CN108531598A - ROS1 Gene Fusions detection primer, method and kit - Google Patents
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Abstract
The invention discloses ROS1 Gene Fusions detection primer, method and kit, which contains SEQ ID NO:Primer sets and probe shown in 1~24.The present invention can detect up to 13 kinds of ROS1 Gene Fusion types.Primer and probe is by optimization and screening, and sensitivity is up to 100copies/ μ l, and specificity is high.Internal standard is monitored for system reverse transcription and amplification, ensures that detection is normally carried out.In addition, kit also includes negative and positive control.Multiple Quality Control avoids testing result from false positive and false negative occur.
Description
Technical field
The invention belongs to biotechnologies, and in particular to ROS1 Gene Fusions detection primer, method and kit.
Background technology
Lung cancer is to endanger one of maximum malignant tumour, incidence and death to human health and life in the world today
Rate occupies malignant tumour first, wherein about 80%~85% patients with lung cancer is non-small cell lung cancer (non-small cell lung
cancer,NSCLC).In China, lung cancer is also the first big tumour, accounts for the 25.0% of whole mortality of malignant tumors.And incidence and
The death rate is still rising successively, according to statistics, increases cases of lung cancer newly every year up to 500,000~600,000.The World Health Organization (WHO) prediction,
By 2025, newly-increased cases of lung cancer will be more than 1,000,000 every year for China, become the first in the world lung cancer big country.China's lung cancer for many years
5 years survival rates of patient are without significantly improving.However, U.S. FDA and China CFDA approval in recent years is used for NSCLC targeted therapies
EGF-R ELISA tyrosine kinase inhibitor (EGFR-TKI, such as Gefitinib, Conmana and Ao Xi replace Buddhist nun),
ALK kinase inhibitors and ROS1 kinase inhibitors (such as gram azoles replaces Buddhist nun for Buddhist nun, Ai Le) can significantly improve the existence of patient.China
Advanced primary lung cancer specialist common recognition, US National synthesis cancer network (NCCN) clinical practice guideline and European tumour
Society of Internal Medicine (ESMO) lung cancer common recognition is clearly suggested：Advanced NSCLC patients should first detect EGFR, ALK before receiving to treat
With ROS1 genes, corresponding therapeutic strategy is determined according to gene appearance.
The common detection technique of ROS1 Gene Fusions has fluorescence in situ hybridization FISH, immunohistochemistry at present
Immunohistochemistry (IHC) and Real time reverse transcription fluorescent PCR (RT-qPCR).
Fluorescence in situ hybridization FISH specificity is high, but the sample process period is long, (probe is expensive) of high cost, detection
As a result interpretation subjectivity and strongly professional.IHC is there is also the sample process period is long, and result receptor 2 fusion protein expression quantity and antibody
Specificity and sensibility influence it is big, as a result judge uncertain big.RT-qPCR has required sample few, easy to operate, consumption
When it is short, it is at low cost, as a result judge the advantages that simple, play an important roll in clinical detection.But ROS1 on the market melts at present
Conjunction detection kit sensitivity is low, and detectable fused type is few.
Invention content
The purpose of the present invention is to provide ROS1 Gene Fusions detection primer and kits.
Another object of the present invention is to provide ROS1 Gene Fusion detection methods.
The technical solution used in the present invention is：
ROS1 Gene Fusion detection primer groups, the nucleotide sequence of primer sets are as follows：
F1：TCCTTGGAGCAAAAGCCCA(SEQ ID NO:1)
F2：AGTGGGGCCCGGGCAGG(SEQ ID NO:2)
F3：CCAGCACTGTGCAGGGCAGCAACA(SEQ ID NO:3)
F4：CGGATTTCTCTACTTTTTCGT(SEQ ID NO:4)
R1：GTATTGAATTTTTACTCCCTT(SEQ ID NO:5)
F5：TCCTTGGAGCAAAAGCCCA(SEQ ID NO:7)
F6：GGAGTTGATGCTGCGGCT(SEQ ID NO:8)
F7：AGTGGGGCCCGGGCAGG(SEQ ID NO:9)
F8：CCAGCACTGTGCAGGGCAGCAACA(SEQ ID NO:10)
F9：CGGATTTCTCTACTTTTTCGT(SEQ ID NO:11)
R2：AAATATTCCAACTATAAT(SEQ ID NO:12)
F10：CCAAGCTGGAAAAGACAATT(SEQ ID NO:14)
F11：CAGGTGCTGGATTTTTCTTACC(SEQ ID NO:15)
F12：AAAGACACAAGTGGGGAAATCAAA(SEQ ID NO:16)
R3：TTATAAGCACTGTCACCCC(SEQ ID NO:17)
F13：TATATGGGGCGAGACTAGCT(SEQ ID NO:19)
R4：AGAGTCAGTTTTTCCCGAGGG(SEQ ID NO:20).
A kind of ROS1 Gene Fusions detection kit, the kit contain primer described above.
Further, which also contains detection probe group, and nucleotide sequence is as follows：
P1：CCCAAATAAACCAGG(SEQ ID NO:6)
P2：TTTTTGGATACCAGAAAC(SEQ ID NO:13)
P3：GATTAAAGAATCAAAAA(SEQ ID NO:18)
P4：AGAGGAGATTGAAAAT(SEQ ID NO:21)
Or the nucleotide reverse complementary sequence for those sequences.
Further, the fluorophor of the end of probe sequence 5 ' label is in FAM, JOE, HEX, VIC, CY5, TET
The quenching group of one kind, the end of probe sequence 3 ' label is a kind of in TAMRA, MGB, BHQ.
Further, which also contains reverse transcriptase, RT Buffer, quantitative fluorescent PCR reaction buffer,
DNTPs, DNA polymerase, negative controls, positive reference substance, interior label primer F14 (SEQ ID NO:22)、R5(SEQ
ID NO:And internal standard probe P5 (SEQ ID NO 23):24).
A kind of ROS1 Gene Fusions detection method, includes the following steps：
1) RNA is extracted from sample；
2) RNA extracted is subjected to reverse transcription reaction, obtains cDNA, primer shown in above-mentioned is contained in reverse transcription system
R1~R5；
3) fluorescent quantitative PCR reaction is carried out using primer sets described above as template using cDNA and collects fluorescence letter
Number；
4) interpretation of result：Judge that sample whether there is corresponding ROS1 Gene Fusions type according to fluorescence signal；
The above method is not used in the diagnosing and treating of disease.
Further, reverse transcription reaction system is in step 2)：
Further, in step 3), fluorescent quantitative PCR reaction system is divided into 4 reaction systems and carries out, and is denoted as respectively
System A~D, specifying information are as follows：
System A：
System B：
System C：
System D：
Further, fluorescent quantitative PCR response procedures are in step 3)：94~96 DEG C, 4~7min；94~96 DEG C,
13~17s, 58~62 DEG C, 28~32s, 71~73 DEG C, 13~16s recycle 40~50 times, are collected simultaneously fluorescence signal.
Further, the detailed process of interpretation of result is as follows in step 4)：
1. positive：When negative control group is without amplification curve and specific positive controls are with apparent amplification curve, detection
The amplification curve of sample has apparent Exponential growth stage, and illustrating the sample, there are ROS1 Gene Fusions；
2. negative：When negative control group has apparent amplification curve without amplification curve and specific positive controls, but examine
Test sample this without Ct values and curve is without apparent Exponential growth stage, illustrating the sample, there is no ROS1 in ROS1 Gene Fusions or sample
The content of Gene Fusion type is less than kit minimum detection limit or is the ROS1 fusions other than kit covering scope.
The beneficial effects of the invention are as follows：
Different fusions can occur with several genes for ROS1 genes, and the present invention can detect up to 13 kinds of ROS1 Gene Fusions
Type.Primer and probe is by optimization and screening, and sensitivity is up to 100copies/ μ l, and specificity is high.
Description of the drawings
Fig. 1 is testing result of the present invention to the positive RNA sample of 13 kinds of ROS1 Gene Fusion types；
Fig. 2 is the present invention to a concentration of 102The testing result of the positive RNA sample of 13 kinds of copies/ μ l；
Fig. 3 is testing result of the method for the present invention to clinical FFPE samples.
Specific implementation mode
With reference to specific embodiment, the present invention is further illustrated.
1 ROS1 Gene Fusions detection primer of embodiment and probe
ROS1 genes can be merged with several genes, as CD74, EZR, SDC4, SLC34A2, TPM3, LRIG3,
GOPC etc..Early period of the invention devises a large amount of primer and probes by design of primers principle and in conjunction with actual conditions, then passes through
A large amount of primer and probes are optimized and screened, highly sensitive and high specific multiple RT-qPCR primer sets and inspection are filtered out
Probing needle can detect up to 13 kinds of ROS1 Gene Fusion types.Primer sets and the probe sequence difference obtained is as shown in table 1.
1 ROS1 Gene Fusions detection primer of table and probe sequence
2 ROS1 Gene Fusion detection kits of embodiment
The present embodiment detection kit includes：
1) ROS1 Gene Fusions detection primer F1~F13, R1~R4 and probe P1~P4 (particular sequence is shown in Table 1)；
2) interior label primer F14, R5 and probe P5 (particular sequence is shown in Table 1)；
3) kit also includes that (negative control is 2 to negative controls, is ddH respectively2O and wild type 293T cells
RNA, hereinafter referred to as 293RNA) and the positive reference substance (RNA sequence and ROS1 of ROS1 Gene Fusions type in artificial synthesized table 1
Fusion plasmid).Multiple Quality Control avoids the false positive and false negative of testing result.
4) reverse transcriptase, RT Buffer, DNA polymerase, dNTP and real-time fluorescence PCR reaction buffer.
3 ROS1 Gene Fusion detection methods of embodiment
1) RNA is extracted from sample；
2) by the RNA extracted progress reverse transcription reaction, (reverse transcription system reagent only praises biotechnology purchased from Nanjing promise to be had
Limit company), cDNA is obtained, contains primer R1~R5 in reverse transcription system；
Reverse transcription system is：
Reverse transcription reaction program is：50℃、15min；85 DEG C, 2min, 10 DEG C of heat preservations.
3) fluorescent quantitative PCR reaction is carried out as template using cDNA and collects fluorescence signal；
PCR reaction systems (PCR reaction reagents are purchased from Nanjing Vazyme Biotechnology Co., Ltd.) point 4 system (bodies
It is A~D) PCR detections are carried out, system A~D is specific as follows：
System A：
System B：
System C：
System D：
Pcr amplification reaction program is：PCR amplification, PCR conditions are carried out using real-time fluorescence quantitative PCR instrument (ABI 7500)
For：95℃、5min；95 DEG C, 15s, 60 DEG C, 30s (collecting fluorescence signal), 72 DEG C, 15s are recycled 45 times.
4) interpretation of result：
1. positive：When negative control group is without amplification curve and specific positive controls are with apparent amplification curve, detection
The amplification curve of sample has apparent Exponential growth stage, and illustrating the sample, there are ROS1 Gene Fusions；
2. negative：When negative control group has apparent amplification curve without amplification curve and specific positive controls, but examine
Test sample this without Ct values and curve is without apparent Exponential growth stage, illustrate that above-mentioned 13 kinds of ROS1 Gene Fusion types are not present in the sample
The content of 13 kinds of ROS1 Gene Fusion types of this in sample less than kit minimum detection limit or for kit covering scope with
Outer ROS1 fusions.
4 positive reference substance of embodiment detects
1) RNA sequence of 13 kinds of ROS1 Gene Fusion types in the artificial table 1 for closing object is subjected to reverse transcription respectively and obtains 13
Kind cDNA, reverse transcription system and response procedures are the same as embodiment 3.
2) PCR detections are carried out by template of above-mentioned 13 kinds of cDNA respectively；PCR reaction systems and response procedures are the same as embodiment 3.
Positive test symbol is as shown in Figure 1, it can be seen that the positive RNA sample of 13 kinds of ROS1 Gene Fusion types of the present invention couple is equal
Apparent amplification curve can be obtained.Simultaneously to negative controls ddH2The RNA of O (NTC) and wild type 293T cells is (referred to as
Do not have amplification curve 293RNA).
5 sensitivity technique of embodiment
Above-mentioned positive reference substance (the positive RNA sample of 13 kinds of ROS1 Gene Fusion types) is subjected to gradient dilution, is made dilute
The RNA concentration of positive reference substance after releasing is respectively 104、103、102、101Copies/ μ l, according to the inspection of the above embodiments 3
Survey method carries out sensitivity technique experiment.NTC non-templates control (ddH is set simultaneously2O negative controls) and wild type 293T it is thin
The negative control (293RNA) of born of the same parents RNA.
Testing result as shown in Fig. 2, the present invention to a concentration of 102The positive RNA sample of 13 kinds of copies/ μ l all has very
Good detection result, and do not have amplification curve to NTC and 293RNA (wild type rna).Illustrate the sensitivity of the method for the present invention
Up to 102copies/μl。
6 clinical sample of embodiment detects
By 3 parts of clinic FFPE samples respectively according to method described in embodiment 3, extracts RNA and carry out fluorogenic quantitative detection.
Positive controls and NTC non-templates control (ddH are set simultaneously2O negative controls) and wild type 293T cell RNAs the moon
Property control.
Testing result as shown in figure 3, the method for the present invention all has good detection result to above-mentioned 3 parts of clinical sample RNA,
There is apparent amplification curve, does not have amplification curve to NTC and 293T cell RNAs (293RNA).Illustrate 3 parts of clinical samples
There is ROS1 Gene Fusions；In addition, confirmed that this 3 parts clinic FFPE samples have ROS1 genes really by the sequencing of two generations
Fusion, and fused type is respectively SDC4exon 4：ROS1exon 34、EZR exon 10：ROS1exon 34、CD74exon
6：34 Gene Fusion types of ROS1exon illustrate that the method for the present invention has good accuracy and applicability, can be used for clinical sample
The quick detection of product.In conclusion the present invention can detect up to 13 kinds of ROS1 Gene Fusion types.Primer and probe is by optimization
And screening, sensitivity is up to 100copies/ μ l, and specificity is high.Internal standard is monitored for system reverse transcription and amplification, ensures inspection
Survey is normally carried out.In addition, kit also includes negative and positive control.Multiple Quality Control avoids testing result from false positive and vacation occur
It is negative.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from the spirit and principles of the present invention made by changes, modifications, substitutions, combinations, simplifications,
Equivalent substitute mode is should be, is included within the scope of the present invention.
SEQUENCE LISTING
<110>Guangzhou steps scape gene medical science and technology Co., Ltd
<120>ROS1 Gene Fusions detection primer, method and kit
<130>
<160> 24
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213>Artificial sequence
<400> 1
tccttggagc aaaagccca 19
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agtggggccc gggcagg 17
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<213>Artificial sequence
<400> 3
ccagcactgt gcagggcagc aaca 24
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<212> DNA
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<400> 4
cggatttctc tactttttcg t 21
<210> 5
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<213>Artificial sequence
<400> 5
gtattgaatt tttactccct t 21
<210> 6
<211> 15
<212> DNA
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cccaaataaa ccagg 15
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<212> DNA
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<400> 7
tccttggagc aaaagccca 19
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<212> DNA
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ggagttgatg ctgcggct 18
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<212> DNA
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<400> 9
agtggggccc gggcagg 17
<210> 10
<211> 24
<212> DNA
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<400> 10
ccagcactgt gcagggcagc aaca 24
<210> 11
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<212> DNA
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<400> 11
cggatttctc tactttttcg t 21
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aaatattcca actataat 18
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tttttggata ccagaaac 18
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ccaagctgga aaagacaatt 20
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caggtgctgg atttttctta cc 22
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aaagacacaa gtggggaaat caaa 24
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ttataagcac tgtcacccc 19
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gattaaagaa tcaaaaa 17
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tatatggggc gagactagct 20
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agagtcagtt tttcccgagg g 21
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agaggagatt gaaaat 16
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tttgttggat ttgaaattcc aga 23
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Claims (10)
1.ROS1 Gene Fusion detection primer groups, the nucleotide sequence of primer sets are as follows：
F1：TCCTTGGAGCAAAAGCCCA(SEQ ID NO:1)
F2：AGTGGGGCCCGGGCAGG(SEQ ID NO:2)
F3：CCAGCACTGTGCAGGGCAGCAACA(SEQ ID NO:3)
F4：CGGATTTCTCTACTTTTTCGT(SEQ ID NO:4)
R1：GTATTGAATTTTTACTCCCTT(SEQ ID NO:5)
F5：TCCTTGGAGCAAAAGCCCA(SEQ ID NO:7)
F6：GGAGTTGATGCTGCGGCT(SEQ ID NO:8)
F7：AGTGGGGCCCGGGCAGG(SEQ ID NO:9)
F8：CCAGCACTGTGCAGGGCAGCAACA(SEQ ID NO:10)
F9：CGGATTTCTCTACTTTTTCGT(SEQ ID NO:11)
R2：AAATATTCCAACTATAAT(SEQ ID NO:12)
F10：CCAAGCTGGAAAAGACAATT(SEQ ID NO:14)
F11：CAGGTGCTGGATTTTTCTTACC(SEQ ID NO:15)
F12：AAAGACACAAGTGGGGAAATCAAA(SEQ ID NO:16)
R3：TTATAAGCACTGTCACCCC(SEQ ID NO:17)
F13：TATATGGGGCGAGACTAGCT(SEQ ID NO:19)
R4：AGAGTCAGTTTTTCCCGAGGG(SEQ ID NO:20).
2. a kind of ROS1 Gene Fusions detection kit, which is characterized in that the kit contains primer described in claim 1.
3. kit according to claim 2, it is characterised in that：The kit also contains detection probe group, nucleotide
Sequence is as follows：
P1：CCCAAATAAACCAGG(SEQ ID NO:6)
P2：TTTTTGGATACCAGAAAC(SEQ ID NO:13)
P3：GATTAAAGAATCAAAAA(SEQ ID NO:18)
P4：AGAGGAGATTGAAAAT(SEQ ID NO:21)
Or the nucleotide reverse complementary sequence for those sequences.
4. kit according to claim 3, which is characterized in that the fluorophor of the end of probe sequence 5 ' label is selected from
A kind of in FAM, JOE, HEX, VIC, CY5, TET, the quenching group of the end of probe sequence 3 ' label is one in TAMRA, MGB, BHQ
Kind.
5. kit according to claim 2, which is characterized in that the kit also contains reverse transcriptase, reverse transcription buffering
Liquid, quantitative fluorescent PCR reaction buffer, dNTPs, DNA polymerase, negative controls, positive reference substance, interior label primer
F14(SEQ ID NO:22)、R5(SEQ ID NO:And internal standard probe P5 (SEQ ID NO 23):24).
6. a kind of ROS1 Gene Fusions detection method, which is characterized in that include the following steps：
1) RNA is extracted from sample；
2) RNA extracted is subjected to reverse transcription reaction, obtains cDNA, contains in reverse transcription system as shown in claim 1
Primer R1~R5；
3) fluorescent quantitative PCR reaction is carried out using primer sets described in claim 1 as template using cDNA and collects fluorescence
Signal；
4) interpretation of result：Judge that sample whether there is corresponding ROS1 Gene Fusions type according to fluorescence signal；
The above method is not used in the diagnosing and treating of disease.
7. according to the method described in claim 6, it is characterized in that：Reverse transcription reaction system is in step 2)：
8. according to the method described in claim 6, it is characterized in that：In step 3), fluorescent quantitative PCR reaction system is divided into
4 reaction systems carry out, and are denoted as system A~D respectively, specifying information is as follows：
System A：
System B：
System C：
System D：
9. according to the method described in claim 6, it is characterized in that：Fluorescent quantitative PCR response procedures are in step 3)：94
~96 DEG C, 4~7min；94~96 DEG C, 13~17s, 58~62 DEG C, 28~32s, 71~73 DEG C, 13~16s, cycle 40~50
It is secondary, it is collected simultaneously fluorescence signal.
10. according to the method described in claim 6, it is characterized in that：The detailed process of interpretation of result is as follows in step 4)：
1. positive：When negative control group is without amplification curve and specific positive controls are with apparent amplification curve, detection sample
Amplification curve have apparent Exponential growth stage, illustrating the sample, there are ROS1 Gene Fusions；
2. negative：When negative control group has apparent amplification curve without amplification curve and specific positive controls, but detect sample
This is without Ct values and curve is without apparent Exponential growth stage, and illustrating the sample, there is no ROS1 genes in ROS1 Gene Fusions or sample
The content of fused type is less than kit minimum detection limit or is the ROS1 fusions other than kit covering scope.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109666746A (en) * | 2019-02-19 | 2019-04-23 | 合肥欧创基因生物科技有限公司 | For detecting primer, probe and the kit and its detection method of the mutation of mankind's ROS1 Gene Fusion |
| CN110964830A (en) * | 2019-12-31 | 2020-04-07 | 中山大学达安基因股份有限公司 | Kit and method for multiple detection of ROS1 gene mutation |
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