CN108374008A - Combination product, composition, kit and its application of primer pair and probe for detecting KRAS mutation - Google Patents
Combination product, composition, kit and its application of primer pair and probe for detecting KRAS mutation Download PDFInfo
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- CN108374008A CN108374008A CN201810424855.5A CN201810424855A CN108374008A CN 108374008 A CN108374008 A CN 108374008A CN 201810424855 A CN201810424855 A CN 201810424855A CN 108374008 A CN108374008 A CN 108374008A
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Abstract
The present invention relates to molecular biology fields, in particular to a kind of combination product, composition, kit and its application for detecting the primer pair and probe of KRAS mutation.The combination product includes the LNA fluorescence probes and primer for detecting different KRAS Exon 2s saltant types；At least there is a lock nucleic acid in the forward primer sequence of the primer pair；And/or；At least there is a lock nucleic acid in the LNA fluorescence probes sequence.Primer pair and probe combinations product provided by the present invention has the advantages that fast and convenient, high specificity, sensibility height, good reliability, testing cost is low, the time is short, detection efficiency is high, accuracy is high, false positive is low etc..Kit of the present invention can detect KRAS gene mutation type, and can provide guidance for the clinical diagnosis and treatment of KRAS mutation cancer, there is good application prospect.
Description
Technical field
The present invention relates to molecular biology field, in particular to a kind of primer pair for detecting KRAS mutation and
Combination product, composition, kit and its application of probe.
Background technology
Molecular targeted therapy is one of the hot spot in cancer Therapy study field, and epidermal growth factor receptor inhibitor is that targeting is controlled
Treat one kind in drug.Studies have shown that the effect of mutation status and epidermal growth factor receptor inhibitor of mankind's KRAS genes
There are apparent correlations.
Circulating tumor DNA (circulating tumor DNA, ctDNA) is novel tumor molecular marker, is had higher
Recall rate, specificity and the features such as consistency higher with histologic results.The side of non-intruding may be implemented by ctDNA
Formula is detected tumour, helps clinically to judge tumor tissues KRAS gene mutation state.But due to blood plasma
In cycle dissociative DNA content it is few, and ctDNA contents therein are even more fewer and fewer, and the ctDNA contents of many patients only account for entirely
The 0.01%~1% of DNA fragmentation (cell-free DNA, the cfDNA) content dissociated in portion's blood, therefore conventional gene is prominent
Become detection method to be difficult to meet needs.
Method currently used for ctDNA in detection clinical serum sample is mainly based on PCR amplification.Target gene passes through
PCR amplification is mutually distinguishable with molecular beacon, but since existing molecular beacon essence is section of DNA sequence, it is easily by nuclease
Decompose and easily with single-stranded DNA binding protein (Single-stranded DNA-binding protein, SSB or SSBP) non-spy
The opposite sex combines.Therefore, there is an urgent need to a kind of molecular beacons of high sensitivity by user.Lock nucleic acid (locked nucleicacid,
LNA be) a kind of novel double-ring oligonucleotide derivative, with DNA and RNA in structure phosphate backbone having the same,
And basepairing rule is followed, there is very strong affinity, thermal stability and antienzyme to cut ability, good water solubility and in vivo nontoxicity
Effect.Lock nucleic acid decorating molecule beacon (locked nucleic acid molecular beacon, LNA-MB) can be preferable
The shortcomings that making up molecular beacon.However there is no the LNA-MB detection methods about KRAS gene mutation in the prior art.
In view of this, special propose the present invention.
Invention content
The present inventor has obtained can be used in detecting different KRAS genes prominent by performing creative labour and a large amount of experiment
Become the primer pair and probe of type, the primer pair and probe specificity be strong, high sensitivity, uses it for KRAS gene mutation class
Type is easy to operate when detecting.Thus provide following inventions.
One aspect of the present invention is related to a kind of combination product of primer pair and probe, including the different KRAS genes of detection the
The LNA fluorescence probes and primer of 2 exons mutation types；
The nucleotide sequence of the LNA fluorescence probes such as SEQ ID NO：Shown in 1；
The primer is one or more pairs of in following primer pair：
Wherein, at least there is a lock nucleic acid in the forward primer sequence of the primer pair；
And/or；
At least there is a lock nucleic acid in the LNA fluorescence probes sequence.
Another aspect of the present invention further relates to a kind of composition, the combination production containing primer pair as described above and probe
Product.
Another aspect of the present invention further relates to a kind of kit, and it includes the combination of primer pair as described above and probe productions
Product preferably further include quantitative fluorescent PCR reaction buffer, sample treatment liquid, seedless sour water, archaeal dna polymerase, KRAS genes the 2nd
It is one or more in exons mutation Type Positive control sequence and wild type control sequence.
According to an aspect of the present invention, the invention further relates to the combination product of primer pair as described above and probe, combinations
The application of object or kit in preparing reagent or the kit for diagnosing KRAS mutation cancer.
Compared with prior art, beneficial effects of the present invention are：
Primer pair and probe combinations product provided by the present invention has fast and convenient, high specificity, sensibility high, reliable
The advantages such as property is good, testing cost is low, the time is short, detection efficiency is high, accuracy is high, false positive is low.Kit of the present invention can detect
KRAS gene mutation type, and guidance can be provided for the clinical diagnosis and treatment of KRAS mutation cancer, before having application well
Scape.
Specific implementation mode
One aspect of the present invention is related to a kind of combination product of primer pair and probe, including the different KRAS genes of detection the
The LNA fluorescence probes and primer of 2 exons mutation types；
The nucleotide sequence of the LNA fluorescence probes such as SEQ ID NO：Shown in 1；
The primer is one or more pairs of in following primer pair：
Wherein, at least there is a lock nucleic acid in the forward primer sequence of the primer pair；Can select 2,3,4,5,6,
7,8 or more lock nucleic acid, most preferably 1；
And/or；
At least there is a lock nucleic acid in the LNA fluorescence probes sequence；It can select 1,2,3,4,5,6,7,8,9 or more
Multiple lock nucleic acids, most preferably 4.
The primer and probe can be synthesized according to the state of the art, and the corporation of profession can also be entrusted standby.
The combination product can be by SEQ ID NO：Nucleotide shown in 1-9 is separately stored in different containers, such as EP
Guan Zhong, can also be by the forward primer and reverse primer mixed storage in each pair of primer pair.
Preferably, the combination product of primer pair as described above and probe, the 3' of each forward primer of the primer pair
First, end nucleotide is lock nucleic acid.
Preferably, the combination product of primer pair as described above and probe, the ends LNA fluorescence probes 5' the 9th, 11,
18,23 nucleotide are lock nucleic acid.
Preferably, the combination product of primer pair as described above and probe, 5 ' end mark fluorescents of the LNA fluorescence probes
Emit group, 3 ' end label quenching groups.
Preferably, the combination product of primer pair as described above and probe, the fluorescent emission group be selected from FAM, HEX,
Any one of VIC or JOE, more preferably FAM.
Preferably, the combination product of primer pair as described above and probe, the quenching group be selected from TAMRA, BHQ or
Any one of MGB, more preferably BHQ, most preferably BHQ3.
Preferably, the combination product of primer pair as described above and probe, the combination product further include for expanding
The Exon2 wild primers of KRAS Exon 2 wild-type sequences；Preferably, the forward direction of the Exon2 wild primers
Primer and reverse primer nucleotide sequence are respectively such as SEQ ID NO：10 and SEQ ID NO：Shown in 11.
Another aspect of the present invention further relates to a kind of composition, the combination production containing primer pair as described above and probe
Product.
The composition can exist as a solution, and solvent is preferably water；Can also include pH in the composition
Buffer reagent and enzyme etc..
The composition can be used in detecting KRAS gene mutation type.
Another aspect of the present invention further relates to a kind of kit, and it includes the combination of primer pair as described above and probe productions
Product preferably further include quantitative fluorescent PCR reaction buffer, sample treatment liquid, seedless sour water, archaeal dna polymerase, KRAS genes the 2nd
It is one or more in exons mutation Type Positive control sequence and wild type control sequence.
Preferably, the archaeal dna polymerase can be added in the quantitative fluorescent PCR reaction buffer, form quantitative fluorescent PCR
Reaction solution carries out integration packaging.
It is furthermore preferred that the archaeal dna polymerase be selected from Taq, Bst, Vent, Phi29, Pfu, Tru, Tth, Tl1, Tac, Tne,
Any in Tma, Tih, Tf1, Pwo, Kod, Sac, Sso, Poc, Pab, Mth, Pho, ES4DNA polymerase, Klenow segments
Kind；
It is highly preferred that the archaeal dna polymerase is Taq archaeal dna polymerases.
It is furthermore preferred that the sample handled by the sample treatment liquid include the Nasal swabs of patient, blood, serum, blood plasma,
Tissue fluid, lymph, sperm, cell or tissue；More preferably blood plasma.
Preferably, the KRAS Exon 2s mutation type positive control sequence and wild type control sequence can
The carrier pack in the form of plasmid.
Preferably, seedless sour water can also be added in the kit, using as negative control.
According to an aspect of the present invention, the invention further relates to the combination product of primer pair as described above and probe, combinations
The application of object or kit in preparing reagent or the kit for diagnosing KRAS mutation cancer.
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
It can be with conventional products that are commercially available.
Embodiment
1. the design of primer and probe
It is inquired to obtain the wild type and mutated genes sequence of mankind's KRAS genes according to Genbank, with software Primer
Premier5 designs allele specific amplification primer and hydrolysis probes.The ends allele-specific sense primer 3' are prominent
Become base and uses lock nucleic acid.The ends hydrolysis probes 5' flag F AM, the ends 3' mark BHQ3.
(1) KRAS sequences
Normal human subject KRAS Exon 2s and peripheral sequence are SEQ ID NO：Shown in 14.
(2) primer and probe
Mankind's KRAS common gene mutations types are the corresponding forward direction of G12A, G12C, G12D, G12R, G12S, G12V and G13D
And reverse primer see the table below respectively.
Wherein, SEQ ID NO：3, first, 4,5,6,7,8,9 ends 3' nucleotide is lock nucleic acid；
SEQ ID NO：9th, 11,18,23 nucleotide at 1 ends 5' is lock nucleic acid.
2. the extraction of sample
Every part of sample 1.5-2mL blood plasma, the sample less than 2mL supply 2mL with PBS, with QIAamp DNA Blood Midi
Kit (Qiagen, Germany) purifies cDNA, uses Quant-iTTM dsDNA BR kit(Invitrogen,Carlsbad,CA)
Measure DNA concentration after purification.Sample packing after be stored in -20 DEG C it is spare.
3.ASLNAqPCR reacts
(1) reaction system：Use FastStart Universal Probe Master with ROX (Roche
Applied Science, Mannheim, Germany) ASLNAqPCR reactions are carried out, reaction system is with reference to the kit explanation
Book.
(2) platform is reacted：ABI 7500(Applied Biosystem,Foster City,CA)
(3) reaction condition:It see the table below.
| Temperature | Time | Recurring number |
| 50℃ | 2 seconds | 1 |
| 95℃ | 10 seconds | 1 |
| 95℃ | 30 seconds | 38 |
| 60℃ | 30 seconds | 38 |
| 72℃ | 30 seconds | 38 |
(4) result judges:The copy number of related mutation gene is calculated by △ Ct methods.
4. sensitivity experiment
Each mutant plasmid standard items of structure are quantified respectively and are diluted to 1 × 105Copies/uL. then wild type is used
Template dilutes saltant type template, and the content for preparing saltant type template is respectively 0%, 0.01%, 0.1%, 1%, 10% and 100%
And total DNA template concentrations are 1 × 105The serial sample of copies/uL.Then various kinds is detected respectively with mutant primer detection architecture
This, 3 progress of experiment point set 3 multiple holes every time.
5. confirmatory experiment
150 samples are verified using generation sequencing, and are compared with the result of ASLNAqPCR.According to
The standard specification of GenomeLab DTCS Kit (Beckman Coulter, Inc., Fullerton, CA, U.S.A.) carries out
The result of ASLNAqPCR is verified.
6. verification result
The present invention modifies 3 ' ends of forward primer with LNA, is allowed on the basis of conventional allele specific amplification
It is complementary with mutant nucleotide sequence, since LNA is insensitive to exonuclease, when 3 ' end of primer and template mismatch, Taq
Archaeal dna polymerase can not cut off the LNA bases of 3 ' end of primer to inhibit to extend, and only be exactly matched therewith when primer encounters
Mutant nucleotide sequence is could to extend.Meanwhile we introduce the probe of a LNA modifications on the basis of locus specific amplification, carry
The sensitivity of high mutational site detection and accuracy.
(1) sensitivity
According to the above sensitivity experiment, the Monitoring lower-cut of KRAS mutation reaches 0.01%.
(2) accuracy
The use of generation sequencing is 264bp to the amplified production of KRAS exons.In 150 samples, by a generation sequencing and
It is 150 that ASLNAqPCR, which reacts amplifiable sample number,.A generation is sequenced and the detectable mutation of ASLNAqPCR are respectively 56
Example and 50.In all catastrophes, the mutational site that wherein G12F, Q61H and Q61L do not embody in this invention shares 6
Example mutation can not be detected by ASLNAqPCR, other abrupt climatic change situations are consistent with generation sequence verification result, such as following table institute
Show：
| KRAS gene mutation type | A generation is sequenced | ASLNAqPCR |
| G12A | 3/150 | 3/150 |
| G12C | 4/150 | 4/150 |
| G12D | 20/150 | 20/150 |
| G12R | 2/150 | 2/150 |
| G12S | 2/150 | 2/150 |
| G12V | 11/150 | 11/150 |
| G13D | 8/150 | 8/150 |
| G12F | 1/150 | 0/150 |
| Q61H | 3/150 | 0/150 |
| Q61L | 2/150 | 0/150 |
| All catastrophes | 56/150 | 50/150 |
Finally it should be noted that：The above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Present invention has been described in detail with reference to the aforementioned embodiments for pipe, but it will be understood by those of ordinary skill in the art that：Its
It still can be with technical scheme described in the above embodiments is modified, either to which part or all technical features
Carry out equivalent replacement；And these modifications or replacements, various embodiments of the present invention skill that it does not separate the essence of the corresponding technical solution
The range of art scheme.
SEQUENCE LISTING
<110>It asks and attains medical science and technology（Beijing）Co., Ltd
<120>Combination product, composition, kit and its application of primer pair and probe for detecting KRAS mutation
<160> 14
<170> PatentIn version 3.3
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gtagttggag ctggtga 17
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actggtggag tatttga 17
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tacagataaa ggtttctc 18
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aaggtgagtt tgtattaaaa ggtactgg 28
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ttggagctgg tggcgtaggc aagagtgcct tgacgataca gctaattcag aatcattttg 180
tggacgaata tgatccaaca atagaggtaa atcttgtttt aatatgcata ttactggtgc 240
aggaccattc tttgatacag ataaaggttt ctctgaccat tttcatgagt 290
Claims (10)
1. the combination product of a kind of primer pair and probe includes the LNA fluorescence of the different KRAS Exon 2s saltant types of detection
Probe and primer；
The nucleotide sequence of the LNA fluorescence probes such as SEQ ID NO：Shown in 1；
The primer is one or more pairs of in following primer pair：
Wherein, at least there is a lock nucleic acid in the forward primer sequence of the primer pair；
And/or；
At least there is a lock nucleic acid in the LNA fluorescence probes sequence.
2. the combination product of primer pair according to claim 1 and probe, which is characterized in that the primer pair it is each just
It is lock nucleic acid to first, the ends 3' of primer nucleotide.
3. the combination product of primer pair according to claim 1 and probe, which is characterized in that the LNA fluorescence probes 5'
9th, 11,18,23 nucleotide at end is lock nucleic acid.
4. the combination product of primer pair according to claim 1 and probe, which is characterized in that the LNA fluorescence probes
5 ' end mark fluorescents emit group, 3 ' end label quenching groups.
5. the combination product of primer pair according to claim 4 and probe, which is characterized in that the fluorescent emission group choosing
From any one of FAM, HEX, VIC or JOE, preferably FAM.
6. the combination product of primer pair according to claim 4 and probe, which is characterized in that the quenching group is selected from
Any one of TAMRA, BHQ or MGB, preferably BHQ, more preferably BHQ3.
7. according to the combination product of claim 1-6 any one of them primer pair and probe, which is characterized in that the combination production
Product further include the Exon2 wild primers for expanding KRAS Exon 2 wild-type sequences；Preferably, the Exon2
The forward primer and reverse primer nucleotide sequence of wild primers are respectively such as SEQ ID NO：10 and SEQ ID NO：Shown in 11.
8. a kind of composition, the combination product containing claim 1-7 any one of them primer pair and probe.
9. a kind of kit, it includes the combination products of claim 1-7 any one of them primer pair and probe, preferably also wrap
Include quantitative fluorescent PCR reaction buffer, sample treatment liquid, seedless sour water, archaeal dna polymerase, KRAS Exon 2s mutation class
It is one or more in type positive control sequence and wild type control sequence.
10. the combination product of claim 1-7 any one of them primer pair and probe, composition according to any one of claims 8 or
Application of the kit in preparing reagent or the kit for diagnosing KRAS mutation cancer described in claim 9.
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Cited By (2)
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| CN109439756A (en) * | 2018-11-30 | 2019-03-08 | 广东腾飞基因科技股份有限公司 | It is a kind of for detecting the kit of KRAS correlation drug resistance |
| CN110910957A (en) * | 2019-12-31 | 2020-03-24 | 求臻医学科技（北京）有限公司 | Single-tumor-sample-based high-throughput sequencing microsatellite instability detection site screening method |
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Cited By (3)
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| CN109439756A (en) * | 2018-11-30 | 2019-03-08 | 广东腾飞基因科技股份有限公司 | It is a kind of for detecting the kit of KRAS correlation drug resistance |
| CN110910957A (en) * | 2019-12-31 | 2020-03-24 | 求臻医学科技（北京）有限公司 | Single-tumor-sample-based high-throughput sequencing microsatellite instability detection site screening method |
| CN110910957B (en) * | 2019-12-31 | 2023-06-27 | 求臻医学科技(浙江)有限公司 | Single-tumor-sample-based high-throughput sequencing microsatellite instability detection site screening method |
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