CN107523621A - A kind of primer sets and its kit for being used to detect fragile X mental retardation - Google Patents
A kind of primer sets and its kit for being used to detect fragile X mental retardation Download PDFInfo
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- CN107523621A CN107523621A CN201710787429.3A CN201710787429A CN107523621A CN 107523621 A CN107523621 A CN 107523621A CN 201710787429 A CN201710787429 A CN 201710787429A CN 107523621 A CN107523621 A CN 107523621A
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
The invention discloses a kind of primer sets and its kit for being used to detect fragile X mental retardation.The kit includes the primer sets of detection fragile X mental retardation, and the primer sets include being used for the sense primer F and anti-sense primer R for detecting CGG repeat numbers, and for detecting the sense primer F and anti-sense primer M of AGG insertion information；The sequence of the sense primer F such as SEQ ID NO：Shown in 1, its 5 ' end band FAM fluorescence；Anti-sense primer R sequence such as SEQ ID NO：Shown in 2；Anti-sense primer M sequence such as SEQ ID NO：Shown in 3.Kit of the invention can detect whether sample is that premutation carries or full mutation carries, and avoid missing inspection, methods described is simple, convenient fast, cost is low, and has stronger specific and higher sensitivity, has larger application prospect.
Description
Technical field
The invention belongs to technical field of medical detection.More particularly, to a kind of primer for being used to detect fragile X mental retardation
Group and its kit.
Background technology
Fragile X mental retardation is the most common heredity dysnoesia type for being only second to mongolism, the incidence of disease 1/
2500 ~ 1/5000, the incidence of disease is about 6.3% in China's Hypophrenia.There are obvious gender differences in disease morbidity, man
Property the incidence of disease it is higher, according to statistics, male's incidence of disease be 1：4000, the women incidence of disease is 1:8000~1:6000.
Fragile X mental retardation is X chromosome DNA Hypermethylations or changes the caused chain incomplete dominant lnheritances of X completely
Disease.FXS Disease-causing gene is No. 1 gene of fragile X mental retardation（FMR1）, most FXS are due to FMR1's（CGG）N weights
Complex sequences hyper expanded cause gene methylation and caused by, only only a few case is either complete by FMR1 point mutations
Lack and cause entirely.The CGG repeat numbers of normal person are 6 ~ 44, and most commonly 30 times, repeat number is 55 ~ 200 as " preceding prominent
Become ", repeat number more than 200 for " full mutation ", and it 45 ~ 54 is " gray area " that repeat number, which is, between " premutation " and " just
Between often ", without clinical manifestation.Influence（CGG）The principal element of the unstable amplification of n repetitive sequences has（CGG）N repeat numbers
（Repeat number is bigger, and amplification is higher for the risk being mutated entirely）, AGG numbers and AGG embedded models（AGG can strengthen stability）Deng.
In normal person,（CGG）N repeat numbers increase, AGG insertions number also increases therewith, and the AGG numbers in fragile X mental retardation patient
Ratio is significant lower, prompts AGG reduction may be relevant with illness.It can make when there is AGG insertions（CGG）The chemistry of n sequences is special
Property change, insert 1 AGG's（CGG）Tm values decline 2 DEG C when n sequences are relatively free of AGG insertions, and 2 AGG insert Tm values
Decline 5 DEG C, illustrate that AGG insertions can not only influence（CGG）The stability of n sequences, while thermodynamic stability is significantly reduced,
Offspring easily causes on continuing（CGG）The extension of n repetitive sequences and ultimately result in fragile X mental retardation.As can be seen here, AGG's is slotting
Enter to be advantageous to（CGG）The stable heredity of n repetitive sequences.
Fragile X mental retardation is mainly characterized by moderate to severe feeblemindedness, and the state of an illness has exacerbation with age
Trend, other clinical manifestations are unusual facies, such as long face or protruding ears, occur big testis after puberty, many patients go back table
It is now impulsion, more dynamic, anxiety, the frightened social, Autism behavior such as language is stiff.FXS seriously endangers upgrowth and development of children, early hair
Existing, early treatment, early intervention can significantly improve infant prognosis, and can provide prenatal genetic consulting for family heredity, avoid in family
Occurs same patient again, social population Quality strengthening happy to happy family life is of great importance, and has immeasurable society
Can benefit and economic benefit.The present invention is applied to CGG repeat numbers and the AGG insertion information of clinical detection fragile X mental retardation.
Existing fragile X mental retardation detection method can rapidly detect result using round pcr, avoid fluorescence spy
The technical operation difficulty such as pin, MLPA, Southern Blot are high, detect the shortcomings of time-consuming, cost is high.But due to G/C content very
Height, cause detection specificity and sensitivity of the technology for the carrying of women fragile X mental retardation or chimera etc. relatively low, easily go out
Existing false negative.Chinese patent（CN103981253A）Disclose it is a kind of be used for detect fragile X syndrome CGG tuples and
AGG inserts the PCR kit of information, and the patent is S+ (CGG) 4AGG, S+ (GCG) 4GAG, S for the primer of AGG insertion information
+ (GGC) 4GGA or their reverse complementary sequence S+ (GCC) 4TCC, S+ (CGC) 4CTC, S+ (CCG) 4CCT, these primers are complete
Portion is the primer designed in AGG sequence, can only identify the sample of fragile X mental retardation wild type, and fragile X mental retardation sample
DNA CGG repeat number it is too high and when being inserted without AGG, the primer sequence sample that information is inserted without AGG can not be judged be
No is fragile X mental retardation or the failure of an experiment, and follow-up calculation formula is also not suitable for.Therefore the patent can only judge normal sample
CGG repeat numbers, and fragile X mental retardation carrier can not be determined whether, there is larger limitation.
The content of the invention
The technical problems to be solved by the invention are to overcome the defects of fragile X mental retardation detection method is present in the prior art
A kind of and deficiency, there is provided detection kit of the CGG tuples for being used to detect fragile X syndrome and AGG insertion information.Institute
State kit it is simple, convenient it is quick, cost is low, and has stronger specific and higher sensitivity, and existing
Detection CGG tuples and the kit of AGG insertion information are compared, and the present invention can either identify the CGG repeat numbers of normal sample,
The carrier for belonging to fragile X mental retardation premutation or being mutated entirely is capable of deciding whether, there is larger application prospect.
It is an object of the invention to provide a kind of primer sets for being used to detect fragile X syndrome.
It is a further object of the present invention to provide a kind of kit for detecting fragile X syndrome.
The above-mentioned purpose of the present invention is to give realization by the following technical programs：
A kind of primer sets for being used to detect fragile X mental retardation, including sense primer F and downstream for detecting CGG repeat numbers draw
Thing R, and for detecting the sense primer F and anti-sense primer M of AGG insertion information；The sequence of the sense primer F such as SEQ ID
NO：Shown in 1, its 5 ' end band FAM fluorescence；Anti-sense primer R sequence such as SEQ ID NO：Shown in 2；Anti-sense primer M sequence is such as
SEQ ID NO：Shown in 3；Shown in specific as follows：
F：FAM-5’-TCAGGCGCTCAGCTCCGTTTCGGTTTCA-3’（SEQ ID NO：1）
R：5’-AAGCGCCATTGGAGCCCCGCACTTCC-3’（SEQ ID NO：2）
M：5’-AGCGTCTACTGTCTCGGCACTTGCCCGCCGCCGCCG-3’（SEQ ID NO：3）
The present invention is designed for augmentation detection target fragment at the both ends of FMR1 gene C GG repetitive sequences（Duplicate block containing CGG）'s
Primers F, R, the primer M of augmentation detection CGG repetitive sequences is designed in CGG repeat regions, by carrying out two reactants to sample
The fluorescent PCR amplification of system, a system are amplification CGG repeat number fragments, and another system is expands the AGG information of insertion, so
Detected afterwards using capillary electrophoresis, can enough detect whether sample is fragile X with and can while quick detection fragile X mental retardation
Syndrome carries so that the detection method of fragile X mental retardation is more perfect.The present invention for AGG insertion information primer 5 '-
AGCGTCTACTGTCTCGGCACTTGCCCGCCGCCGCCG-3’（SEQ ID NO：3）For non genome+（CCG）4, main pin
To CGG repetitive sequence, for the sample inserted without AGG, the peak type is continuous peak, for the normal sample for having AGG information to insert
This or premutation carry sample, and peak type is discontinuous peak, and AGG number of insertion is judged according to discontinuous breach quantity,
Judge specific CGG repeat numbers by the amplified fragments of first reaction again, can either identify that the CGG of normal sample is repeated
Number, is also capable of deciding whether the carrier for belonging to fragile X mental retardation premutation or being mutated entirely.
Meanwhile application of the above-mentioned primer sets in fragile X mental retardation detection kit is prepared is also in the scope of the present invention
It is interior.
A kind of kit for detecting fragile X mental retardation, the kit include above-mentioned detection primer group.
Preferably, the kit also includes archaeal dna polymerase, PCR reinforcing agents, PCR buffer solutions and DNA molecular amount standard
Product.
Preferably, the amplification reaction system of the kit is 2 × GC Buffer I 10 μ L, 10mmol/L dNTPs
0.4 μ L, PCR reinforcing agent 5.2 μ L, each μ L of 0.3 μ L, 5U/ μ L archaeal dna polymerases 0.3 of 10 μm of ol/L upstream and downstream primers, template DNA
80ng, ddH2O complements to 20 μ L.
Preferably, the amplification reaction condition of the kit is 98 DEG C of pre-degeneration 10min；97 DEG C of denaturation 35s, 62 DEG C of annealing
35s, 68 DEG C of extension 4min, 33 circulations；68 DEG C extend 10min eventually.
The application method of mentioned reagent box, comprises the following steps：
S1. sample genomic dna is extracted；
S2. using genomic DNA described in step S1 as template, pcr amplification reaction is carried out respectively using the primer sets in kit；
S3. after reaction terminates, reaction product is detected with capillary electrophoresis respectively, the model of Direct Analysis CGG repeat numbers
Enclose and AGG insertion number；The CGG repeat numbers n=（Sample to be tested fragment length-control normal sample fragment length）/3+
30；The AGG insertions number is the breach number at discontinuous peak.
Preferably, the capillary electrophoresis be by 1 μ L pcr amplification products add 8.5 μ L Hi-Di Formamide and
The LIZ size standard of 0.5 μ L GeneScan 600,98 DEG C of denaturation 5min, put 2min on ice, with hair immediately after mixing
Tubule Genetic Analyser is analyzed.
The present invention is pointed to the primers F of CGG duplicate blocks flank using one, R is used to expand the fragment containing CGG duplicate blocks,
Interpretation of result is carried out to the fragment using detection device simultaneously；Establish the mathematical modeling on CGG repeat numbers and analyze sample according to this
The CGG repeat numbers of product.Anti-sense primer M is provided simultaneously to be used to expand CGG repeat regions, utilizes expansion of the detection device to the region
Increase fragment to be analyzed, establish the mathematical modeling on AGG insertion information and analyze the AGG insertion information of sample according to this.
Compared with prior art, the present invention has the advantages that：
（1）The present invention accurately can quickly analyze sample CGG repeat numbers and AGG insertion information, while can detect sample and be
It is no to be carried for premutation or be mutated carrying entirely, avoid missing inspection；
（2）The present invention can break through existing fragile X mental retardation detection method using fluorescence probe, MLPA, SouthernBlot etc.
The barrier that difficulty is high, time-consuming, cost is high, specificity and sensitivity are relatively low；
（3）The present invention can be directly according to the scope of formula analysis CGG repeat numbers and AGG insertion by Capillary Electrophoresis result
Number is so as to quick diagnosis sample characteristic；
（4）The present invention obtains product separating degree using high-resolution equipment separation product, passes through mathematical formulae（Repeat number n=（Treat
Test sample this fragment length-control normal sample fragment length）/3+30）Obtain CGG repeat numbers and number AGG insertion infomation detection knots
The number at the peak of fruit obtains AGG insertion information, and determination methods are convenient and swift to be easily understood, and has larger application value.
Brief description of the drawings
Fig. 1 is PCR schematic diagrams of the present invention.
Fig. 2 is that the CGG repeat numbers of normal male sample in the embodiment of the present invention 2 detect electrophoretogram.
Fig. 3 is that the AGG of normal male sample in the embodiment of the present invention 2 inserts infomation detection electrophoretogram.
Fig. 4 is that the CGG repeat numbers of normal female sample in the embodiment of the present invention 3 detect electrophoretogram.
Fig. 5 is that the AGG of normal female sample in the embodiment of the present invention 3 inserts infomation detection electrophoretogram.
Fig. 6 is the CGG repeat numbers detection electrophoretogram that premutation carries sample in the embodiment of the present invention 4.
Fig. 7 is the AGG insertion infomation detection electrophoretograms that premutation carries sample in the embodiment of the present invention 4.
Embodiment
The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
Limit in any form.Unless stated otherwise, the reagent of the invention used, method and apparatus routinely try for the art
Agent, method and apparatus.
Unless stated otherwise, following examples agents useful for same and material are purchased in market.
The design of the fragile X mental retardation detection primer of embodiment 1
（1）Augmentation detection target fragment is designed at the both ends of FMR1 gene C GG repetitive sequences（Duplicate block containing CGG）Draw
Thing F, R, it is as shown in Figure 1 in the primer M of CGG repeat regions design augmentation detection CGG repetitive sequences, the amplification principle of primer；Institute
State detection CGG repeat numbers and detect the sense primer F of AGG insertion information sequence such as SEQ ID NO：Shown in 1, its 5 ' end band
FAM fluorescence；Detect the anti-sense primer R of CGG repeat numbers sequence such as SEQ ID NO：Shown in 2；Detect the downstream of AGG insertion information
Primer M sequence such as SEQ ID NO：Shown in 3；Specific primer sequence is as shown in table 1：
Table 1
| Sequence number | Primer（3’-5’） |
| SEQ ID NO:1 | FAM-TCAGGCGCTCAGCTCCGTTTCGGTTTCA |
| SEQ ID NO:2 | AAGCGCCATTGGAGCCCCGCACTTCC |
| SEQ ID NO:3 | AGCGTCTACTGTCTCGGCACTTGCCCGCCGCCGCCG |
（2）Reaction system：10 μ L 2 × GC Buffer I, 0.4 μ L dNTPs（10mmol/L）, 5.2 μ L PCR reinforcing agents,
10 μm of ol/L each 0.3 μ L of upstream and downstream primer, archaeal dna polymerase（5U/μL）0.3 μ L, template amount are 80ng, are finally mended with ddH2O
Enough to 20 μ L.
（3）Reaction condition：After 98 DEG C of pre-degeneration 10min, 33 circulations are carried out：97 DEG C of denaturation 35s, 62 DEG C of 35s that anneal, 68
DEG C extension 4min, then 68 DEG C eventually extension 10min.
（4）Result judgement：Calculated according to mathematical formulae, CGG repeat numbers n=（Sample to be tested fragment length-control normal sample
This fragment length）/3+30）Obtain CGG repeat numbers；AGG inserts the number that information inserts the peak of infomation detection result by several AGG
Obtain AGG insertion information（The CGG repeat numbers at first peak be 4, behind be continuous peak, show there is AGG to insert when there is breach
Enter）.
The normal male sample CGG repeat numbers of embodiment 2 detect and AGG insertion infomation detections
（1）DNA is extracted：Under the conditions of I agrees to or its guardian knows, its peripheral blood is gathered.Using the base of QIAGEN companies
Because of a group DNA extraction kit extraction, it is 40ng/ μ L to survey its concentration with fluorescent quantitation instrument.
（2）Prepare PCR reaction systems using the primer sets of embodiment 1, by the glimmering of sample two reaction systems of progress
Light PCR is expanded, and a system is amplification CGG repeat number fragments, and another system is the AGG information of amplification insertion；Two systems
In addition to primer pair is different, reaction system and response procedures are all identical.The PCR that 20 μ L are prepared in 200 μ L thin-walled PCR pipe is anti-
Answer system, PCR amplification system is as follows：
2×GC BufferⅠ 10μL
dNTPs（10mmol/L） 0.4μL
The μ L of PCR reinforcing agents 5.2
Sense primer F（10μmol/L） 0.3μL
Anti-sense primer R（10μmol/L） 0.3μL
DNA 80ng
Archaeal dna polymerase（5U/μL） 0.3μL
ddH2O complements to 20 μ L
Primer SEQIDNO is used respectively：1 and SEQIDNO：2 detection sample CGG repeat numbers；With primer SEQIDNO：1 and SEQIDNO：
3 detection AGG insertion information.
The composition of PCR reinforcing agents：Glycine betaine（5mol/L）4μL、DMSO 1.2μL.
PCR response procedures：First after 98 DEG C of pre-degeneration 10min, 33 circulations are carried out：97 DEG C of denaturation 35s, 62 DEG C of annealing 35s,
68 DEG C of extension 4min, then 68 DEG C extend 10min eventually.
（3）After reaction terminates, detected using ABI3500Dx capillary Genetic Analysers.Take 1 μ L PCR amplification productions
Thing, add 8.5 μ LHi-Di Formamide and the LIZ size standard of 0.5 μ L GeneScan 600,98 after mixing
DEG C denaturation 5min, put 2min on ice, upper machine testing immediately.
（4）Normal male sample CGG repeat numbers testing result is as shown in Fig. 2 the sample clinical detection result repeat number is
30, from the results, it was seen that purpose fragment is 312bp, it is normal sample；Its AGG insert infomation detection result as shown in figure 3,
From the results, it was seen that 2 AGG, profile 10A9A9 are inserted on the X chromosome of the sample in CGG sequences.
The normal female sample CGG repeat numbers of embodiment 3 detect and AGG insertion infomation detections
（1）DNA is extracted：Under the conditions of I agrees to or its guardian knows, its peripheral blood is gathered.Using the base of QIAGEN companies
Because of a group DNA extraction kit extraction, it is 40ng/ μ L to survey its concentration with fluorescent quantitation instrument.
（2）Prepare PCR reaction systems using the primer sets of embodiment 1, by the glimmering of sample two reaction systems of progress
Light PCR is expanded, and a system is amplification CGG repeat number fragments, and another system is the AGG information of amplification insertion；Two systems
In addition to primer pair is different, reaction system and response procedures are all identical.The PCR that 20 μ L are prepared in 200 μ L thin-walled PCR pipe is anti-
Answer system, PCR amplification system is as follows：
2×GC BufferⅠ 10μL
dNTPs（10mmol/L） 0.4μL
The μ L of PCR reinforcing agents 5.2
Sense primer F（10μmol/L） 0.3μL
Anti-sense primer R（10μmol/L） 0.3μL
DNA 80ng
Archaeal dna polymerase（5U/μL） 0.3μL
ddH2O complements to 20 μ L
Primer SEQIDNO is used respectively：1 and SEQIDNO：2 detection sample CGG repeat numbers；With primer SEQIDNO：1 and SEQIDNO：
3 detection AGG insertion information.
The composition of PCR reinforcing agents：Glycine betaine（5mol/L）4μL、DMSO 1.2μL.
PCR response procedures：First after 98 DEG C of pre-degeneration 10min, 33 circulations are carried out：97 DEG C of denaturation 35s, 62 DEG C of annealing 35s,
68 DEG C of extension 4min, then 68 DEG C extend 10min eventually.
（3）After reaction terminates, detected using ABI3500Dx capillary Genetic Analysers.Take 1 μ L PCR amplification productions
Thing, add 8.5 μ LHi-Di Formamide and the LIZ size standard of 0.5 μ L GeneScan 600,98 after mixing
DEG C denaturation 5min, put 2min on ice, upper machine testing immediately.
（4）Normal female sample CGG repeat numbers testing result is as shown in figure 4, from the results, it was seen that two purpose fragments
Respectively 309bp and 330bp, repeat number n1=（309-312）/ 3+30=29, n2=（330-312）/ 3+30=36, it is normal sample
This, this result is identical with clinical detection result；Its AGG inserts infomation detection result as shown in figure 5, from the results, it was seen that should
2 AGG are respectively inserted in CGG sequences on two X chromosomes of sample, profile is respectively 9A9A9 and 9A9A16.
The mutation of embodiment 4 carries sample CGG repeat numbers detection and AGG insertion infomation detections
1st, premutation pattern detection
（1）DNA is extracted：Under the conditions of I agrees to or its guardian knows, its peripheral blood is gathered.Using the base of QIAGEN companies
Because of a group DNA extraction kit extraction, it is 40ng/ μ L to survey its concentration with fluorescent quantitation instrument.
（2）Prepare PCR reaction systems using the primer sets of embodiment 1, by the glimmering of sample two reaction systems of progress
Light PCR is expanded, and a system is amplification CGG repeat number fragments, and another system is the AGG information of amplification insertion；Two systems
In addition to primer pair is different, reaction system and response procedures are all identical.The PCR that 20 μ L are prepared in 200 μ L thin-walled PCR pipe is anti-
Answer system, PCR amplification system is as follows：
2×GC BufferⅠ 10μL
dNTPs（10mmol/L） 0.4μL
The μ L of PCR reinforcing agents 5.2
Sense primer F（10μmol/L） 0.3μL
Anti-sense primer R（10μmol/L） 0.3μL
DNA 80ng
Archaeal dna polymerase（5U/μL） 0.3μL
ddH2O complements to 20 μ L
Primer SEQIDNO is used respectively：1 and SEQIDNO：2 detection sample CGG repeat numbers；With primer SEQIDNO：1 and SEQIDNO：
3 detection AGG insertion information.
The composition of PCR reinforcing agents：Glycine betaine（5mol/L）4μL、DMSO 1.2μL.
PCR response procedures：First after 98 DEG C of pre-degeneration 10min, 33 circulations are carried out：97 DEG C of denaturation 35s, 62 DEG C of annealing 35s,
68 DEG C of extension 4min, then 68 DEG C extend 10min eventually.
（3）After reaction terminates, detected using ABI3500Dx capillary Genetic Analysers.Take 1 μ L PCR amplification productions
Thing, add 8.5 μ LHi-Di Formamide and the LIZ size standard of 0.5 μ L GeneScan 600,98 after mixing
DEG C denaturation 5min, put 2min on ice, upper machine testing immediately.
（4）Premutation carries sample CGG repeat numbers testing result as shown in fig. 6, from the results, it was seen that one of mesh
Fragment be 312bp, another purpose fragment is 390bp, repeat number n1=（312-312）/ 3+30=30, n2=（390-312）/3
+ 30=56, sample is carried for premutation, this result is identical with clinical detection result；AGG insertion infomation detection result such as Fig. 7 institutes
Show, from the results, it was seen that two X chromosomes of the sample, wherein 2 AGG are inserted in a CGG sequence, one in addition
There is no AGG insertions, profile is respectively 9A9A10 and 56.
2nd, in addition, when result is：If CGG repeat numbers testing result according to formula n=（Sample to be tested fragment length-control is just
Normal sample fragment length）/3+30）, calculate n>When 200, and the number of AGG insertion infomation detection results peaks is more than 200, then the sample
This CGG repeat numbers are more than 200, and the sample for drawing detection is full mutation；AGG insertion information is the individual of the peak of testing result
Number, the peak type of AGG insertion information is discontinuous peak, and AGG number of insertion can be judged according to discontinuous breach quantity.
Because because full mutation probability is smaller, temporarily there is no the sample being mutated entirely can use, therefore do not do complete prominent
Become the detection checking of sample, but it can be seen from the Cleaning Principle of the present invention, premutation and the detection process being mutated entirely and judgement
Method is consistent.
Although full mutation probability is smaller, still there is a small amount of presence, therefore, primer sets and detection kit pair of the invention
Also there is important application value and meaning in the detection of full sudden change sample.
A kind of kit for detecting fragile X mental retardation of embodiment 5
A kind of kit for detecting fragile X mental retardation, including for detecting primer pair F, R of CGG repeat numbers and for detecting AGG
Insert primer pair F, M of information；The sense primer F of the detection CGG repeat numbers and detection AGG insertion information sequence such as SEQ
ID NO：Shown in 1, its 5 ' end band FAM fluorescence；Detect the anti-sense primer R of CGG repeat numbers sequence such as SEQ ID NO：Shown in 2；
Detect the anti-sense primer M of AGG insertion information sequence such as SEQ ID NO：Shown in 3；Shown in specific as follows：
F：FAM-5’-TCAGGCGCTCAGCTCCGTTTCGGTTTCA-3’（SEQ ID NO：1）
R：5’-AAGCGCCATTGGAGCCCCGCACTTCC-3’（SEQ ID NO：2）
M：5’-AGCGTCTACTGTCTCGGCACTTGCCCGCCGCCGCCG-3’（SEQ ID NO：3）
Meanwhile the kit also includes archaeal dna polymerase, PCR reinforcing agents, buffer system and molecular weight standard.
The application method of the detection kit comprises the following steps：
Using the method for mentioned reagent box detection fragile X mental retardation, comprise the following steps：
S1. sample DNA is extracted；
S2. using DNA described in step S1 as template, pcr amplification reaction is carried out respectively using the primer sets in kit；
S3. reaction terminate after, reaction product is detected with capillary electrophoresis, the scope of Direct Analysis CGG repeat numbers and
AGG insertion number.
The PCR reaction systems are μ L, the PCR reinforcing agents 5.2 of 2 × GC Buffer, I 10 μ L, 10mmol/L dNTPs 0.4
μ L, 10 μm of ol/L upstream and downstream primers each μ L of 0.3 μ L, 5U/ μ L archaeal dna polymerases 0.3, template DNA 80ng, ddH2O complements to 20 μ
L。
The PCR response procedures are 98 DEG C of pre-degeneration 10min；97 DEG C of denaturation 35s, 62 DEG C of annealing 35s, 68 DEG C extend
4min, 33 circulations；68 DEG C extend 10min eventually.
The capillary electrophoresis is that 1 μ L pcr amplification products are added into 8.5 μ L Hi-Di Formamide and 0.5 μ L
The LIZ size standard of GeneScan 600,98 DEG C of denaturation 5min, put 2min on ice, with capillary base immediately after mixing
Because analyzer is analyzed.
Sequence table
<110>Guangzhou Da Rui Biotechnology Ltd.
<120>A kind of primer sets and its kit for being used to detect fragile X mental retardation
<130> 1712809ZBSH042
<141> 2017-09-04
<160> 3
<170> SIPOSequenceListing 1.0
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<211> 28
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<213>People (Homo sapiens)
<400> 1
tcaggcgctc agctccgttt cggtttca 28
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<211> 26
<212> DNA
<213>People (Homo sapiens)
<400> 2
aagcgccatt ggagccccgc acttcc 26
<210> 3
<211> 36
<212> DNA
<213>People (Homo sapiens)
<400> 3
agcgtctact gtctcggcac ttgcccgccg ccgccg 36
Claims (8)
1. a kind of primer sets for being used to detect fragile X mental retardation, it is characterised in that including the upstream for detecting CGG repeat numbers
Primers F and anti-sense primer R, and for detecting the sense primer F and anti-sense primer M of AGG insertion information；The sense primer F
Sequence such as SEQ ID NO：Shown in 1, its 5 ' end band FAM fluorescence；Anti-sense primer R sequence such as SEQ ID NO：Shown in 2；Downstream
Primer M sequence such as SEQ ID NO：Shown in 3.
2. application of the primer sets described in claim 1 in fragile X mental retardation detection kit is prepared.
3. a kind of kit for detecting fragile X mental retardation, it is characterised in that include primer sets described in claim 1.
4. kit according to claim 3, it is characterised in that also comprising archaeal dna polymerase, PCR reinforcing agents, PCR bufferings
Liquid and DNA molecular amount standard items.
5. kit according to claim 3, it is characterised in that the amplification reaction system of the kit is 2 × GC
I 10 μ L, 10mmol/L dNTPs of Buffer, 0.4 μ L, PCR reinforcing agents 5.2 μ L, each 0.3 μ L of 10 μm of ol/L upstream and downstream primers,
5U/ μ L archaeal dna polymerases 0.3 μ L, template DNA 80ng, ddH2O complements to 20 μ L.
6. kit according to claim 3, it is characterised in that the amplification reaction condition of the kit is 98 DEG C of pre- changes
Property 10min；97 DEG C of denaturation 35s, 62 DEG C of annealing 35s, 68 DEG C of extension 4min, 33 circulate；68 DEG C extend 10min eventually.
7. the application method of any one of claim 3~6 kit, it is characterised in that comprise the following steps：
S1. sample genomic dna is extracted；
S2. using genomic DNA described in step S1 as template, pcr amplification reaction is carried out respectively using the primer sets in kit；
S3. after reaction terminates, reaction product is detected with capillary electrophoresis respectively, the model of Direct Analysis CGG repeat numbers
Enclose and AGG insertion number；The CGG repeat numbers n=（Sample to be tested fragment length-control normal sample fragment length）/3+
30；The AGG insertions number is the breach number at discontinuous peak.
8. according to the method for claim 7, it is characterised in that the capillary electrophoresis is by 1 μ L pcr amplification products
Add 8.5 μ L Hi-Di Formamide and the LIZ size standard of 0.5 μ L GeneScan 600,98 DEG C after mixing
5min is denatured, 2min on ice is put immediately, is analyzed with capillary Genetic Analyser.
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| CN108531576A (en) * | 2018-04-12 | 2018-09-14 | 北京信诺佰世医学检验所有限公司 | Detect the kit and system of fragile X syndrome |
| CN109536593A (en) * | 2019-01-30 | 2019-03-29 | 北京华瑞康源生物科技发展有限公司 | System, kit and the application that the area 5`UTR of FXS syndrome related gene FMR1 is detected |
| CN110157782A (en) * | 2019-03-08 | 2019-08-23 | 北京华瑞康源生物科技发展有限公司 | A kind of primer group and PCR kit for quickly detecting FMR1 gene C GG repetitive sequence |
| CN112522384A (en) * | 2020-12-10 | 2021-03-19 | 北京华瑞康源生物科技发展有限公司 | Kit for determining AGG insertion quantity and position in CGG repetitive sequence of FMR1 gene and using method thereof |
| CN114645084A (en) * | 2020-12-17 | 2022-06-21 | 浙江大学医学院附属儿童医院 | Primer and kit for detecting CGG repeat number of FMR1 gene |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108531576A (en) * | 2018-04-12 | 2018-09-14 | 北京信诺佰世医学检验所有限公司 | Detect the kit and system of fragile X syndrome |
| CN109536593A (en) * | 2019-01-30 | 2019-03-29 | 北京华瑞康源生物科技发展有限公司 | System, kit and the application that the area 5`UTR of FXS syndrome related gene FMR1 is detected |
| CN109536593B (en) * | 2019-01-30 | 2022-02-25 | 北京华瑞康源生物科技发展有限公司 | System and kit for detecting 5' UTR region of FXS syndrome related gene FMR1 and application |
| CN110157782A (en) * | 2019-03-08 | 2019-08-23 | 北京华瑞康源生物科技发展有限公司 | A kind of primer group and PCR kit for quickly detecting FMR1 gene C GG repetitive sequence |
| CN112522384A (en) * | 2020-12-10 | 2021-03-19 | 北京华瑞康源生物科技发展有限公司 | Kit for determining AGG insertion quantity and position in CGG repetitive sequence of FMR1 gene and using method thereof |
| CN114645084A (en) * | 2020-12-17 | 2022-06-21 | 浙江大学医学院附属儿童医院 | Primer and kit for detecting CGG repeat number of FMR1 gene |
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Application publication date: 20171229 |