Detection of 29 mutations in exons 18-21
Instructions For Use
Instructions Version:
P2.2
Date of Revision: April 2012
Store at -20±2 oC
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For： ADx-EG01
Background
Due to its association with malignancies, epidermal growth factor receptor (EGFR) has become the target of an
expanding class of anticancer therapies, such as gefitinib (Iressa) and erlotinib (Tarceva), which are tyrosine kinase
inhibitors (TKIs). These drugs work best on patients whose cancer is driven by abnormal EGFR signaling. Lung
cancer patients who experienced rapid, durable, complete or partial responses to TKIs therapy have been found to
harbor somatic mutations in the EGFR gene. Cancer patients with somatic EGFR mutations have shown an
impressive 60% response rate, much higher than that for conventional chemotherapy. Therefore, detection of the
EGFR mutation status in tumor tissue is key to offering tailored, personalized treatment to cancer patients.
Resistance to therapy, either in the primary tumor or acquired after TKI treatment, is also associated with somatic
mutations.
The AmoyDxTM EGFR 29 Mutations Detection Kit is highly selective and sensitive, detecting 29 of the most
common somatic mutations (both activating and resistance-related) in the EGFR gene. AmoyDx’s patented
technology allows detection of 1% mutant DNA in a background of 99% normal DNA, while ensuring that false
negatives are minimized. The procedure is easily adapted for use in high-throughput sample processing. The
purpose of the kit is to aid physicians and clinical researchers in identifying non-small cell lung cancer patients
whose tumors harbor EGFR mutations.
Intended Use
AmoyDxTM EGFR 29Mutations Detection Kit is a highly sensitive real-time PCR-based test designed to accurately
identify 29 EGFR mutations in Exons 18-21 (Table 1). It is SFDA approved for clinical use in China and CE
marked for IVD use in Europe.
Table 1 Details of 29 Somatic mutations in EGFR gene
Name
Mutation
Exon
Base Change
Ex18-mutant-1
G719A
18
2156G>C
6239
Ex18-mutant-2
G719S
18
2155G>A
6252
Ex18-mutant-3
G719C
18
2155G>T
6253
Ex19-mutant-1
E746_A750del (1)
19
2235_2249del15
6223
Ex19-mutant-2
E746_A750del (2)
19
2236_2250del15
6225
Ex19-mutant-3
L747_P753>S
19
2240_2257del18
12370
Ex19-mutant-4
E746_T751>I
19
2235_2252>AAT(complex)
13551
Ex19-mutant-5
E746_T751del
19
2236_2253del18
12728
Ex19-mutant-6
E746_T751>A
19
2237_2251del15
12678
Ex19-mutant-7
E746_S752>A
19
2237_2254del18
12367
Ex19-mutant-8
E746_S752>V
19
2237_2250>T(complex)
12384
Ex19-mutant-9
E746_S752>D
19
2238_2255del18
6220
Ex19-mutant-10
L747_A750>P
19
2238_2248>GC(complex)
12422
Ex19-mutant-11
L747_T751>Q
19
2238_2252>GCA(complex)
12419
Ex19-mutant-12
L747_E749del
19
2239_2247del9
6218
Ex19-mutant-13
L747_T751del
19
2239_2253del15
6254
Ex19-mutant-14
L747_S752del
19
2239_2256del18
6255
Ex19-mutant-15
L747_A750>P
19
2239_2248TTAAGAGAAG>C(complex)
12382
Ex19-mutant-16
L747_P753>Q
19
2239_2258>CA(complex)
12387
Ex19-mutant-17
L747_T751>S
19
2240_2251del12
6210
Ex19-mutant-18
L747_T751del
19
2240_2254del15
12369
Ex19-mutant-19
L747_T751>P
19
2239_2251>C(complex)
12383
Ex20-mutant-1
T790M
20
2369C>T
6240
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Cosmic ID
For： ADx-EG01
Ex20-mutant-2
S768I
20
2303G>T
6241
Ex20-mutant-3
H773_V774insH
20
2319_2320insCAC
12377
Ex20-mutant-4
D770_N771insG
20
2310_2311insGGT
12378
Ex20-mutant-5
V769_D770insASV
20
2307_2308insgccagcgtg
12376
Ex21-mutant-1
L858R
21
2573T>G
6224
Ex21-mutant-2
L861Q
21
2582T>A
6213
Kit Contents
This kit contains sufficient reagents to carry out 12 tests (Table 2). For example, 10 samples, a positive control and
a no template control.
Table 2. Kit Contents
Contents
Volume
Strips
12 strips
EGFR Taq DNA Polymerase
45 µL
EGFR Mixed Standard
250 µL
One 8-tube strip will be employed for each sample or control (Table 3). The 19-Del reaction can detect the
presence of any of 19 deletions in exon 19. The Insertions reaction can detect the presence of any of 3 insertions in
exon 20. The G719X reaction well can detect the presence of G719S, G719A and G719C.
Table 3. Mutation information for each strip
Tube
1
2
3
4
5
6
7
8
Name of Mutation
19-Del
L858R
T790M
Insertions
G719X
S768I
L861Q
External Control
Channel
FAM, HEX/VIC
FAM, HEX/VIC
FAM, HEX/VIC
FAM, HEX/VIC
FAM, HEX/VIC
FAM, HEX/VIC
FAM, HEX/VIC
FAM
The reagents are pre-loaded into strips that are compatible with ABI7300, ABI7500, ABI7900, ABI StepOne,
Stratagene Mx3000P™ , Stratagene Mx3005P™, LightCycler480 I and II, or BioRad-CFX96 machines.
The order of the wells (“1” to “8”) for each strip can be determined as follows:
For the ABI Stepone strip, the wells are identified with numbers on the top of each tube.
•
•
For other instruments, there is a pinhole at either end of the 8-tube strip.
For the No.1 tube,the pinhole lays at the corner of the tab.
•
For the No.8 tube,the pinhole appears in the middle.The strips are general except for LightCycler480
•
and BioRad-CFX96,whose strips are lower.
Equipment and Reagents Not Supplied With Kit
1.
Compatible PCR instruments are: ABI7300, ABI7500, ABI7900, ABI StepOne, Stratagene Mx3000P™ ,
Stratagene Mx3005P™ and LightCycler480, BioRad-CFX96.
a) This kit is compatible with LightCycler480 II instrument. Fluorescence calibration is required for
LightCycler480 I instrument. If the fluorescence crossover occurs in the LightCycler480 II instrument,
the fluorescence calibration is also needed prior to the operation. To run the assays on a LightCycler
machine, please use the Roche 480 adaptor, available from BIOplastics, cat No. B79480.
b) To run the assays on a ABI7900 machine, please use the ABI7900 adaptor, available from BIOplastics,
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For： ADx-EG01
2.
3.
4.
cat No. B7900RNA.
Sterile, nuclease-free tubes.
Dedicated pipette and filtered pipette tips for handling DNA.
Sterile, nuclease-free H2O.
Shipping and Storage
The kit requires cold-chain-transportation. The shelf-life of the kit is six months when the kit is stored immediately
upon receipt at -20±2 oC in a constant-temperature freezer and protected from light.
Specimen Material
Human genomic DNA must be extracted from tissue or blood, or fixed paraffin-embedded tissue prior to use and
stored at -20±2 ℃. Good DNA quality is essential and we recommend use of Qiagen DNA extraction kit (QIAamp
DNA FFPE Tissue Kit, cat No. 56404, for paraffin embedded specimens; DNeasy Blood & Tissue kit, cat. No.
69504 or 69506, for tissue and blood specimens). The OD value of DNA samples should be measured using the
spectrophotometer after extraction. The Thermo Fisher NanoDrop 1000 /2000 spectrophotometer is recommended.
Make sure A260/A230 value is greater than 2.0 and A260/A280 value between 1.8 and 2.0.
Technological Principles
The kit uses novel, proprietary primers and probes in a real-time PCR assay to detect EGFR mutations in human
genomic DNA. The mutant EGFR gene DNA is amplified by the specific primers, and detected by the novel
probes. A highly validated procedure based on EGFR Taq DNA polymerase contributes to outstanding assay
sensitivity and selectivity.
Notes on Protocol
1. The reaction buffer, dNTPs, specific oligos and novel probes are pre-loaded in the PCR tubes.
2. Tubes 1 to 7 contain reagents for detecting mutations in the EGFR gene (FAM) and generating an internal
control signal (HEX/VIC). The internal control system is designed to detect the presence of inhibitors, which
may lead to false negative results. The No. 8 tube is used as an external control: the FAM signal in this tube is
used to calculate the ΔCt values.
3. The threshold at which the signal is detected above background fluorescence is called the Cycle threshold (Ct).
The Ct values used to determine if a sample is positive or negative are based on extensive validation. If the Ct
value falls within the appointed range (see below), the sample is classed as mutation positive. If the Ct value
is outside the appointed range, the sample is classed as negative or below the detection limit of the kit. Weak
positives can be confirmed by calculating ΔCt values and referring to Table 5 below.
4. The EGFR mixed standard contains 29 EGFR mutations, combined with normal human genomic DNA.
5. The 8 reactions for each sample must be analyzed within the same PCR run to avoid run-to-run variations in
threshold settings. It is recommended that the EGFR mixed standard should be analyzed during each PCR run,
along with no-template controls.
Protocol
1. Thaw the EGFR mixed standard.
2. Briefly centrifuge EGFR Taq DNA polymerase and EGFR mixed standard.
3. Add 2.7 µL EGFR Taq DNA polymerase into the following solutions individually:
a)
42.3 µL, each test sample (see below for sample DNA concentrations).
i.
The contents of this tube will be aliquoted into eight PCR tubes, 5 µL per tube.
ii.
The amount of DNA per 5 µL aliquot depends on the tissue source, as described below.
b)
42.3 µL, EGFR mixed standard.
c)
42.3 µL, no-template controls (sterile water).
d)
Since Taq DNA polymerase is viscous, please pay attention to the centrifugation and pipetting process.
Minimize the contact interface between the pipette tip and Taq DNA polymerase to avoid adding
excessive enzyme.
4. Mix each solution by gently pipetting up and down with fresh PCR tips.
5. Centrifuge briefly.
6. Samples can be divided into two groups: paraffin embedded and non-paraffin embedded specimens.
a)
Non-paraffin embedded specimens include fresh tissue, frozen pathological sections, non-heparin
anticoagulant blood plasma, blood serum and non-heparin anticoagulant blood.
i.
For non-paraffin embedded samples, the recommended DNA amount in each PCR tube is
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For： ADx-EG01
2 ~ 5 ng.
For paraffin embedded samples, we recommend use of 10 or 15 ng template DNA in each PCR tube
depending on storage times.
i.
Use 10 ng of template DNA for samples with less than 3 years storage time.
ii.
Use 15 ng of template DNA for samples with more than 3 years storage time.
c)
We recommend use of TE (pH = 8.0) for extracted DNA dilution.
7. Transfer 5 µL of above DNA mixtures to the appropriate PCR tube of the 8-tube strip. Add the solution to the
side of the tube wall above the reagents in the tube.
a)
Please refer to Appendix 1 for an example of a test plate layout.
8. Seal the strips.
9. Spin the PCR tubes gently to collect the reagents at the bottom of wells.
a)
NOTE: This spin step is critical for proper mixing of the reagents!
10. Place the PCR tubes into the real-time PCR instrument.
11. Carry out real-time PCR using the cycling conditions described in Table 4.
b)
Table 4 Cycling Parameters
Temperature
Stage 1
95℃
Stage 2
95℃
64℃
72℃
Stage 3
93℃
60℃
72℃
Time
Cycles
5min
1
25s
20s
20s
15
25s
35s ☆Data collection of FAM and HEX/VIC
20s
31
Sample Data Analysis
1.
The FAM signals of tubes 1 through 7 indicate the mutation status of sample.
2.
The HEX/VIC signals of tubes 1 through 7 indicate the internal control status.
3.
The FAM signal of tube 8 is used to calculate ΔCt values. The PCR reaction in this tube amplifies and
detects a region of genomic DNA adjacent to the EGFR gene.
4.
Check the FAM signal from the external control assay, tube 8 - it should give a positive result:
i.
The Ct value should be between 15 ~ 21 for paraffin embedded specimens; and between 13~19
for non-paraffin embedded specimens.
ii.
If the requirements of i) are satisfied, further analysis should be carried out. However, if Ct value
is below the indicated range, the DNA is overloaded. The procedure should be repeated with
reduced DNA.
iii.
If the external control assay has failed, the DNA template contains PCR inhibitors, indicting that
the DNA needs to be re-extracted.
5.
The HEX/VIC signals in tubes 1 through 7 are also used as controls. If the HEX/VIC signal assay has failed
but the FAM test has worked well, continue with the analysis. If both the HEX/VIC and FAM signal tests
have failed, the data should be discarded and the experiment should be repeated.
6.
Instrument set-up: Ensure the calibration fluorescence is unselected, and select single mutation detection for
each tube accordingly. It is necessary to choose reaction holes for positive control, no-template reference and
samples simultaneously. Then, users may adjust the Threshold of FAM amplification curve, and obtain the
Ct value of mutant group.
7.
The EGFR mixed standard FAM Ct value should be less than 20, but variation may occur due to different
threshold settings on different instruments.
Analysis of mutation assay results. See Table 5.
1. Check the FAM Ct value for each sample. Based on different mutant Ct values, the detection results are
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2.
3.
4.
divided into strong positive, weak positive or negative
Strong Positive: If the sample FAM Ct value is less than the Ct value shown in the “Strong Positive” row in
Table 5, the sample is classified as strong positive.
Weak Positive: If the sample FAM Ct value is in the range shown in the “Weak Positive” row in Table 5, the
sample is provisionally classified as weak positive.
a)
If the FAM Ct value is in the “Weak Positive” range, the ∆Ct of the reaction tube is calculated to
confirm the result.
b)
If the ∆Ct value is less than the corresponding Cut-off value of ∆Ct, the sample is confirmed as
weak positive.
c)
If the ∆Ct value is greater than the Cut-off ∆Ct value, the sample is classified as negative or below
the limits of the kit.
The calculation of ∆Ct:
Formula 1. ∆Ct = mutant FAM Ct value – external control FAM Ct value.
a)
5.
6.
Where:
i) The “mutant FAM Ct value” indicates the Ct value of the mutant FAM signal from a sample.
ii) The “external control FAM Ct value” indicates the Ct value of the FAM signal in tube 8.
One sample may contain two or more mutant types, and the corresponding sample will have two or more ∆Ct
values.
Negative: If the sample FAM Ct value is greater than or equal to the critical negative value shown in the
“Negative” row in Table 5, the sample is classified as negative or below the detection limit of the kit.
Table 5 Results Determination
Tube No.
1
2
3
4
5
6
7
Name of Mutation
19-Del
L858R
T790M
Insertions
G719X
S768I
L861Q
Ct<26
Ct<26
Ct<26
Ct<26
Ct<26
Ct<26
Ct<26
>5%
>5%
>5%
>5%
>5%
>5%
>5%
26≤Ct <29
26≤Ct <29
26≤Ct <28
26≤Ct <29
26≤Ct <29
26≤Ct <29
26≤Ct <29
11
11
7
9
7
8
8
1%～5%
1%～5%
1%～5%
1%～5%
1%～5%
1%～5%
1%～5%
Ct≥29
Ct ≥29
Ct ≥28
Ct ≥29
Ct ≥29
Ct ≥29
Ct ≥29
Strong
Positive
Mutant Ct
Value
Mutant Content
Mutant Ct
Week
Positive
Value
∆Ct Cut-off
value
Mutant Content
Negative
Mutant Ct
Value
Warnings and Precautions
1.
Please read the instruction carefully and become familiar with all components of the kit prior to use.
2.
Do not exchange and mix up the kit contents with different batches.
3.
The kit and its contents cannot be resold or modified for resale without the written approval of AmoyDx.
4.
Using other sources of reagents is not recommended. Strictly distinguish the reagents from mixed standard to
avoid contamination. Otherwise, false positive may be produced.
5.
Do the experiments with attention to prevent exogenous DNA contamination to reagents. It is recommended
that users have separate, dedicated pipettes and filter pipette tips to add DNA template and during the
preparation of reagents.
6.
To optimize the activity and performance, mixtures should always be protected from light to avoid photo
bleaching.
7.
All the chemicals are potential hazard, only trained professionals should use this kit. Please wear suitable lab
coat and disposable gloves. The used kit should be disposed of properly.
8.
The product is CE-marked according to the European Union In Vitro Diagnostic Medical Devices Directive
98/79/EC.
Website：www.amoydx.com/en
Tel：+86 592 6806835
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9.
10.
AmoyDx grants customer a non-exclusive and non-transferable license to use AmoyDx technologies.
AmoyDx assumes no responsibility for any errors that may appear in this document. The information in this
document is subject to change.
Notes
1.
2.
3.
4.
5.
6.
Symbol for "In Vitro Diagnostic Medical Device".
Symbol for "Authorized Representative in the European Community".
Symbol for "Batch Code".
Symbol for "Used By", it indicates that the reagent should not be used after the end of the
date as shown on box.
Symbol for "Attention, see instructions for use".
Symbol for "Temperature Limitation", the kits should be stored at -20±2℃.
Information of European Authorised Representative
Wellkang Ltd t/a Wellkang Tech Consulting Suite B, 29 Harley Street, London W1G 9QR United Kingdom
Website：www.amoydx.com/en
Tel：+86 592 6806835
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For： ADx-EG01
Appendix 1 - Suggested PCR Plate Layout
96 well layout
Assay
1
2
3
4
5
6
7
8
9
10
11
12
19-del
Sample1
Sample2
Sample3
Sample4
Sample5
Sample6
Sample7
Sample8
Sample9
Sample10
STD
NTC
L858R
Sample1
Sample2
Sample3
Sample4
Sample5
Sample6
Sample7
Sample8
Sample9
Sample10
STD
NTC
T790M
Sample1
Sample2
Sample3
Sample4
Sample5
Sample6
Sample7
Sample8
Sample9
Sample10
STD
NTC
Insertions
Sample1
Sample2
Sample3
Sample4
Sample5
Sample6
Sample7
Sample8
Sample9
Sample10
STD
NTC
G719X
Sample1
Sample2
Sample3
Sample4
Sample5
Sample6
Sample7
Sample8
Sample9
Sample10
STD
NTC
S768I
Sample1
Sample2
Sample3
Sample4
Sample5
Sample6
Sample7
Sample8
Sample9
Sample10
STD
NTC
L861Q
Sample1
Sample2
Sample3
Sample4
Sample5
Sample6
Sample7
Sample8
Sample9
Sample10
STD
NTC
Sample1
Sample2
Sample3
Sample4
Sample5
Sample6
Sample7
Sample8
Sample9
Sample10
STD
NTC
External
Control
References
1.
2.
Herbst RS, 2004. Review of epidermal growth factor receptor biology. Int. J. Radiat. Oncol. Biol. Phys. 59 (2 Suppl): 21–6.
Zhang H, Berezov A, Wang Q, Zhang G, Drebin J, Murali R, Greene MI, 2007. ErbB receptors: from oncogenes to targeted cancer
therapies. J. Clin. Invest. 117 (8): 2051–8.
3.
Oda K, Matsuoka Y, Funahashi A, Kitano H, 2005. A comprehensive pathway map of epidermal growth factor receptor signaling.
Mol. Syst. Biol. 1: 2005.0010.
4.
Lynch TJ, Bell DW, Sordella R, et al, 2004. Activating mutations in the epidermal growth factor receptor underlying
responsiveness of non-small-cell lung cancer to gefitinib. N. Engl. J. Med. 350 (21): 2129–39.
5.
Seth D, Shaw K, Jazayeri J and Leedman PJ, 1999. Complex post-transcriptional regulation of EGF-receptor expression by EGF
and TGF-α in human prostate cancer cells. Br J Cancer 80(5-6):657-69.
6.
Pao W, Miller VA, Politi KA, Riely GJ, Somwar R, Zakowski MF, Kris MG and Varmus H, 2005. Acquired resistance of Lung
Adenocarcinomas to Gefitinib or Erlotinib is associates with a second mutation in the EGFR kinase domain. Plos Medicine
2(3):1-11.
Website：www.amoydx.com/en
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