For: ADx-AR01
AmoyDx® ALK Gene Fusions and ROS1 Gene Fusions
Detection Kit
For qualitative detection of 26 ALK gene fusions and 14 ROS1 gene fusions
Instruction for Use
Instruction Version:
P1.1
Revision Date:
June 2014
Store at -20±5℃
Manufacturer: Amoy Diagnostics Co., LTD
Website: http://www.amoydx.com/en
E-mail: sales@amoydx.com
Tel: +86 592 6806835
Fax: +86 592 6806839
Address: 5th Floor, No.4 Plant, Kechuang Center Building, 289 Wengjiao Road, Xinyang Street, Haicang District,
Xiamen, Fujian 361026, PR China
For: ADx-AR01
Background
ALK gene fusions and ROS1 gene fusions define unique molecular subsets of non–small-cell lung cancer (NSCLC).
EML4, KIF5B, TFG, KLC1 etc., are identified as fusion partners of ALK; SLC34A2, CD74, SDC4, EZR etc., are
identified as fusion partners of ROS1. These fusions lead to constitutive kinase activity and activation of
downstream pathways, leading to carcinogenesis. It has been reported that the presence of the ALK gene fusions
and ROS1 gene fusions are correlated with the efficacy of TKI therapy. Based on analysis of tumor messenger
RNA, ALK gene fusions and ROS1 gene fusions can be detected by real-time PCR method.
Intended Use
The AmoyDx® ALK Gene Fusions and ROS1 Gene Fusions Detection Kit is an in vitro nucleic acid amplification
test intended for qualitative detection of 26 ALK gene fusions and 14 ROS1 gene fusions in human NSCLC
formalin-fixed paraffin-embedded (FFPE) tissue samples.
Kit Contents
The kit contains sufficient reagents to carry out 8 tests (Table 1).
Table 1 Kit Contents
Reagents Supplied
Volume
ALK&ROS1 PCR Reaction Mix
12 strips
ALK RT Reaction Mix (Red)
8×18.5 μL
ROS1 RT Reaction Mix (Green)
8×18.5 μL
ALK&ROS1 Reverse Transcriptase
12 μL
ALK&ROS1 Enzyme Mix
45 µL
ALK&ROS1 Positive Control
250 µL
One 8-tube strip is employed for each sample or control (Table 2). The ALK gene fusions and ROS1 gene fusions
in Tubes ①~⑧ are indicated by FAM signal and reference gene in Tube ④/⑧ is indicated by HEX(VIC) signal.
Note:
•
Distinguish Tube ⑧ from Tube ① according to the right middle hole of strip edge, as depicted following.
˙①②③④⑤⑥⑦⑧·
•
ALK RT Reaction Mix in the Red PCR tubes, and ROS1 RT Reaction Mix in the Green PCR tubes.
Table 2 Detection Information for Each Strip
Tube No.
Detected target
Volume (µL)
Channel
①
ALK-1
35
FAM
②
ALK-2
35
FAM
③
ALK-3
35
FAM
④
ALK-4 & HPRT1
35
FAM & HEX/VIC
⑤
ROS1-1
35
FAM
⑥
ROS1-2
35
FAM
⑦
ROS1-3
35
FAM
⑧
ROS1-4 & HPRT1
35
FAM & HEX/VIC
Manufacturer: Amoy Diagnostics Co., LTD
Website: http://www.amoydx.com/en
E-mail: sales@amoydx.com
Tel: +86 592 6806835
Fax: +86 592 6806839
Address: 5th Floor, No.4 Plant, Kechuang Center Building, 289 Wengjiao Road, Xinyang Street, Haicang District,
Xiamen, Fujian 361026, PR China
1/7
For: ADx-AR01
Equipment and Reagents Not Supplied With the Kit
1.
Compatible PCR instruments: Stratagene Mx3000P™, ABI7500, ABI7900HT and LightCycler480.
(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 ABI7900 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™, if there’s low net fluorescence signal (dR) but high background signal (R),
please reduces the signal gain setting of instrument properly.
(6)
We recommend that all PCR instruments in use should be conducted fluorescence calibration once a
year.
2.
Nuclease-free tubes.
3.
Dedicated pipette and filter pipette tips for handling RNA&DNA template.
4.
Nuclease-free water.
5.
RNA extraction reagents.
Shipping and Storage
The kit requires cold-chain-transportation. All contents of the kit should be stored immediately upon receipt at
-20±5℃ in the dark in a constant temperature freezer. 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 eight 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 total RNA should be extracted from NSCLC FFPE samples with 5-10 µm thickness. The FFPE samples
should be prepared and stored less than two years following proper procedures. Before extraction of RNA, it is
very important to make sure that there is at least 30% tumor cells in the FFPE tissue samples. RNA should be
stored below -70℃ for no more than one week if no used immediately. High quality RNA is essential for the kit
so we recommend use of AmoyDx RNA extraction kit (AmoyDx® FFPE RNA Kit, Cat No. ADx-FF04, for
paraffin embedded specimens). The OD value of RNA samples should be measured using the spectrophotometer
after extraction.
We recommend:
•
Total RNA concentration should be between 50 and 800 ng/μL.
•
OD260/OD280 value should be between 1.8 and 2.1.
(Optional) Store the cDNA
Please store the cDNA at -20±5℃ less than one week after reverse transcription if not proceeded to next step.
Manufacturer: Amoy Diagnostics Co., LTD
Website: http://www.amoydx.com/en
E-mail: sales@amoydx.com
Tel: +86 592 6806835
Fax: +86 592 6806839
Address: 5th Floor, No.4 Plant, Kechuang Center Building, 289 Wengjiao Road, Xinyang Street, Haicang District,
Xiamen, Fujian 361026, PR China
2/7
For: ADx-AR01
Technological Principles
The kit is intended for the qualitative detection of ALK gene fusions and ROS1 gene fusions in NSCLC FFPE
samples.
The kit is based on three major processes: (1) specimen preparation to isolate total RNA from NSCLC FFPE
samples; (2) reverse transcription of target RNA to generate complementary DNA (cDNA); (3) simultaneous PCR
amplification of target cDNA to detect ALK gene fusions and ROS1 gene fusions with specific primers and
fluorescent probes.
Reverse Transcription
ALK RT Reaction Mix contains primers specific for reverse transcription of ALK RNA and reference gene RNA
into cDNA, ROS1 RT Reaction Mix contains primers specific for reverse transcription of ROS1 RNA and
reference gene RNA into cDNA.
Real-time PCR
The ALK&ROS1 PCR Reaction Mix ①-④ contain primers and FAM-labeled probes specific for ALK gene
fusions and the ALK&ROS1 PCR Reaction Mix ⑤-⑧ contain primers and FAM-labeled probes specific for
ROS1 gene fusions, the ALK&ROS1 PCR Reaction Mix ④/⑧ also contains primers and HEX(VIC)-labeled
probe for detection of housekeeping gene HPRT1 as reference gene to reveal the presence of PCR inhibitors or
compromised RNA integrity that may lead to false negative results. ALK&ROS1 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.
Protocol
1.
Reverse Transcription
(1)
Thaw ALK RT Reaction Mix and ROS1 RT Reaction Mix at room temperature.
(2)
Centrifuge ALK&ROS1 Reverse Transcriptase, ALK RT Reaction Mix and ROS1 RT Reaction
Mix prior to use.
(3)
For each RNA sample, transfer 0.5 μL ALK&ROS1 Reverse Transcriptase into a ALK RT Reaction
Mix tube and a ROS1 RT Reaction Mix tube respectively.
(4)
Add 6 μL sample RNA into ALK RT Reaction Mix tube and ROS1 RT Reaction Mix tube
respectively; mix well by gently pipetting up and down, and then centrifuge briefly.
(5)
Incubate the tubes at 42℃ for one hour.
(6)
Heat the tubes at 95℃ for 5 minutes, then transfer them to ice. The resulting ALK cDNA and ROS1
cDNA solutions are ready for PCR amplification.
2.
PCR amplification
(1)
Thaw ALK&ROS1 Positive Control (PC) at room temperature.
(2)
Centrifuge ALK&ROS1 Enzyme Mix and ALK&ROS1 Positive Control (PC) prior to use.
(3)
Transfer 1.5 μL ALK&ROS1 Enzyme Mix into ALK cDNA and ROS1 cDNA respectively. Transfer
3μL ALK&ROS1 Enzyme Mix into 50 μL ALK&ROS1 Positive Control (PC) and 50 μL ddH2O
(NTC) respectively.
(4)
Mix well by inverting 10 times and centrifuge briefly.
(5)
Add 5 μL sample ALK cDNA Mix into ALK&ROS1 PCR Reaction Mix ①-④, 5 μL sample ROS1
cDNA Mix into ALK&ROS1 PCR Reaction Mix ⑤-⑧. Add 5 μL ALK&ROS1 Positive Control Mix
and 5 μL ddH2O Mix into a strip of ALK&ROS1 PCR Reaction Mix ①-⑧ respectively.
Manufacturer: Amoy Diagnostics Co., LTD
Website: http://www.amoydx.com/en
E-mail: sales@amoydx.com
Tel: +86 592 6806835
Fax: +86 592 6806839
Address: 5th Floor, No.4 Plant, Kechuang Center Building, 289 Wengjiao Road, Xinyang Street, Haicang District,
Xiamen, Fujian 361026, PR China
3/7
For: ADx-AR01
(6)
Seal the PCR tubes and spin the PCR tubes gently to collect the reagents at the bottom of tubes.
Note: this spin step is essential for proper mixing of the reagents.
(7)
Immediately place the PCR tubes into the real-time PCR instrument. A recommended plate layout is
shown in Table 3.
Table 3 Plate Layout (example for 8 tests)
Tube
No.
(8)
1
2
3
4
5
6
7
8
9
10
①
Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8
PC
NTC
②
Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8
PC
NTC
③
Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8
PC
NTC
④
Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8
PC
NTC
⑤
Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8
PC
NTC
⑥
Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8
PC
NTC
⑦
Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8
PC
NTC
⑧
Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8
PC
NTC
Carry out real-time PCR procedure 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 PCR Cycling Parameters
Stage
Temperature
Time
Cycles
1
95℃
5 min
1
95℃
25s
64℃
20s
72℃
20s
93℃
25s
60℃
35s ☆Data collection of FAM and HEX(VIC)
72℃
20s
2
3
15
31
Sample Data Analysis
1.
The FAM signal indicates the sample ALK gene fusions and ROS1 gene fusions status in Tube ①~⑧ and
the HEX(VIC) signal indicates the reference gene RNA status in Tube ④/⑧.
2.
Ensure the calibration fluorescence is unselected. Select the sample and controls as a group, and then adjust
the threshold separately for FAM and HEX/VIC amplification curves to obtain the Ct values of the samples.
3.
Assess each NTC Ct value to ensure that there is no a positive amplification, if the NTC has positive
amplification, the data must be discarded as there may be contamination.
4.
Assess each Positive Control (PC) Ct value to ensure there is a positive amplification, and the Ct value
should be less than 24. Otherwise, the data must be discarded.
5.
Analysis of the sample reference gene assay (HEX/VIC) in Tube ④/⑧, there are 3 possible outcomes:
1)
If both the sample reference gene Ct values in Tube ④/⑧ ≤20, then continue with the analysis.
Manufacturer: Amoy Diagnostics Co., LTD
Website: http://www.amoydx.com/en
E-mail: sales@amoydx.com
Tel: +86 592 6806835
Fax: +86 592 6806839
Address: 5th Floor, No.4 Plant, Kechuang Center Building, 289 Wengjiao Road, Xinyang Street, Haicang District,
Xiamen, Fujian 361026, PR China
4/7
For: ADx-AR01
2)
If any of the sample reference gene Ct value >20 which indicates the partial fragmentation or
degradation of RNA, but if the FAM Ct value located in positive area, the result is believable,
otherwise, we suggest re-extracting the RNA and doing this experiment again.
3)
If any of the sample reference signal assay fails, the data must be discarded as there may be inhibitors
present, or the sample RNA has been fragmented or degraded which is not suitable for the kit. Please
re-extract the RNA and do this experiment again.
6.
7.
Analysis of fusion assay results, there are 2 possible outcomes:
1)
If the sample FAM Ct value ≥30, the sample contains no ALK gene fusions and ROS1 gene fusions.
2)
If the sample FAM Ct value <30, the sample contains gene fusion.
a)
If ①~④ FAM Ct value <30, the sample contains ALK gene fusion;
b)
If ⑤~⑧ FAM Ct value <30, the sample contains ROS1 gene fusion.
The sample may contain two or more fusion patterns simultaneously.
Performance Characteristics
The performance characteristics of this kit were validated on Stratagene Mx3000P™, ABI7500, ABI7900HT and
LightCycler480.
1.
Sensitivity: the kit allows detection of 25 copies/μL ALK gene fusions plasmid DNA and ROS1 gene fusions
plasmid DNA.
2.
Specificity: the kits have been validated of non-specificity amplification at a background of 500 ng wild-type
DNA or 9600 ng wild-type RNA.
3.
Accuracy: Accuracy of the kit was established by testing ALK&ROS1 fusion-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 ALK&ROS1 fusion-positive reference
control for 10 repeats, CV (coefficient of variation) ≤10%.
5.
Productivity: the kit can be used to analysis 8 samples maximum (see Table 5).
Table 5 Sample Qty detected with the Kit
PCR Run(s)
Control Qty
Total samples can be detected
1 run
2 (2 controls/run * 1)
8
2 runs
4 (2 controls/run * 2)
8
3 runs
6 (2 controls/run * 3)
6
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 re-sale without the written approval of the
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 RNase contamination to reagents. It is
recommended that users have separate, dedicated pipettes and filter pipette tips to add RNA template during
the preparation of reagents.
Manufacturer: Amoy Diagnostics Co., LTD
Website: http://www.amoydx.com/en
E-mail: sales@amoydx.com
Tel: +86 592 6806835
Fax: +86 592 6806839
Address: 5th Floor, No.4 Plant, Kechuang Center Building, 289 Wengjiao Road, Xinyang Street, Haicang District,
Xiamen, Fujian 361026, PR China
5/7
For: ADx-AR01
7.
To optimize the activity and performance, mixtures should always be protected from light to avoid photo
bleaching.
8.
Only trained professionals could use this kit. Please wear suitable lab coat and disposable gloves. The used
kit should be disposed of properly.
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.
Sasaki T et al., Eur J Cancer. 2010;46:1773-80.
2.
OU SH et al., Oncologist. 2012;17:1351-1375.
3.
Bergethon K et al., J Clin Oncol.2012;46:863-70.
4.
Tan J et al., Expert Rev Anticancer Ther.2012;12:447-56.
Manufacturer: Amoy Diagnostics Co., LTD
Website: http://www.amoydx.com/en
E-mail: sales@amoydx.com
Tel: +86 592 6806835
Fax: +86 592 6806839
Address: 5th Floor, No.4 Plant, Kechuang Center Building, 289 Wengjiao Road, Xinyang Street, Haicang District,
Xiamen, Fujian 361026, PR China
6/7
For: ADx-AR01
Appendix I
ALK gene fusions and ROS1 gene fusions detected with the Kit
Tube No.
①
②
Detection Target
ALK-1
ALK-2
Fusion patterns
EML4-ALK
EML4-ALK
KIF5B-ALK
Abbreviation of Fusion patterns
E6ins33;A20;
E13;A20
E20;A20
E6;ins18A20
E6;A19
E13;ins69A20
E6;A20
E20;ins18A20
E14ins11;del49A20
E14;del14A20
KI15;A20
E2;A20
E2;ins117A20
③
ALK-3
EML4-ALK
E3;ins53A20
E17ins61;ins34A20
E17del58;ins39A20
④
⑤
ALK-4 & HPRT1
ROS1-1
KIF5B-ALK
KI17;A20
KLCL1-ALK
KI24;A20
TFG-ALK
T4;A20
SLC34A2-ROS1
SL4;R32
CD74-ROS1
SL14del;R32
SDC4
CD6;R32
SLC34A2-ROS1
⑥
ROS1-2
CD74-ROS1
SDC4-ROS1
EZR-ROS1
⑦
⑧
ROS1-3
ROS1-4 & HPRT1
SL4;R34
SL14del;R34
CD6;R34
TPM3-ROS1
TP8;R35
LRIG3-ROS1
LR16;R35
GOPC-ROS1
GO8;R35
GOPC-ROS1
GO4;R36
E15del60;del71A20
E14;del38A20
E17;ins68A20
E18;A20
E17;ins30A20
E17ins65;A20
KL9;A20
SD2;R32
SD4;R32
SD4;R34
EZ10;R34
Manufacturer: Amoy Diagnostics Co., LTD
Website: http://www.amoydx.com/en
E-mail: sales@amoydx.com
Tel: +86 592 6806835
Fax: +86 592 6806839
Address: 5th Floor, No.4 Plant, Kechuang Center Building, 289 Wengjiao Road, Xinyang Street, Haicang District,
Xiamen, Fujian 361026, PR China
7/7