CN106399487A - Method and kit for detecting human fructosediphosphate aldolase B gene - Google Patents
Method and kit for detecting human fructosediphosphate aldolase B gene Download PDFInfo
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- CN106399487A CN106399487A CN201610799420.XA CN201610799420A CN106399487A CN 106399487 A CN106399487 A CN 106399487A CN 201610799420 A CN201610799420 A CN 201610799420A CN 106399487 A CN106399487 A CN 106399487A
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Abstract
The invention provides a method and a kit for detecting the human fructosediphosphate aldolase B gene. The detection kit comprises a primer pair for specifically amplifying three polymorphic sites on the human fructosediphosphate aldolase B gene, wherein the three polymorphic sites are respectively A150P, A175D and N335K, the primer pair comprises a common upstream primer and genotyping downstream primers, and the genotyping downstream primers comprise a wild type specific primer and a mutant type specific primer. With the adoption of the kit, the genotypes of the three polymorphic sites on the human fructosediphosphate aldolase B gene can be rapidly and efficiently detected, and the kit belongs to the kit for screening the human fructosediphosphate aldolase B gene, which is rapid, easy and convenient to operate, and is economical and efficient.
Description
Technical field
The present invention relates to gene engineering technology field, detect examination particularly to a kind of people's fructosediphosphate aldolase 1 B gene
Agent box, and its detection method.
Background technology
Hereditary fructose intolerance (Hereditary Fructose Intolerance, abbreviation HFI) is for Fructose generation
The one kind declined office, invitation, etc. on account of illness, is autosomal recessive hereditary diseasess, is to cause Type B aldolase to lack because ALD-B gene (ALDOB gene) is mutated
Weary or active reduction, thus lead to patient the exception of fructose metabolism.European sickness rate about 1:26100, and China's sickness rate
It is not immediately clear.With the raising of living standards of the people, sugared consumption is got more and more, HFI sickness rate can gradually increase.
Clinically hereditary fructose intolerance infant is more rare, mostly contains sucrose due in various formulas, so
It is that agalactous new life infant may occur in which that in 2-3 days vomiting, diarrhoea, dehydration, shock and bleeding tendency etc. are anxious after birth
Property liver failure symptom.The clinical diagnosises of primary disease need to rely on blood biochemistry checking and fructose tolerance test, but both sides
Method is all abrasive.It is good to have the advantages that to damage little, feasibility using the method auxiliary diagnosis of molecular biology.
Now the method for detection people's fructosediphosphate aldolase 1 B gene polymorphism mainly has direct Sequencing and allele specific
Property oligonucleotide analysis method (ASO) auxiliary detection.Although direct Sequencing accuracy rate is higher, time-consuming, high cost.Allele
Specific oligonucleotide analytic process be using pcr amplification product after, degeneration point sample on nylon membrane, fixing, then drawn with labelling
Thing hybridizes, and finally needs autoradiography.The shortcoming of this method is complex operation, high to the technical requirements of operator, needs
Want radiography, resolution is not high, and time-consuming.
Content of the invention
The present invention is intended to provide a kind of high accuracy, low cost, high flux, quick detection kit, can be used for detecting
People's fructosediphosphate aldolase 1 B gene whether there is mutation.
The technical solution used in the present invention is：
A kind of people's fructosediphosphate aldolase 1 B gene detection kit, described detection kit includes expanding for specificity
Increase the primer pair of 3 pleomorphism sites in people's fructosediphosphate aldolase 1 B gene, 3 pleomorphism sites be respectively A150P,
A175D, N335K, described primer pair includes common upstream primer and typing downstream primer, and described typing downstream primer includes wild
Type special primer and saltant type special primer, the common upstream primer of corresponding 3 pleomorphism sites, wild type special primer and prominent
Modification special primer is as shown in the table：
Table 1, the primer sequence of each pleomorphism site
F represents common upstream primer, and W represents wild type special primer, and M represents saltant type special primer.
Preferably, the 5' end of each common upstream primer is marked with fluorescent dye.Described fluorescent dye be FAM, HEX,
One or more of TAMRA, ROX, Cy3, Cy5, JOE, VIC, PET.
People's fructosediphosphate aldolase 1 B gene detection kit of the present invention also includes crt gene sequence, described right
Include normal sequence and mutant nucleotide sequence according to gene order.
Described normal sequence：
CAAAGGGAGAACTCCTTCCCTTTATTAGAAGCCCCATGGATCAGGTACAAAGGTACAAAGAAGCCTTTCTCTCTTTT
GTGACTTGCAGGGCTTGATGGCCTCTCAGAGCGCTGTGCTCAGTACAAGAAAGATGGTGTTGACTTTGGGAAGTGGC
GTGCTGTGCTGAGGATTGCCGACCAGTGTCCATCCAGCCTCGCTATCCAGGAAAACGCCAACGCCCTGGCTCGCTAC
GCCAGCATCTGTCAGCAGGTGCTCTGCCTTCCCCTTGGGCTGAAAAAGAGTAGGCTAGAGTTTTCTTCAGAGCTTTT
CTTTTCAATTATACTATAACTACAAATGGACCTCCTTTTCCCAGAAGGGGATGGTATCCCCAGCAATATTCAGCAAC
ATTGCTGTAAAAAGAAGAAAATCTGAGTGAAGGTTTGACTGGTTTCCCATGAGAGGCAGACAGGGTCAAGGTGGGGT
CACATTTACTCTAACCAGTCTCCTCTCTCATATTTGTCTTCTAGGCTAACTGCCAGGCGGCCAAAGGACAGTATGTT
CACACGGGTTCTTCTGGGGCTGCTTCCACCCAGTCGCTCTTCACAGCCTGCTATACCTACTAGGGTCCAATGCCCGC
CAGCCTAGCTCCAGTGCTTCTAGTAGGAGGGCTGAAAGGGAGCAACTTTTCCTCCAATCCTGGAAATTCGACACAAT
TAGATTT(SEQ ID NO.10)
Described mutant nucleotide sequence：
CAAAGGGAGAACTCCTTCCCTTTATTAGAAGCCCCATGGATCAGGTACAAAGGTACAAAGAAGCCTTTCTCTCTTTT
GTGACTTGCAGGGCTTGATGGCCTCTCAGAGCGCTGTGCTCAGTACAAGAAAGATGGTGTTGACTTTGGGAAGTGGC
GTCCTGTGCTGAGGATTGCCGACCAGTGTCCATCCAGCCTCGCTATCCAGGAAAACGCCAACGCCCTGGCTCGCTAC
GACAGCATCTGTCAGCAGGTGCTCTGCCTTCCCCTTGGGCTGAAAAAGAGTAGGCTAGAGTTTTCTTCAGAGCTTTT
CTTTTCAATTATACTATAACTACAAATGGACCTCCTTTTCCCAGAAGGGGATGGTATCCCCAGCAATATTCAGCAAC
ATTGCTGTAAAAAGAAGAAAATCTGAGTGAAGGTTTGACTGGTTTCCCATGAGAGGCAGACAGGGTCAAGGTGGGGT
CACATTTACTCTAACCAGTCTCCTCTCTCATATTTGTCTTCTAGGCTAAGTGCCAGGCGGCCAAAGGACAGTATGTT
CACACGGGTTCTTCTGGGGCTGCTTCCACCCAGTCGCTCTTCACAGCCTGCTATACCTACTAGGGTCCAATGCCCGC
CAGCCTAGCTCCAGTGCTTCTAGTAGGAGGGCTGAAAGGGAGCAACTTTTCCTCCAATCCTGGAAATTCGACACAAT
TAGATTT(SEQ ID NO.11)
Described people's fructosediphosphate aldolase 1 B gene detection kit also includes PCR buffer, hot start Taq polymerase, surpasses
Pure water, fluorescence molecule amount internal standard and allelic ladder.
Present invention also offers using the using method of above-mentioned people's fructosediphosphate aldolase 1 B gene detection kit, bag
Include the amplification to 3 pleomorphism sites of people's fructosediphosphate aldolase 1 B gene in sample and adopt polymerase chain reaction, its expansion
The detection of volume increase thing adopts high performance liquid chromatography, multi-fluorescence capillary electrophoresis or polyacrylamide gel electrophoresis.
Wherein, the amplification program of polymerase chain reaction is：95 DEG C 2 minutes；94 DEG C 30 seconds, 60 DEG C 30 seconds, 72 DEG C 30 seconds,
Totally 10 circulations；94 DEG C 30 seconds, 59 DEG C 30 seconds, 72 DEG C 30 seconds, totally 18 circulation；72 DEG C 10 minutes.
The invention has the beneficial effects as follows：
(1) it is based on polymerase chain reaction, improve the template amount of pleomorphism site detection, substantially reduce false positive；
(2) common upstream primer passes through to be equipped with different fluorophors, at most can detect 4-96 under an electrophoresis
The gene type of individual sample, has the characteristics that efficient economizing；
(3) polymerase chain reaction completes in 1 hour, capillary electrophoresis detection process also less than 1 hour, entirely
Detection process completed in 3 hours, had that the used time is few, quick and feature that accuracy is high；
(4) DNA extraction steps can be saved, saliva card, FTA card and blood filter paper can directly expand, and decrease operation step
Suddenly, simplify its operating process；
(5) present invention can accurately judge normal and sudden change sample gene type.
Brief description
Fig. 1 is 3 site primer collection of illustrative plates that in test kit, normal sequence compares wild type special primer；
Fig. 2 is 3 site primer collection of illustrative plates that in test kit, mutant nucleotide sequence compares saltant type special primer；
Fig. 3 is the detection collection of illustrative plates of embodiment 1；
Fig. 4 is the detection collection of illustrative plates of embodiment 2；
Fig. 5 is the detection collection of illustrative plates of embodiment 3；
Fig. 6 is the detection collection of illustrative plates of embodiment 4；
Fig. 7 is the detection collection of illustrative plates of embodiment 5；
Fig. 8 is the detection collection of illustrative plates of embodiment 6.
Specific embodiment
Combine accompanying drawing below by specific embodiment the present invention is described in further detail.But those skilled in the art
It will be understood that, the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Unreceipted in embodiment
Particular technique or condition person, are carried out according to the technology described by document in the art or condition or according to product description.
Agents useful for same or the unreceipted production firm person of instrument, be can by city available from conventional products.
The sequence such as following table institute that the pleomorphism site of 3 people's fructosediphosphate aldolase 1 B gene that the present invention is directed to is located
Show：
Table 2, the sequence of each pleomorphism site
W represents wild-type sequence, and M represents mutant sequences.
People's fructosediphosphate aldolase 1 B gene detection kit, described detection kit is included for specific amplification people
The primer pair of 3 pleomorphism sites in fructosediphosphate aldolase 1 B gene, 3 pleomorphism sites be respectively A150P, A175D,
N335K, described primer pair includes common upstream primer and typing downstream primer, and it is special that described typing downstream primer includes wild type
Primer and saltant type special primer, wherein forward primer are general primer, including fluorescent labeling and target sequence specific bond area；Under
Trip primer include typing recognition site and length adjustment area, described fluorescent dye be FAM, HEX, TAMRA, ROX, Cy3, Cy5,
One or more of JOE, VIC, PET.Also include crt gene sequence, PCR buffer, hot start Taq polymerase, ultra-pure water, glimmering
Optical molecule amount internal standard and allelic ladder, described crt gene sequence includes normal sequence and mutant nucleotide sequence.
Specially as shown in the table：
Table 3, the composition of test kit
After amplification
The specifically used method of test kit of the present invention：
1st, the extraction of sample DNA
According to the practical situation of sample, suitable method is selected to extract sample DNA, common method has：Paramagnetic particle method, cloudy from
Sub-exchange resin method and alkaline lysises etc..If sample is blood filter paper, FTA card, saliva card etc., step can be simplified, directly expand.
2nd, PCR reaction
The amplification system of PCR reaction is as follows：
The amplification program of PCR reaction is as follows：
3rd, genetic analyzer fluorescence electrophoresis detection
12 μ L deionized formamides are mixed with 0.5 μ L molecular weight internal standard (AGCU Marker SIZ-500).Add 1 μ L
PCR product or ALDOB Allelic Ladder, are vortexed and mix 3500r/min centrifugation 2min, 95 DEG C of degeneration 3min, ice bath
3min, is tested and analyzed with genetic analyzer ABI3130XL.
Embodiment 1：
1. the foundation of standard diagram
Detected by above-mentioned using method crt gene sequence, the normal sequence comparison obtaining as illustrated in fig. 1 and 2 is wild
3 site primer collection of illustrative plates of type special primer and 3 site primer collection of illustrative plates of mutant nucleotide sequence comparison saltant type special primer；
2. sample detection and comparison
By above-mentioned using method, a number sample is detected, obtain collection of illustrative plates as shown in Figure 3 it is known that a sample is
A150P site single-site mutant.
Embodiment 2：
Its overall flow is same as Example 1, and the testing result of No. two samples is as shown in Figure 4 it is known that No. two samples are
A175D site single-site mutant.
Embodiment 3：
Its overall flow is same as Example 1, and the testing result of No. three samples is as shown in Figure 5 it is known that No. three samples are
N335K site single-site mutant.
Embodiment 4：
Its overall flow is same as Example 1, and the testing result of No. four samples is as shown in Figure 6 it is known that No. four samples are
A150P and A175D site double-site mutant.
Embodiment 5：
Its overall flow is same as Example 1, and the testing result of No. five samples is as shown in Figure 7 it is known that No. five samples are
A150P and N335K site double-site mutant.
Embodiment 6：
Its overall flow is same as Example 1, and the testing result of No. six samples is as shown in Figure 8 it is known that No. six samples are
A175D and N335K site double-site mutant.
Above the better embodiment of the present invention is illustrated, but the invention is not limited to described enforcement
Example, those of ordinary skill in the art also can make a variety of equivalent modifications without prejudice on the premise of present invention spirit or replace
Change, these equivalent modifications or replacement are all contained in the application claim limited range.
SEQUENCE LISTING
<110>Guangdong magnificent Zhong Yuan bio tech ltd
<120>A kind of people's fructosediphosphate aldolase 1 B gene detection method and test kit
<130> 2016
<160> 17
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence
<400> 1
cttgcagggc ttgatggcct c 21
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
tcggcaatcc tcagcacagc 20
<210> 3
<211> 24
<212> DNA
<213>Artificial sequence
<400> 3
ctaatcggca atcctcagca cagg 24
<210> 4
<211> 21
<212> DNA
<213>Artificial sequence
<400> 4
cttgcagggc ttgatggcct c 21
<210> 5
<211> 24
<212> DNA
<213>Artificial sequence
<400> 5
taatcacctg ctgacagatg ctgg 24
<210> 6
<211> 20
<212> DNA
<213>Artificial sequence
<400> 6
cacctgctga cagatgttgt 20
<210> 7
<211> 23
<212> DNA
<213>Artificial sequence
<400> 7
tggtttccca tgagaggcag aca 23
<210> 8
<211> 17
<212> DNA
<213>Artificial sequence
<400> 8
ctttggccgc ctggcag 17
<210> 9
<211> 21
<212> DNA
<213>Artificial sequence
<400> 9
tatactttgg ccgcctggca c 21
<210> 10
<211> 700
<212> DNA
<213>Artificial sequence
<400> 10
caaagggaga actccttccc tttattagaa gccccatgga tcaggtacaa aggtacaaag 60
aagcctttct ctcttttgtg acttgcaggg cttgatggcc tctcagagcg ctgtgctcag 120
tacaagaaag atggtgttga ctttgggaag tggcgtgctg tgctgaggat tgccgaccag 180
tgtccatcca gcctcgctat ccaggaaaac gccaacgccc tggctcgcta cgccagcatc 240
tgtcagcagg tgctctgcct tccccttggg ctgaaaaaga gtaggctaga gttttcttca 300
gagcttttct tttcaattat actataacta caaatggacc tccttttccc agaaggggat 360
ggtatcccca gcaatattca gcaacattgc tgtaaaaaga agaaaatctg agtgaaggtt 420
tgactggttt cccatgagag gcagacaggg tcaaggtggg gtcacattta ctctaaccag 480
tctcctctct catatttgtc ttctaggcta actgccaggc ggccaaagga cagtatgttc 540
acacgggttc ttctggggct gcttccaccc agtcgctctt cacagcctgc tatacctact 600
agggtccaat gcccgccagc ctagctccag tgcttctagt aggagggctg aaagggagca 660
acttttcctc caatcctgga aattcgacac aattagattt 700
<210> 11
<211> 700
<212> DNA
<213>Artificial sequence
<400> 11
caaagggaga actccttccc tttattagaa gccccatgga tcaggtacaa aggtacaaag 60
aagcctttct ctcttttgtg acttgcaggg cttgatggcc tctcagagcg ctgtgctcag 120
tacaagaaag atggtgttga ctttgggaag tggcgtcctg tgctgaggat tgccgaccag 180
tgtccatcca gcctcgctat ccaggaaaac gccaacgccc tggctcgcta cgacagcatc 240
tgtcagcagg tgctctgcct tccccttggg ctgaaaaaga gtaggctaga gttttcttca 300
gagcttttct tttcaattat actataacta caaatggacc tccttttccc agaaggggat 360
ggtatcccca gcaatattca gcaacattgc tgtaaaaaga agaaaatctg agtgaaggtt 420
tgactggttt cccatgagag gcagacaggg tcaaggtggg gtcacattta ctctaaccag 480
tctcctctct catatttgtc ttctaggcta agtgccaggc ggccaaagga cagtatgttc 540
acacgggttc ttctggggct gcttccaccc agtcgctctt cacagcctgc tatacctact 600
agggtccaat gcccgccagc ctagctccag tgcttctagt aggagggctg aaagggagca 660
acttttcctc caatcctgga aattcgacac aattagattt 700
<210> 12
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<212> DNA
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aagtggcgtg ctgtgctga 19
<210> 13
<211> 19
<212> DNA
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<400> 13
aagtggcgtc ctgtgctga 19
<210> 14
<211> 17
<212> DNA
<213>Unknown
<400> 14
tcgctacgcc agcatct 17
<210> 15
<211> 17
<212> DNA
<213>Unknown
<400> 15
tcgctacgac agcatct 17
<210> 16
<211> 16
<212> DNA
<213>Unknown
<400> 16
gctaactgcc aggcgg 16
<210> 17
<211> 16
<212> DNA
<213>Unknown
<400> 17
gctaagtgcc aggcgg 16
Claims (7)
1. a kind of people's fructosediphosphate aldolase 1 B gene detection kit it is characterised in that：Described detection kit include for
The primer pair of 3 pleomorphism sites in specific amplification people's fructosediphosphate aldolase 1 B gene, 3 pleomorphism sites are respectively
A150P, A175D, N335K, described primer pair includes common upstream primer and typing downstream primer, described typing downstream primer bag
Include wild type special primer and saltant type special primer, the common upstream primer of corresponding 3 pleomorphism sites, wild type specifically draw
Thing and saltant type special primer are as shown in the table：
F represents common upstream primer, and W represents wild type special primer, and M represents saltant type special primer.
2. according to people's fructosediphosphate aldolase 1 B gene detection kit as claimed in claim 1 it is characterised in that：Each
The 5' end of common upstream primer is marked with fluorescent dye.
3. according to people's fructosediphosphate aldolase 1 B gene detection kit as claimed in claim 2 it is characterised in that：Described
Fluorescent dye is one or more of FAM, HEX, TAMRA, ROX, Cy3, Cy5, JOE, VIC, PET.
4. according to people's fructosediphosphate aldolase 1 B gene detection kit as claimed in claim 1 it is characterised in that：Also wrap
Include crt gene sequence, described crt gene sequence includes normal sequence and mutant nucleotide sequence.
5. according to people's fructosediphosphate aldolase 1 B gene detection kit as claimed in claim 4 it is characterised in that：Also wrap
Include PCR buffer, hot start Taq polymerase, ultra-pure water, fluorescence molecule amount internal standard and allelic ladder.
6. a kind of people's fructosediphosphate aldolase 1 B gene detection method it is characterised in that：Using such as any one of Claims 1 to 5
Described people's fructosediphosphate aldolase 1 B gene detection kit is to more than 3 of people's fructosediphosphate aldolase 1 B gene in sample
The amplification in state property site adopts polymerase chain reaction, and the detection of its amplified production adopts high performance liquid chromatography, multi-fluorescence hair
Cons electrophoresis or polyacrylamide gel electrophoresis.
7. people's fructosediphosphate aldolase 1 B gene detection method according to claim 6 it is characterised in that：Polymerase chain
Formula reaction amplification program be：95 DEG C 2 minutes；94 DEG C 30 seconds, 60 DEG C 30 seconds, 72 DEG C 30 seconds, totally 10 circulation；94 DEG C 30 seconds,
59 DEG C 30 seconds, 72 DEG C 30 seconds, totally 18 circulation；72 DEG C 10 minutes.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610799420.XA CN106399487B (en) | 2016-08-31 | 2016-08-31 | A kind of people's fructosediphosphate aldolase 1 B gene detection method and kit |
| PCT/CN2016/107577 WO2018040320A1 (en) | 2016-08-31 | 2016-11-29 | Detection method and kit for human fructose bisphosphate aldolase b gene |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610799420.XA CN106399487B (en) | 2016-08-31 | 2016-08-31 | A kind of people's fructosediphosphate aldolase 1 B gene detection method and kit |
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| Publication Number | Publication Date |
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| CN106399487A true CN106399487A (en) | 2017-02-15 |
| CN106399487B CN106399487B (en) | 2019-07-23 |
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| CN201610799420.XA Active CN106399487B (en) | 2016-08-31 | 2016-08-31 | A kind of people's fructosediphosphate aldolase 1 B gene detection method and kit |
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| WO (1) | WO2018040320A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107312834A (en) * | 2017-06-07 | 2017-11-03 | 广东华美众源生物科技有限公司 | Glucocerebrosidase gene detecting kit and its detection method |
| CN107828879A (en) * | 2017-10-19 | 2018-03-23 | 广东华美众源生物科技有限公司 | A kind of PKP2 gene mutation detection kits and its detection method |
| CN112126685A (en) * | 2020-09-10 | 2020-12-25 | 长沙金域医学检验实验室有限公司 | Kit and method for identifying position of ALDOB gene containing two mutation sites simultaneously |
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| CN103184268A (en) * | 2011-12-28 | 2013-07-03 | 协和干细胞基因工程有限公司 | Kit for detecting aldehyde dehydrogenase 2 gene polymorphism and amplification method and detection method thereof |
| CN104774933A (en) * | 2015-03-30 | 2015-07-15 | 深圳市第二人民医院 | Primers, kit and method for detection of polymorphism of human IL28B gene |
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| CN107312834A (en) * | 2017-06-07 | 2017-11-03 | 广东华美众源生物科技有限公司 | Glucocerebrosidase gene detecting kit and its detection method |
| CN107312834B (en) * | 2017-06-07 | 2021-02-02 | 广东华美众源生物科技有限公司 | Glucocerebrosidase gene detection kit and detection method thereof |
| CN107828879A (en) * | 2017-10-19 | 2018-03-23 | 广东华美众源生物科技有限公司 | A kind of PKP2 gene mutation detection kits and its detection method |
| CN107828879B (en) * | 2017-10-19 | 2021-05-11 | 广东华美众源生物科技有限公司 | PKP2 gene mutation detection kit and detection method thereof |
| CN112126685A (en) * | 2020-09-10 | 2020-12-25 | 长沙金域医学检验实验室有限公司 | Kit and method for identifying position of ALDOB gene containing two mutation sites simultaneously |
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| Publication number | Publication date |
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| CN106399487B (en) | 2019-07-23 |
| WO2018040320A1 (en) | 2018-03-08 |
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