For: ADx-NR02
AmoyDx® NRAS Mutation Detection Kit
Detection of 16 mutations in NRAS codons 12, 13, 59, 61, 117 and 146
Instruction for Use
Instruction Version:
P1.3
Revision Date:
July 2016
Store at -20±5℃
Manufacturer: Amoy Diagnostics Co., Ltd.
Website: http://www.amoydx.com
E-mail: [email protected]
Tel: +86 592 6806835
Address: 39 Dingshan Road, Haicang District, Xiamen 361027, P. R. China
Fax: +86 592 6806839
For: ADx-NR02
Background
The RAS proto-oncogenes (HRAS, KRAS and NRAS) encode a family of GDP/GTP-regulated switches that convey
extracellular signals to regulate the growth and survival properties of cells. GTP-bound RAS transmits its signal
through downstream EGFR signaling pathways, for example the RAF→MEK→ERK and PI3K→AKT cascades.
RAS family members are frequently found in their mutated, oncogenic forms in human tumors. In total, activating
mutations in the NRAS genes occur in 13~25% of cutaneous melanomas, 1~6% of colorectal cancer and 1% of
lung cancer, mainly in exons 2, 3 or 4. The mutation status of NRAS gene is relevant to drug resistance of
non-small cell lung cancer treated with tyrosine kinase inhibitors. As a result, NRAS mutations detection supplies
evidence for targeted clinical therapy of tumor patients, decreases cost and time of treatment.
The AmoyDx® NRAS Mutation Detection Kit is a real-time PCR test intended for qualitative detection of 16
hotspot somatic mutations in codons 12, 13, 59, 61, 117 and 146 of NRAS gene. Our company’s patented
technology allows detection of 1~2% mutant DNA in a background of 98~99% normal DNA at 10 ng sample DNA
amount, while ensuring that false negatives are minimized. The purpose of the kit is to aid doctors to identify lung
cancer or colorectal cancer patient carrying mutations in NRAS gene.
Intended Use
The AmoyDx® NRAS Mutation Detection Kit is a highly sensitive test designed to accurately identify 16 NRAS
mutations in codons 12, 13, 59, 61, 117 and 146 in human tissue samples.
Kit Contents
This kit contains sufficient reagents to carry out 8 tests (Table1).
Table 1 Kit Contents
Contents
Volume
NRAS PCR Reaction Mix
12 strips
NRAS Enzyme Mix
NRAS Positive Control
45µL
250 µL
One 8-tube strip is employed for each sample (Table 2).
Table 2 Mutation information for each strip
Tube No.
Mutation Name
Volume (µL)
Channel
①
NRAS Reaction Mix 1
35
FAM, HEX/VIC
②
NRAS Reaction Mix 2
35
FAM, HEX/VIC
③
NRAS Reaction Mix 3
35
FAM, HEX/VIC
④
NRAS Reaction Mix 4
35
FAM, HEX/VIC
⑤
NRAS Reaction Mix 5
35
FAM, HEX/VIC
⑥
NRAS Reaction Mix 6
35
FAM, HEX/VIC
⑦
NRAS Reaction Mix 7
35
FAM, HEX/VIC
⑧
NRAS External Control Reaction Mix
35
FAM
Note:
For milky white strip: Distinguish Tube ⑧ from Tube ① according to the right middle hole of strip edge,
described as follows.
˙①②③④⑤⑥⑦⑧·
For other stripes: the number is marked on the side of the tube.
Manufacturer: Amoy Diagnostics Co., Ltd.
Website: http://www.amoydx.com
E-mail: [email protected]
Tel: +86 592 6806835
Address: 39 Dingshan Road, Haicang District, Xiamen 361027, P. R. China
Fax: +86 592 6806839
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For: ADx-NR02
Equipment and Reagents Not Supplied With Kit
1. The compatible PCR instruments are:
Stratagene Mx3000P™/Mx3005P™, ABI7500, LightCycler480, ABI7900HT, Bio-Rad CFX96 and
SLAN-96S.
(1) 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 assay on a LightCycler machine,
please use the Roche 480 adaptor, available from BIOplastics, Cat No. B79480.
(2) For ABI instruments, please set up as follows: Reporter Dye: FAM, VIC; Quencher Dye: TAMRA;
Passive Reference: NONE.
(3) To run the assay on ABI7900HT machine, please use the ABI7900HT adaptor, available from
BIOplastics, Cat No. B7900RAN.
(4) For ABI7900HT, please set up as follows: Instrument: Standard, Ramp speed: Standard, Reaction volume:
40 µL.
(5) For Stratagene Mx3000P™/Mx3005P™, if there’s low net fluorescence signal (dR) but high background
signal (R), please reduce the signal gain setting of instrument properly.
(6) For SLAN-96S, please set up as follows: Probe mode: FAM, VIC. During the result analysis, open the
“Preference” window, in “Chart Options” section, select “Selected Wells” for “Y-Axis Scaling
Auto-adjust By” and “Absolute Fluorescence Value Normalization” for “Amplification Curve”.
(7) We recommend that all PCR instruments in use should be conducted fluorescence calibration once a year.
2. Sterile, nuclease-free tubes.
3. Dedicated pipettes and filtered pipette tips for handling DNA.
4. Sterile, nuclease-free H2O.
Shipping and Storage
The kit requires cold-chain-transportation. All contents of the kit should be stored immediately upon receipt at
-20±5℃. Avoid unnecessary freezing and thawing of the contents of the kit. Do not use the reagent after five
freeze-thaw cycles. Once opened, this reagent is stable at -20±5℃ until the expiry.
Stability
The shelf-life of the kit is 8 months when stored under the recommended conditions and in the original packaging.
Do not use the kit after the stated expiry date.
Specimen Material
Human genomic DNA should be extracted from tissue or formalin-fixed paraffin-embedded (FFPE) samples and
stored at -20±5℃ prior to use. The FFPE samples should be made and stored following proper procedures. The
slides should preferably be less than 3 years. Before extraction of DNA, it is very important to make sure that there
is at least 30% tumor cells in the tissue samples. If the extracted DNA is not used immediately, it should be stored
at below -20℃ for no more than 6 months.
Selection of high quality DNA extraction reagents is essential for the kit. We recommend use of DNA extraction
kit (AmoyDx® FFPE DNA Kit, Cat No. ADx-FF01, for paraffin embedded specimens; AmoyDx® Tissue DNA Kit,
Cat No. ADx-TI01, for tissue and pleural effusion specimens). The OD value of DNA samples should be measured
using the spectrophotometer after extraction. The NanoDrop 1000 /2000 spectrophotometer is recommended.
Make sure A260/A280 value between 1.8 and 2.0.
Manufacturer: Amoy Diagnostics Co., Ltd.
Website: http://www.amoydx.com
E-mail: [email protected]
Tel: +86 592 6806835
Address: 39 Dingshan Road, Haicang District, Xiamen 361027, P. R. China
Fax: +86 592 6806839
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For: ADx-NR02
Technological Principles
The kit uses novel, patented primers and probes to detect mutations in a real-time PCR assay. The mutant DNA is
amplified accurately by the specific primers, and detected by the novel probes. Highly Specific primers and probes,
and a highly validated procedure based on Taq DNA polymerase contribute to outstanding assay sensitivity and
selectivity.
Protocol
1.
The reaction mix tubes contain the reaction buffer, dNTPs, specific oligos and probes.
2.
The NRAS Reaction Mix 1~7 includes a mutation detection system and an internal control system. The
mutation detection system is used to detect the mutation status of NRAS gene (positive or negative). The
internal control system is designed to detect the presence of inhibitors and monitor the accuracy of
experimental operation, which may lead to false negative results.
3.
The NRAS External Control Reaction Mix is used to assess the DNA quality, that is, to reveal the presence
of PCR inhibitors or compromised DNA integrity that may lead to false negative results.
4.
The NRAS Positive Control contains a recombinant NRAS gene with the 16 mutations, and normal human
genomic DNA.
5.
The NRAS Enzyme Mix contains the Taq DNA polymerase for PCR amplification and uracil-N-glycosylase
which works at room temperature to prevent PCR amplicon carryover contamination.
Experimental Procedure
1.
Thaw NRAS Positive Control at room temperature. When the reagents completely thawed, mix the reagents
by inverting the tube 10 times and centrifuge briefly to collect the contents at the bottom of the tube.
2.
Briefly centrifuge NRAS Enzyme Mix prior to use.
3.
Add 2.7 µL NRAS Enzyme Mix into the following solutions individually: 42.3 µL DNA sample (see
following for DNA sample concentrations), 42.3 µL NRAS Positive Control (PC) and 42.3 µL no-template
controls (sterile water, NTC).
Note:

The volumes given for each reaction mix have been optimized and validated. Changing volumes of
any reagent may result in a loss of performance.

Do not store user-prepared mixes, use immediately.

Since Enzyme Mix is viscous, please pay attention to the centrifugation and pipetting process.

Minimize the contact interface between the pipette tip and Enzyme Mix to avoid adding excess
enzyme.
4.
Mix each solution thoroughly by gently pipetting up and down more than 10 times, and then centrifuge
briefly.
Note: avoid vortexing solutions with NRAS Enzyme Mix.
5.
According to different sources, samples can be divided into two groups: paraffin embedded and non-paraffin
embedded specimens.
(1) Non-paraffin embedded specimens include fresh tissue, and frozen pathological sections.
a) For non-paraffin embedded samples, the recommended DNA amount in each PCR tube is 1.88 ~ 4.7
ng (concentration 0.4~1 ng/µL).
(2) For paraffin embedded samples, we recommend use of 7.05~14.1 ng template DNA (concentration 1.5~3
ng/μL) in each PCR tube depending on storage times.
a) Use 7.05 ng template DNA (concentration 1.5 ng/µL) for samples with less than 3 months storage
time.
Manufacturer: Amoy Diagnostics Co., Ltd.
Website: http://www.amoydx.com
E-mail: [email protected]
Tel: +86 592 6806835
Address: 39 Dingshan Road, Haicang District, Xiamen 361027, P. R. China
Fax: +86 592 6806839
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For: ADx-NR02
b) Use 9.4 ng template DNA (concentration 2 ng/µL) for samples with storage time between 3 months
and 1 year.
c) Use 11.75~14.1 ng template DNA (concentration 2.5~3 ng/µL) with storage time between 1 year and
3 years.
Note:

Avoid use DNA for samples with more than 3 years storage time.

We recommend use of TE (pH = 8.0) for extracted DNA dilution. Suggest using at least 5 µL DNA
for dilution within ten-fold series, to ensure the accuracy of final concentration.
6.
Take out the NRAS PCR Reaction Mix strips and centrifuge the strips if there are any reagent droplets in the
caps of the PCR tubes. Then briefly uncover the caps prior to use.
7.
Transfer 5 µL of the above mixed solution to each PCR tube of 8-tube strip. Add the solution to the side of
the tube wall above the reagents in the tube, and then seal the strips.
8.
Spin down the PCR tubes gently or centrifuge them briefly to collect the reagents at the bottom of wells.
Note: this spin or centrifuge step is essential for proper mixing of the reagents.
9.
Place the PCR tubes into the real-time PCR instrument. A recommended plate layout is shown in Table 3.
Note: place the PCR tubes into the real-time PCR instrument and start to run immediately. If not, please store
the PCR tubes at 4℃ for no more than 12 hours.
Table 3 Suggested PCR Plate Layout
8.
Tube
1
2
3
…
8
9
10
11
12
①
Sample1
Sample2
Sample3
…
Sample8
Sample9
Sample10
PC
NTC
②
Sample1
Sample2
Sample3
…
Sample8
Sample9
Sample10
PC
NTC
③
Sample1
Sample2
Sample3
…
Sample8
Sample9
Sample10
PC
NTC
④
Sample1
Sample2
Sample3
…
Sample8
Sample9
Sample10
PC
NTC
⑤
Sample1
Sample2
Sample3
…
Sample8
Sample9
Sample10
PC
NTC
⑥
Sample1
Sample2
Sample3
…
Sample8
Sample9
Sample10
PC
NTC
⑦
Sample1
Sample2
Sample3
…
Sample8
Sample9
Sample10
PC
NTC
⑧
Sample1
Sample2
Sample3
…
Sample8
Sample9
Sample10
PC
NTC
Carry out real-time PCR using the cycling conditions described in Table 4.
Note:

The reaction volume is 40 μL per well.

Please pack the post-PCR tubes with two disposable gloves and discard properly. Do NOT open
the post-PCR tubes to avoid contamination.
Table 4 Cycling Parameters
Stage
Temperature
Time
Cycles
1
95℃
5min
1
95℃
25s
64℃
20s
72℃
20s
93℃
25s
60℃
35s ☆Data collection of FAM and HEX/VIC
72℃
20s
2
3
Manufacturer: Amoy Diagnostics Co., Ltd.
Website: http://www.amoydx.com
E-mail: [email protected]
Tel: +86 592 6806835
Address: 39 Dingshan Road, Haicang District, Xiamen 361027, P. R. China
15
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Sample Data Analysis
1.
The FAM signals of the mutation detection system indicate the mutation status of the sample. The HEX/VIC
signals indicate the internal control status.
2.
Ensure the calibration fluorescence is unselected, and select single mutation detection for each tube
accordingly. It is necessary to choose the reaction wells for positive control, no-template control and sample
simultaneously. Then users could adjust the threshold of FAM amplification curve, and obtain the Ct value
of mutant group.
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, the sample is classed as positive. If the Ct value is outside the
appointed range, the sample is classed as negative or below the detection limit of the kit.
4.
Assess each NTC Ct value to ensure that there is no positive amplification, if the NTC has positive
amplification, the data must be discarded as there is contamination. If the HEX/VIC signal of Tube ①~⑦
and FAM signal of Tube ⑧ occasionally rises, further analysis could be carried out.
5.
The NRAS Positive Control FAM Ct value should be less than 20, but variation may occur due to different
threshold settings on different instruments.
6.
If the HEX/VIC signals of sample fails but the FAM signal works well, continue with the analysis. If both
the HEX/VIC and FAM signals fail, the obtained data must be discarded and the experiment should be
repeated.
7.
Check the FAM signal of the external control assay:
(1) Ct value should be between 15~21 for paraffin embedded specimens, and between 13~19 for
non-paraffin embedded specimens.
(2) If the requirements of item (1) are satisfied, further analysis shall be carried out. However, if Ct value is
below the corresponding range, it indicates the DNA is overloaded, so the amount of DNA should be
reduced. If all the results of seven tubes (①-⑦) are negative, the sample is classified as negative.
(3) If the external control assay fails, it shows that the DNA template contains PCR inhibitors, indicating
that the DNA needs to be re-extracted. If the result in any of the seven tubes (①-⑦) is Strong positive,
the sample is classified as positive.
8.
Analysis of mutation assay results (see Table 5):
Check the FAM Ct value for each sample. Based on different mutant Ct values, the detection results are
divided into strong positive, weak positive or negative.
(1) Strong Positive: if the sample FAM Ct value is less than the Ct value shown in the “Strong Positive” in
Table 5, the sample is classified as strong positive.
(2) Weak Positive: if the sample FAM Ct value is in the range shown in the “Weak Positive” 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. If the ∆Ct value is less than the corresponding Cut-off value of ∆Ct, the sample is
confirmed as weak positive. Otherwise, the sample is classified as negative or below the limits of the
kit.
b) The calculation of ∆Ct: ∆Ct = mutant FAM Ct value – external control FAM Ct value. The
mutant FAM Ct value indicates the Ct value of the mutant FAM signal from a sample. The external
control FAM Ct value indicates the Ct value of the FAM signal in external control tube of that
sample.
Manufacturer: Amoy Diagnostics Co., Ltd.
Website: http://www.amoydx.com
E-mail: [email protected]
Tel: +86 592 6806835
Address: 39 Dingshan Road, Haicang District, Xiamen 361027, P. R. China
Fax: +86 592 6806839
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For: ADx-NR02
(3) Negative: if the sample FAM Ct value is greater than or equal to the critical negative value shown in the
“Negative” in Table 5, the sample is classified as negative or below the detection limit of the kit.
Table 5 Results Determination
①
②
③
④
⑤
⑥
⑦
Mutant Ct Value
Ct <26
Ct <26
Ct <26
Ct <26
Ct <26
Ct <26
Ct <26
Mutant Ct Value
26≤Ct <28
26≤Ct <29
26≤Ct <29
26≤Ct <29
26≤Ct <28
26≤Ct <29
26≤Ct <29
∆Ct Cut-off value
9
10
10
11
9
11
9
Mutant Ct Value
Ct≥28
Ct ≥29
Ct ≥29
Ct ≥29
Ct ≥28
Ct ≥29
Ct ≥29
Tube No.
Strong
Positive
Weak
Positive
Negative
Performance Characteristics
The performance characteristics of this kit were validated on Stratagene Mx3000P™/Mx3005P™, ABI7500,
LightCycler480, ABI StepOnePlus, ABI7900HT, Bio-Rad CFX96.
1.
Sensitivity: the kit allows detection of 1% mutant DNA in a background of 99% normal DNA at 10 ng sample
DNA amount. Except: The sensitivity of NRAS-M1, NRAS-M2 and NRAS-M12 on LightCycler480 and
Bio-Rad CFX96 are 2% at 10 ng sample DNA amount.
2.
Productivity: the kit can be used to analysis 10 samples maximum. (see Table 6)
Table 6 Sample Qty detected with the Kit
3.
PCR Run(s)
Control Qty
Total samples can be detected
1 run
2 (2 controls/run * 1)
10
2 runs
4 (2 controls/run * 2)
8
3 runs
6 (2 controls/run * 3)
6
Accuracy: Accuracy of the kit was established by testing 16 mutant positive reference controls and 10
negative reference controls, the detection concordance rate are 100%.
4.
Precision: Precision of the kit was established by performing a certain mutant positive reference control for 10
repeats; all the controls can be detected.
Warnings and Precautions
1.
Please read the instruction carefully and become familiar with all components of the kit prior to use.
2.
The product specified above does not contain any virus, reagent by-product of the same or metabolic
by-product of Hepatitis A, B, C, D or HIV.
3.
Do not exchange and mix up the kit contents with different batches.
4.
The kit and its contents cannot be resold or modified for resale without the written approval of manufacturer.
5.
Using other sources of reagents is not recommended. Strictly distinguish the reagents from Positive Control
to avoid contamination. Otherwise, false positive may be produced.
6.
Do the experiments with attention to prevent exogenous DNA contamination to reagents. It is recommended
that, use separate, dedicated pipettes and filtered pipette tips to add DNA template during the preparation of
reagents.
7.
To optimize the activity and performance, mixtures should always be protected from light to avoid photo
bleaching.
8.
All the chemicals are potential hazard, only trained professionals could use this kit. Please wear suitable lab
coat and disposable gloves. The used kit should be disposed properly.
Manufacturer: Amoy Diagnostics Co., Ltd.
Website: http://www.amoydx.com
E-mail: [email protected]
Tel: +86 592 6806835
Address: 39 Dingshan Road, Haicang District, Xiamen 361027, P. R. China
Fax: +86 592 6806839
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For: ADx-NR02
Notes
1.
Symbol for "AUTHORISED REPRESENTATIVE IN THE EUROPEAN COMMUNITY"
2.
Symbol for "IN VITRO DIAGNOSTIC MEDICAL DEVICE"
3.
Symbol for "KEEP DRY"
4.
Symbol for "THIS WAY UP"
5.
Symbol for "FRAGILE，HANDLE WITH CARE"
Information of European Authorised Representative
Wellkang Ltd t/a Wellkang Tech Consulting Suite B, 29 Harley Street, London W1G 9QR United Kingdom
References
1.
Brose MS, Volpe P, Feldman M, et al. BRAF and RAS Mutations in Human Lung Cancer and Melanoma. Cancer
Res.2002,62:6997-7000.
2.
Ascierto PA, Schadendorf D,Berking C,et al.MEK162 for patients with advanced melanoma harbouring NRAS or Val600 BRAF
mutations: a non-randomised, open-label phase 2 study. Lancet Oncol. 2013, 14: 249–56.
3.
Kadoaki Ohashi, Lecia V, Sequist, et al. Characteristics of Lung Cancers Harboring NRAS Mutations.Clin Cancer
Res.2013,19:2584–91.
4.
Douillard JY,Oliner KS,Siena S,et al. Panitumumab-FOLFOX4 Treatment and RAS Mutations in Colorectal Cancer.The New
England Journal of Medicine.2013,369 (11):1023-34.
Manufacturer: Amoy Diagnostics Co., Ltd.
Website: http://www.amoydx.com
E-mail: [email protected]
Tel: +86 592 6806835
Address: 39 Dingshan Road, Haicang District, Xiamen 361027, P. R. China
Fax: +86 592 6806839
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For: ADx-NR02
Appendix I
NRAS Mutations detected with the Kit
Tube No.
Reagent Supplied
Exon
1
NRAS Reaction Mix 1
2
2
3
4
5
6
7
NRAS Reaction Mix 2
NRAS Reaction Mix 3
NRAS Reaction Mix 4
NRAS Reaction Mix 5
NRAS Reaction Mix 6
NRAS Reaction Mix 7
2
2
3
Mutation
Base Change
Cosmic ID
Name
G12D
35G>A
564
NRAS-M3
G12S
34G>A
563
NRAS-M10
G13D
38G>A
573
NRAS-M4
G13R
37G>C
569
NRAS-M6
G12C
34G>T
562
NRAS-M7
G12V
35G>T
566
NRAS-M9
G12A
35G>C
565
NRAS-M11
G13V
38G>T
574
NRAS-M14
A59D
176C>A
253327
NRAS-M15
Q61R
182A>G
584
NRAS-M1
Q61K
181C>A
580
NRAS-M2
Q61L
182A>T
583
NRAS-M5
Q61H
183A>C
586
NRAS-M8
K117N
351G>C
\
NRAS-M16
K117N
351G>T
\
NRAS-M17
A146T
436G>A
27174
NRAS-M12
3
4
4
Manufacturer: Amoy Diagnostics Co., Ltd.
Website: http://www.amoydx.com
E-mail: [email protected]
Tel: +86 592 6806835
Address: 39 Dingshan Road, Haicang District, Xiamen 361027, P. R. China
Fax: +86 592 6806839
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