CN104498615A - Primer and probe for detecting mutant KRAS genes - Google Patents
Primer and probe for detecting mutant KRAS genes Download PDFInfo
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Abstract
The invention provides a primer and probe for detecting mutant KRAS genes and further provides a method and kit for detecting mutant KRAS genes by using the primer and the probe. By adopting the primer and probe provided by the invention, the amplification of wild KRAS genes can be inhibited, and the combination of the wild KRAS genes with the probe can be also inhibited, thus less than 1% of mutant KRAS genes can be detected.
Description
Technical field
The invention belongs to biotechnology and diagnosis research field, relating to a kind of primer for detecting KRAS and probe, and to utilize in this primer and probe in detecting sample the method whether containing KRAS and test kit.
Background technology
The gene that RAS gene family is relevant to human tumor has three kinds of-H-RAS, KRAS and N-RAS, is positioned at respectively on 11,12 and No. 1 karyomit(e)s.Just become the oncogene having carcinogenic activity after being activated as the RAS gene of proto-oncogene, RAS gene activates by suddenling change.
KRAS is one of RAS gene experiencing sudden change in kinds cancer.KRAS gene can be standard state (being called wild-type) or error state (ERST) (saltant type).Under normal physiological conditions, when activating the signal paths such as somatomedin after cell is subject to external stimulus, the KRAS of wild-type is by activation of short duration after the tyrosine kinases phosphorylate such as active growth factor, KRAS after activation can activate the downstream signaling proteins in this signal path, then KRAS fast deactivation.KRAS activation/inactivating effect is controlled.Saltant type KRAS albumen causes protein function abnormal, and under without growth factor activation token stimulus, be still in state of activation, its functional status is uncontrollable, leads oncogenic continuous proliferation etc.
The KRAS gene mutation at codon 12 and 13 place participates in tumour and occurs, and has widely used the qualification of KRAS gene mutation at present as cancer diagnosis, such as pancreas, colorectum and nonsmall-cell lung cancer.But detect KRAS gene and mainly contain common sequencing and the PCR based on simple typing probes, wherein the sensitivity of common sequencing is 10%-30% (saltant type/wild-type Kras), PCR sensitivity based on simple typing probes is about 10%, is difficult to the Kras transgenation detecting less than 10%.
Summary of the invention
The present invention is directed to above-mentioned shortcoming, providing a kind of for detecting KRAS gene mutation primer used and probe, to reach the object of the KRAS that can detect less than 1%.
For achieving the above object, the present invention takes following technical proposals to realize:
For detecting a primer for KRAS gene mutation, comprising:
A forward primer, is selected from
F1：GTAAGGTATTTTGAAATAATTTTTCATATAAAGGTGA(SEQ ID NO：1)，
F2：TTATAAGGCC TGCTGAAAAT GACTG(SEQ ID NO：2)，
F3：GGTGGAGTATTTGATAGTGTATTAACCTTA(SEQ ID NO：3)，
F4：GGAGTTTGTAAATGAAGTACAGTT(SEQ ID NO：4)，
F5：TAAGGCCTGCTGAAAATGACTGA(SEQ ID NO：5)
F：6：GAAGTACAGTTCATTACGATACAC(SEQ ID NO：6)，
F7：GTTCATTACGATACACGTCTGC(S EQ ID N O：7)；
With a reverse primer, be selected from
R1：CAAGATTTACCTCTATTGTTGGATCA(SEQ ID NO：8)
R2：GAGAAACCTTTATCTGTATCAAAGAAT(SEQ ID NO：9)
R3：CGTCAAGGCACTCTTGCCTAC(SEQ ID NO：10)
R4：TGTTGGATCATATTCGTCCACAAA(SEQ ID NO：11)
R5：AAATGGTCAGAGAAACCTTTATCTG(SEQ ID NO：12)
R6：GTCAGAGAAACCTTTATCTGTATC(SEQ ID NO：13)
R7：CAACTGGAATTTTCATGATTGAATTTTGTAAG(SEQ ID NO：14)。
The present invention also provides a kind of probe for detecting KRAS gene mutation, at least comprises a probe,
Be selected from (A) group or (B) group, wherein often organize the probe that probe comprises corresponding 12D, 13D, 12V, 12C, 12S, 12A, 12R seven kinds sudden change;
(A)
12D：01 AGTTGGAGCTGATGGC(SEQ ID NO：15)
Or 02TTGGAGCTGATGGC (SEQ ID NO:16),
13D：01 TAGCTGGTGACGTA(SEQ ID NO：17)
Or 02CTGGTGACGTAGGC (SEQ ID NO:18),
12V：01 GTTGGAGCTGTTGGC(SEQ ID NO：19)
Or 02TTGGAGCTGTTGGC (SEQ ID NO:20),
12C：01 TTGGAGCTTGTGGCG(SEQ ID NO：21)
Or 02TTGGAGCTTGTGGC (SEQ ID NO:22),
12S：01 AGTTGGAGCTAGTGGC(SEQ ID NO：23)
Or 02TTGGAGCTAGTGGC (SEQ ID NO:24),
12A：01 TGGAGCTGCTGGC(SEQ ID NO：25)
Or 02TGGAGCTGCTGGCG (SEQ ID NO:26),
12R：01 GTTGGAGCTCGTGGC(SEQ ID NO：27)
Or 02TTGGAGCTCGTGGC (SEQ ID NO:28),
(B)
12D probe sequence is CCTACGCCATCAGCTCCAA (SEQ ID NO:29),
13D probe sequence is CCTACGTCACCAGCTCCAA (SEQ ID NO:3O),
12V probe sequence is CCTACGCCAACAGCTCCAA (SEQ ID NO:31),
12R probe sequence is CCTACGCCACGAGCTCCAA (S EQ ID N O:32),
12C probe sequence is CCTACGCCACAAGCTCCAA (SEQ ID NO:33),
12A probe sequence is CCTACGCCAGCAGCTCCAA (SEQ ID NO:34),
12S probe sequence is CCTACGCCACTAGCTCCAA (SEQ ID NO:35);
Further, the 5 ' end that described (A) group or (B) organize probe is provided with Fam, Vic, he ×, Ro ×, Texas red, a kind of fluorescent substance in Cy3, Cy5 preferably, is Fam.
Preferably, the 3 ' end that described (A) organizes probe is provided with MGBNFQ, distinguishes better to being rich in A/T template.
Preferably, described (B) organize probe 3 ' end be provided with BHQ1.
The present invention also provides a kind of detection method of KRAS gene mutation, uses the following to carry out PCR reaction,
Any one aforementioned positive primer;
Any one aforementioned reverse primer;
At least one aforesaid probe, selects in aforementioned A group or B group probe;
A blocker PNA, is selected from
PNA01：GGAGCTGGTGGCGTA G(SEQ ID NO：36)，
Or PNA02:GGAGCTGGTGGCGT (SEQ ID NO:37).
Preferably, use one group of probe, be selected from (A) group or (B) group, comprise the probe of corresponding 12D, 13D, 12V, 12C, 12S, 12A, 12R seven kinds sudden change owing to often organizing probe, therefore can detect the saltant type of whether suddenling change containing any one codon 12 or 13 place in testing sample.
The present invention also provides a kind of test kit detecting KRAS gene mutation, comprising:
Any one aforementioned positive primer;
Any one aforementioned reverse primer;
At least one aforesaid probe, selects in aforementioned A group or B group probe;
Any one aforementioned blocker PNA.
Preferably, described test kit comprises one group of probe, is selected from (A) group or (B) group.The same probe comprising corresponding 12D, 13D, 12V, 12C, 12S, 12A, 12R seven kinds sudden change owing to often organizing probe, therefore can detect the saltant type of whether suddenling change containing any one codon 12 or 13 place in testing sample.
In addition the present invention also provides aforementioned primer and probe to detect the purposes in the test kit of KRAS gene mutation in preparation.
Beneficial effect of the present invention: compared with prior art, primer for detecting KRAS gene mutation provided by the invention and probe effectively can suppress the amplification of KRAS wild-type and with the structure of probe thus detect less than 1% KRAS type.
Accompanying drawing explanation
Fig. 1 is eight target gene fluorescent quantitative PCR curves to be measured in embodiment 1.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail.
Embodiment 1
The following is used to carry out PCR reaction.
(1) target to be measured (DNA profiling namely in PCR reaction)
Prepare eight groups of targets to be measured, be respectively 1%12DKRAS saltant type+99% wild-type, 1%13DKRAS saltant type+99% wild-type, 1%12VKRAS saltant type+99% wild-type, 1%12CKRAS saltant type+99% wild-type, 1%12SKRAS saltant type+99% wild-type, 1%12AKRAS saltant type+99% wild-type, 1%12RKRAS saltant type+99% wild-type, 100% wild-type and without Template Controls group.
(2) primer
Select any one forward primer, in the present embodiment, select F1,
Select any one reverse primer, in the present embodiment, select R3,
(3) probe
Any selection one group of probe, selects A group probe in the present embodiment, its middle probe 5 ' end mark fluorescent substance Fam, 3 ' holds as M G B NFQ.
Fam is Fluoresceincarboxylic acid, and 5-Fam, 6-Fam all can select, preferred 5-Fam.
MGB and minor groove binding, N FQ and non-fluorescence quenching group, the probe 3 ' of the present embodiment is held on probe and is also connected with MGB (Minor Groove Binder) modification group, can by the Tm value raising about 10 DEG C of probe, thus make probe can be shorter, cost-saving and improve success ratio; Adopt non-fluorescence quenching group (Non-FluorescentQuencher), itself does not produce fluorescence, greatly reduces the intensity of background signal simultaneously.
Following probe is selected: 12D01,13D01,12V01,12C01,12S01,12A01,12R01 in the present embodiment.
(4)blocker PNA
Any selection blocker PNA, selects PNA01 in the present embodiment.
In the present embodiment, PCR reaction system is 50ul, and concrete reaction system is as shown in table 1:
The present embodiment without particular requirement, uses general Tris-Cl damping fluid to PCR damping fluid (PCR buffer).
The significant parameter of PCR reaction: all primer final concentrations are 300-1000nM, and probe final concentration is 100-500nM, PNA final concentration is 1nM-10uM.
PCR reaction conditions is: first enzyme activition, 95 DEG C of 5-10 minute, then 45 circulations: 95 DEG C 15 seconds, 71 DEG C of 15-60 seconds, 55-65 DEG C 50 seconds.
Adopt ABI7500 real-time fluorescence quantitative PCR instrument in the present embodiment, Fig. 1 is eight target gene fluorescent quantitative PCR curves to be measured.As can be seen from Figure 1, the primer selected of the present embodiment and probe and detection method can detect the KRAS gene mutation of 1%.
Embodiment 2
A kind of test kit for detecting KRAS gene mutation
Test kit in the present embodiment comprises: PCR reaction solution, PNA reagent, enzymic digestion liquid, sequencing reaction liquid, magnetic bead and product purification liquid.Wherein PCR reaction solution comprises PCR damping fluid, dNTPs, PCR primer, probe and archaeal dna polymerase.
Preferably, in the present embodiment, in PCR primer, forward primer is F2, and reverse primer is R1, and probe is B group probe, and namely the fluorophor of 5 ' end of described probe is FAM, and the fluorophor of 3 ' end is BHQ1.
Described PNA is PNA02.
In the present embodiment, other compositions do not have particular requirement, and selection is generally general can realization response object.
When using this test kit to detect target gene, comprise the following steps:
(1) target gene fluorescent quantitative PCR: use fluorescence quantitative PCR reaction solution and PNA reagent in test kit, pcr amplification reaction is carried out to testing sample DNA, obtains quantitative result and the PCR primer of goal gene;
(2) according to quantitative result, use the enzymic digestion liquid in test kit, purifying is carried out to PCR primer, obtain PCR primer after purifying;
(3) use the sequencing reaction liquid in test kit, after purifying, PCR primer is carried out order-checking PCR and is reacted, and obtains order-checking PCR primer;
(4) use the magnetic bead in test kit and product purification liquid, carry out purifying to the order-checking PCR primer obtained, check order after obtaining purifying PCR primer;
(5) the PCR primer application of sample that checks order after purifying is carried out sequential analysis to sequenator, order-checking gained gene order and KRAS gene standard sequence are compared, and judge whether to there is KRAS gene mutation.
Although the present invention with preferred embodiment openly as above; but it is not for limiting the present invention; any those skilled in the art without departing from the spirit and scope of the present invention; the Method and Technology content of above-mentioned announcement can be utilized to make possible variation and amendment to technical solution of the present invention; therefore; every content not departing from technical solution of the present invention, any simple modification done above embodiment according to technical spirit of the present invention, equivalent variations and modification all belong to the protection domain of technical solution of the present invention.
Claims (10)
1., for detecting a primer for KRAS gene mutation, comprise
A forward primer, is selected from
GTAAGGTATTTTGAAATAATTTTTCATATAAAGGTGA(SEQ ID NO：1)，
TTATAAGGCC TGCTGAAAAT GACTG(SEQ ID NO：2)，
GGTGGAGTATTTGATAGTGTATTAACCTTA(SEQ ID NO：3)，
GGAGTTTGTAAATGAAGTACAGTT(SEQ ID NO：4)，
TAAGGCCTGCTGAAAATGACTGA(SEQ ID NO：5)
GAAGTACAGTTCATTACGATACAC(SEQ ID NO：6)，
GTTCATTACGATACACGTCTGC(SEQ ID NO：7)；
With a reverse primer, be selected from
CAAGATTTACCTCTATTGTTGGATCA(SEQ ID NO：8)
GAGAAACCTTTATCTGTATCAAAGAAT(SEQ ID NO：9)
CGTCAAGGCACTCTTGCCTAC(SEQ ID NO：10)
TGTTGGATCATATTCGTCCACAAA(SEQ ID NO：11)
AAATGGTCAGAGAAACCTTTATCTG(SEQ ID NO：12)
GTCAGAGAAACCTTTATCTGTATC(SEQ ID NO：13)
CAACTGGAATTTTCATGATTGAATTTTGTAAG(SEQ ID NO：14)。
2. for detecting a probe for KRAS gene mutation, it is characterized in that, at least comprising a probe,
Be selected from (A) group or (B) group, wherein often organize the probe that probe comprises corresponding 12D, 13D, 12V, 12C, 12S, 12A, 12R seven kinds sudden change;
(A) 12D probe sequence is AGTTGGAGCTGATGGC (SEQ ID NO:15)
Or TTGGAGCTGATGGC (SEQ ID NO:16),
13D probe sequence is TAGCTGGTGACGTA (SEQ ID NO:17)
Or CTGGTGACGTAGGC (SEQ ID NO:18),
12V probe sequence is GTTGGAGCTGTTGGC (SEQ ID NO:19)
Or TTGGAGCTGTTGGC (SEQ ID NO:20),
12C probe sequence is TTGGAGCTTGTGGCG (SEQ ID NO:21)
Or TTGGAGCTTGTGGC (SEQ ID NO:22),
12S probe sequence is AGTTGGAGCTAGTGGC (SEQ ID NO:23)
Or TTGGAGCTAGTGGC (SEQ ID NO:24),
12A probe sequence is TGGAGCTGCTGGC (SEQ ID NO:25)
Or TGGAGCTGCTGGCG (SEQ ID NO:26),
12R probe sequence is GTTGGAGCTCGTGGC (SEQ ID NO:27)
Or TTGGAGCTCGTGGC (SEQ ID NO:28),
(B)
12D probe sequence is CCTACGCCATCAGCTCCAA (SEQ ID NO:29),
13D probe sequence is CCTACGTCACCAGCTCCAA (SEQ ID NO:30),
12V probe sequence is CCTACGCCAACAGCTCCAA (SEQ ID NO:31),
12R probe sequence is CCTACGCCACGAGCTCCAA (SEQ ID NO:32),
12C probe sequence is CCTACGCCACAAGCTCCAA (SEQ ID NO:33),
12A probe sequence is CCTACGCCAGCAGCTCCAA (S EQ ID N O:34),
12S probe sequence is CCTACGCCACTAGCTCCAA (S EQ ID N O:35).
3. probe according to claim 1, is characterized in that, the 5 ' end that described (A) group or (B) organize probe is provided with Fam, Vic, hex, Rox, Texas red, a kind of fluorescent substance in Cy3, Cy5.
4. probe according to claim 1, is characterized in that, the 3 ' end that described (A) organizes probe is provided with MG B NFQ.
5. probe according to claim 1, is characterized in that, the 3 ' end that described (B) organizes probe is provided with BHQ1.
6. a detection method for KRAS gene mutation, is characterized in that, uses the following to carry out PCR reaction,
A forward primer as claimed in claim 1;
A reverse primer as claimed in claim 1;
At least one probe as described in claim 2-5 any one;
A blocker PNA, is selected from
GGAGC TGGT GGCGTA G(SEQ ID NO：36)，
Or GGAGCTGGTGGCGT (SEQ ID NO:37).
7. detection method according to claim 1, is characterized in that, uses one group of probe, is selected from (A) group or (B) group.
8. detect a test kit for KRAS gene mutation, it is characterized in that, described test kit comprises:
A forward primer as claimed in claim 1;
A reverse primer as claimed in claim 1;
At least one probe as described in claim 2-5 any one;
A blocker PNA, is selected from
GGAGC TGGT GGCGTA G(SEQ ID NO：36)，
Or GGAGCTGGTGGCGT (SEQ ID NO:37).
9. test kit according to claim 8, is characterized in that, described test kit comprises one group of probe, is selected from (A) group or (B) group.
10. primer as claimed in claim 1 or probe according to claim 2 detect the purposes in the test kit of KRAS gene mutation in preparation.
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Cited By (4)
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| CN106399570A (en) * | 2016-11-30 | 2017-02-15 | 杭州诺辉健康科技有限公司 | Kit for early stage colorectal cancer auxiliary diagnosis and use method and detection system thereof |
| WO2018096349A1 (en) * | 2016-11-25 | 2018-05-31 | Imperial Innovations Limited | Primers, methods and kits for diagnosing and predicting therapy response of cancers by cold-pcr based amplification of mutation-rich regions of kras, egfr and p53 |
| CN109097471A (en) * | 2018-08-21 | 2018-12-28 | 杭州和壹基因科技有限公司 | A kind of kit detected for colorectal cancer and precancerous lesion and its application method |
| CN110396517A (en) * | 2019-05-21 | 2019-11-01 | 珠海圣美生物诊断技术有限公司 | It is a kind of that type oligonucleotide probe and its application being quenched for expanding the non-of anomaly target fragment |
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Cited By (4)
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| WO2018096349A1 (en) * | 2016-11-25 | 2018-05-31 | Imperial Innovations Limited | Primers, methods and kits for diagnosing and predicting therapy response of cancers by cold-pcr based amplification of mutation-rich regions of kras, egfr and p53 |
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| CN109097471A (en) * | 2018-08-21 | 2018-12-28 | 杭州和壹基因科技有限公司 | A kind of kit detected for colorectal cancer and precancerous lesion and its application method |
| CN110396517A (en) * | 2019-05-21 | 2019-11-01 | 珠海圣美生物诊断技术有限公司 | It is a kind of that type oligonucleotide probe and its application being quenched for expanding the non-of anomaly target fragment |
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