CN107267648A - A kind of kit of quick detection thalassemia common mutations gene - Google Patents
A kind of kit of quick detection thalassemia common mutations gene Download PDFInfo
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Abstract
The invention discloses a kind of kit of quick detection thalassemia common mutations gene, it includes being directed toSEA、‑‑THAI、‑α2.4、‑α21.9、HPFH‑SEA、DBT、‑α4.2、‑α3.7、αCSα、αQSα、αWestmeadα、Hb Q‑Thailand、β‑90、β‑29、β‑28、βCap+40‑43、βInt M、βInt CD、βCD14‑15、βCD17、βCD26、β27/28、βIVS‑I‑1、βIVS‑I‑5、βCD37、βCD41‑42、βCD43、βCD71–72、βCD95、βIVS‑II‑654The primer of design and the primer for detecting sex, these primers point 2 pipes packing.The kit can fast and accurately detect typically poor mutator.
Description
Technical field
The present invention relates to a kind of kit for detecting thalassemia, and in particular to a kind of quick detection thalassemia is normal
See the kit of mutator.
Background technology
Thalassemia is tropical and subtropical region single gene inheritance disease occurred frequently.Severe β poor patient need it is conventional defeated
Blood and de-iron treatment, quality of life are low.Guangxi and Guangdong are thalassemia districts occurred frequently, and about 7% population is the poor gene in β ground
Carrier.Due to this area crowd poor pathogenic allele carrying rate it is higher, therefore homotype ground poor allele carrier wedding
The probability matched somebody with somebody is also higher, and the probability of poor neonate's birth is larger with causing severe.Poor patient is born the way of probability with reducing severe
The poor examination in ground of this area's marriage and childbirth crowd is is strengthened in footpath, and the poor gene diagnosis in ground is then whether clear and definite individual carries poor equipotential base
The method of cause.
(Pang Wanrong, dragon colt, Ye Xuehe wait the regional crowd's thalassemia gene mutation of Beibu Bay, guangxis for existing research
Analyze the aristogenesis of China and Journal of Heredity, 2016 (1):39-42.) show, in the crowd of Guangxi α poor gene main Types for-
-SEA、--THAI、-α4.2、-α3.7、-α2.4、-α21.9、αCSα、αQSα and αWestmeadα allele, and β poor gene main Types
For β-90、β-29、β-28、βCap+40-43、βInt M、βInt CD、βCD14-15、βCD17、βCD26、β27/28、βIVS-I-1、βIVS-I-5、βCD37、
βCD41-42、βCD43、βCD71–72、βCD95、βIVS-II-654、HPFH-SEA、Gγ+(Aγδβ)0(abbreviation DBT) allele.It is foregoing non-scarce
Mistake type poor allele abbreviation and HGVS (Human Genome Variation Society, human genome variation association
Meeting) title is as described in Table 1：
Table 1：
The country in the diagnosis of thalassemia, detection thalassemia genotype method be usually Gap-PCR and
PCR-RDB.It is to be expanded by designing across breakpoint formula primer pair sample that Gap-PCR, which diagnoses poor principle, amplified production
Analyzed by agarose gel electrophoresis, and PCR-RDB rules need the processes such as completion amplification, hybridization to complete analysis.
Because both approaches testing conditions and operating method are inconsistent, independent operation must be separated, and PCR-RDB is cumbersome, and
Amplified fragments are shorter, easily produce PCR primer pollution.And the detection range for the kit that early stage is developed also has certain limitation
Property, therefore adjustment and the improvement of detection method to detection range will provide support for the poor preventing and controlling in ground.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of reagent of quick detection thalassemia common mutations gene
Box, the kit can realize two pipe multiple PCR techniques to alpha Thalassemia --SEA、--THAI、-α4.2、-α3.7、-α2.4、-
α21.9、Hb Q-Thailand、αCSα、αQSα and αWestmeadα allele, and beta Thalassemia β-90、β-29、β-28、
βCap+40-43、βInt M、βInt CD、βCD14-15、βCD17、βCD26、β27/28、βIVS-I-1、βIVS-I-5、βCD37、βCD41-42、βCD43、βCD71–72、
βCD95、βIVS-II-654、HPFH-SEA、Gγ+(Aγδβ)0The joint-detection of allele and AMEL locus, and one can be mixed in
The poor detection kit in ground of fragment analysis detection is once completed in individual sequencing pipe.
The kit of quick detection thalassemia common mutations gene of the present invention, including for deletion form it is poor
Gene --SEA、--THAI、-α2.4、-α21.9, HPFH-SEA andGγ+(Aγδβ)0The primer of design, for deletion form poor gene-
α4.2With-α3.7The primer designed using Y1 sections and Y2 sections relative quantitative assay, for the poor allele Hb in non-deletion type ground
Q-Thailand、αCSα、αQSα、αWestmeadα、β-90、β-29、β-28、βCap+40-43、βInt M、βInt CD、βCD14-15、βCD17、βCD26、
β27/28、βIVS-I-1、βIVS-I-5、βCD37、βCD41-42、βCD43、βCD71–72、βCD95、βIVS-II-654The primer of design, for non-deletion type
The poor allele α in groundCSα、αQSα、αWestmeadα、β-28、βCD17、βCD26、β27/28、βIVS-I-1、βCD41-42、βCD43、βCD71–72、
βIVS-II-654The normal control primer of design, and detect the AMXY primers of sex, 2 detection pipes packing of these primers point, 2
Detection pipe is designated as tube1 and tube2 respectively, wherein：
The Primer and sequence that tube1 detection pipes are included are as follows：
Beta1-FAM-F：5’-6FAM-TCCTAAGCCAGTGCCAGAAGAGC-3’；
Beta2-FAM-R：5’-6FAM-CCTGAGACTTCCACACTGATGC-3’；
Beta3-FAM-R：5’-6FAM-AGTTGGACTTAGGGAACAAAGGAAC-3’；
CQW-co-ROX-R：5’-ROX-ACCTCCATTGTTGGCACATTCC-3’；
AMXY-6FAM-F：5’-6-FAM-CCCTGGGCTCTGTAAAGAATAGTG-3’；
AMXY–R：5’-ATCAGAGCTTAAACTGGGAAGCTG-3’；
CD37M-R：5’-GGACTCAAAGAACCTCTGGGACT-3’；
IntM-R：5’-AAAAAAACAAAAATCCTCAGGAGTCAGTTGCACT-3’；
-90M-R：5’-GTAGATTGGCCAACCCAAGGGTA-3’；
-28M-R：5’-CAAAACAATAGATGGCTCTGCCCTGACTAC-3’；
-29M-R：5’-GATGGCTCTGCCCTGCCTTTC-3’；
IntCD-R：5’-CCTCAGGAGTCAGATGCATCC-3’；
CD17M-R：5’-AAAAACAACTTCATCCACGTTCGCCTA-3’；
Cap+40-43M-R：5’-AAAAGATGCACCATGGTGTCTGAGG-3’；
CD14-15(+G)M-R：5’-CCACGTTCACCTTGCTCTACCA-3’；
CD26M-R：5’-ACCAACCTGCACAGGGTCTT-3’；
IVS-Ⅰ-1M-R：5’-CTGTCTTGTAACCTTGATGCCAAA-3’；
IVS-Ⅰ-5M-R：5’-AACTTAAACCTGTCTTGTAACCTTGACAG-3’；
CD27/28(+C)M-R：5’-TTTTGATACCAACCTGACCAGGAGC-3’；
CD71-72(+A)M-F：5’-CAAGAAAGTGCTCGGTGCCTGTAA-3’；
CD95M-F：5’-GTGAGCTGCACTGTGACGAAG-3’；
CD43M-F：5’-CCTTGGACCCAGAGGTTCGTTT-3’；
CD41-42M-F：5’-CAAAAAAAAAACAAAACTACCCTTGGACCCAGAT GTTG-3’；
654M-F：5’-CAGTGATAATTTCTGGGTTGAGGT-3’；
WS-N-F：5’-AGTTCACCTCTGCGGTGCAC-3’；
CS-N-F：5’-CGTGCTGACCTCCAAATACTGTT-3’；
QS-N-F：5’-CTGCGGTGCAAGCCTACCT-3’；
Y1-Y2-HEX-F：5’-HEX-CAGGGATGCACCCACTGGCA-3’；
Y1-Y2-R：5’-CTCGACACGCATCTGCTCAGGGGTGAGGAAGGAAGG GGTG-3’；
The Primer and sequence that Tube2 detection pipes are included are as follows：
Beta1-TAMRA-F：5’-TAMRA-TCCTAAGCCAGTGCCAGAAGAGC-3’；
Beta2-TAMRA-R：5’-TAMRA-CCTGAGACTTCCACACTGATGC-3’；
Beta3-TAMRA-R：5’-TAMRA-AGTTGGACTTAGGGAACAAAGGAAC-3’；
CQW-co-HEX-R：5’-HEX-ACCTCCATTGTTGGCACATTCC-3’；
-28N-R：5’-TAGATGGCTCTGCCCTGACATT-3’；
CD17N-R：5’-CAACTTCATCCACGTTCACCAT-3’；
CD26N-R：5’-ACCAACCTGCTCAGGGACTC-3’；
IVS-Ⅰ-1N-R：5’-CTGTCTTGTAACCTTGATACCATC-3’；
CD27/28N-R：5’-AAAATGATACCAACCTGCCCAGCGC-3’；
CD71-72N-F：5’-CAAGAAAGTGCTCGGTGCCTTGAG-3’；
43/41-42N-F：5’-CCTTGGACCCAGAGGTTCCTTG-3’；
654N-F：5’-CAAAAAACAAAAAAAAAAAACAGTGATAAGTTCTGGG TTAAGGC-3’；
WS-M-F：5’-GAGTTCACCCCTGCGGTTCAG-3’；
CS-M-F：5’-CGTGCTGACCTCCAAATACAGTC-3’；
QS-M-F：5’-CTGCGGTGCACGGCTCACC-3’；
A1-74M-HEX-F：5’-HEX-AACGCCGTGGCGCACTTGC-3’；
A1-QT-R：5’-CAAAACAGCAGGCAGTGGCTTAGGAG-3’；
THAI-HEX-R：5’-HEX-CTTGGATCTGCACCTCTGGGTAGG-3’；
THAI-F：5’-GAATAAAGCGAGAGGAATCACATTCCTC-3’；
SEA-HEX-F：5’-HEX-AGAAGCTGAGTGATGGGTCCG-3’；
SEA-R：5’-TGGACTTAAGTGATCCTCCTGCCC-3’；
HPFH-F：5’-AAAACCAGCCTCATGGTAGCAGAATC-3’；
HPFH-M-FAM-R：5’-6FAM-TGGTATCTGCAGCAGTTGCC-3’；
DBT-M-FAM-F：5’-6FAM-CCAGAAATTGCCTCATGTCTCT-3’；
DBT-R：5’-CACATATAAAATGCTGCTAATGCTTCATTAC-3’；
2.4-HEX-F：5’-HEX-ACCACGACCTCTAGGCCAGT-3’；
2.4R：5’-CAACCAGCCCTCTGCTGTAC-3’；
21.9-F：5’-GGAGCTTTTCCTTCCCTGGAACG-3’；
21.9-HEX-R：5’-HEX-TGTGGTTGGAGAATGGAGGTGG-3’；
In above-mentioned primer, " M " represents mutational site, and " N " represents normal control site, and FAM refers to hydroxyl fluorescein, ROX
Refer to carboxy-X-rhodamine, HEX refers to chlordene -6- methylfluoresceins, and TA MRA refer to carboxyl tetramethylrhodamine.It is above-mentioned to draw
The fluorescence labeling combination of thing can be substituted according to the type of sequenator and setting using other fluorescent dyes, such as FAM, HEX,
TAMRA and ROX is a kind of combination of E5 dyestuffs in ABI sequenators, and FAM (blueness), the VIC in can also being set from G5 are (green
Color), NED (yellow) and PET (red) be combination, or other process spectrum corrections fluorochrome combinations.In the application only
One of which combination is listed, but is not limited only to above-mentioned one kind, can also be realized using other dye combinations；Selected fluorescence
Mark is also not necessarily limited to above color, is combined using the differentiable fluorescence color through spectrum correction.
In above-mentioned technical proposal, the sequence of the Y1 sections is：5’-
GTGATTTCTCAGGCTGTTTTCTCCTCAGTACCATCCCCCCAAAAAACATCACTTTTCATGCACAGGGATGCACCCAC
TGGCACTCCTGCACCTCCCACCCTTCCCCAGAAGTCCACCCCTTCCTTCCTCACCCTGCAGGAGCTGGCCAGCCTCA
TCACCCCAACATCTCCCCACCTCCATTCTCCAACCACAGGGCCCTTGTCTCCTCTGTCCTTTCCCCTCCCCGAGCCA
AGCCTCCTCCCTCCTCCACCTCCTCCACCTAATACATATCCTTAAGTCTCACCTCCTCCAGGAAGCCCTCAGACTAA
CCCTGGTCACCTTGAATGCCTCGTCCACACCTCCAGACTTCCTCAGGGCCTGTGATGAGGTCTGCACCTCTGTGTGT
ACTTGTGTGATGGTTAGAGGACTGCCTACCTCCCAGAGGAGGTTGAATGCTCCAGCCGGTTCCAGCTATTGCTTTGT
TTACCTGTTTAACCAGTATTTACCTAGCAAGTCTTCCATCAGATAG-3’；The sequence of Y2 sections is：5’-
CACCGAGCCTGGCCAAACCATCACTTTTCATGAGCAGGGATGCACCCACTGGCACTCCTGCACCTCCCACCCTCCCC
CTCGCCAAGTCCACCCCTTCCTTCCTCACCCCACATCCCCTCACCTACATTCTGCAACCACAGGGGCCTTCTCTCCC
CTGTCCTTTCCCTACCCAGAGCCAAGTTTGTTTATCTGTTTACAACCAGTATTTACCTAGCAAGTCTTCCATCAGAT
AG-3’。
The kit of quick detection thalassemia common mutations gene of the present invention, in addition to some available reagents
Conventional and necessary component, such as buffer solution, enzyme liquid, Mg in box2+(MgCl2) and dNTP.Specifically, enzyme liquid is Taq polymerase body
System, including available for PCR thermal starting enzyme systems reacted etc.；The buffer solution is conventional PCR buffer solutions.When enzyme liquid selection is adopted
With health by produce in century GoldStar Taq DNA Polymerase when, buffer solution then be preferably and GoldStar Taq
Buffer solution supporting DNA Polymerase.
Method using mentioned reagent box quick detection thalassemia common mutations gene comprises the following steps：
1) sample genomic dna is extracted, DNA profiling is prepared using existing conventional method；
2) reaction system is prepared, is specially：
Tube1 detection pipes：Take each primer included by DNA, Tube1 detection pipe to be detected, PCR buffer solutions, enzyme liquid,
MgCl2, dNTP, water and DNA profiling be configured to reaction system；Wherein, the concentration of each component is usually：DNA to be detected：20-
50ng；Each primer：0.038~0.36 μm of ol/L；Mg2+：1.6~1.8mmol/L；The final volume of reaction system can be 10 μ L,
20 μ L or 25 μ L；
Tube2 detection pipes：Take each primer included by DNA, Tube2 detection pipe to be detected, PCR buffer solutions, enzyme liquid,
MgCl2, dNTP, water and DNA profiling be configured to reaction system；Wherein, the concentration of each component is usually：DNA to be detected：20~
50ng；Each primer：0.08~0.54 μm of ol/L；Mg2+：1.6~1.8mmol/L；The final volume of reaction system can be 10 μ L, 20
μ L or 25 μ L；
3) pattern detection：2 detection pipes are separately operable PCR response procedures, take each 0.4 μ of the PCR primer in 2 detection pipes
L, mixes the molecular weight internal reference of sequencing system, and mixing sequencing loading carrier deionized formamide (HiDi) 10 μ L are used after mixing
Sequenator carries out loading sequencing, using slice parsing method testing result；
4) data analysis and result judgement：
Digital independent and analysis, the equipotential base wherein corresponding to Tube1 PCR primer are carried out using GeneMapper softwares
Cause, final PCR primer fluorescence color and product length are as shown in table 2；It is allele corresponding to Tube2 PCR primer, final
PCR primer fluorescence color and product length are as shown in table 3；Table 4 is then-α3.7With-α4.2Basis for estimation.
Table 2：
Table 3：
Table 4：
Because 2 pipe PCR primers (Tube1 and Tube2) are to be mixed in a sequencing pipe to carry out fragment analysis, therefore single sample
The interpretation of genotype can be completed after a sequencing reaction.Specifically interpretation mode is：
M represents mutational site, and N represents normal control site, 3 kinds of non-deletion type α ground poor allele and 9 kinds of non-deletion types
The poor allele β in β ground-28、βCD17、βCD26、β27/28、βIVS-I-1、βCD41-42、βCD43、βCD71–72、βIVS-II-654It is normal right to be designed with
According to SNP detection primers (βCD41-42、βCD431 normal control primer is shared, therefore poor allele is normally right with setting 8 β altogether
Illuminate the way thing).
A, is provided with the detection site of normal control, when not detecting the color corresponding to M, length signals, then the sample
Grade mutation position gene is not carried；When detecting the color corresponding to M, N, length signals simultaneously, the sample is in the detection site
Allele for mutation heterozygous；As only detection M corresponding to color, length signals, but do not detect the color corresponding to N,
During length signals, the sample is non-deletion type homozygote in the detection site.
B, is not provided with the poor detection site in non-deletion type ground of normal control, when not detecting the color corresponding to M, length letter
Number when, then the sample do not carry the grade mutation position gene；The poor detection site in non-deletion type ground of normal control is not provided with, works as inspection
When going out color corresponding to M, length signals, then the sample carries grade mutation position gene, due to being not provided with normal control
Non-deletion type poor genotype shared ratio in crowd it is relatively low, so general when detecting the color corresponding to M, length signals
For the heterozygous mutation in the site, but preferably confirm by sequencing.
C, deletion form poor determination methods be：When deletion form it is poor correspondence fluorescent value product length detecting signal when, sentence
The sample that breaks carries the deletion form, but must the poor normal control site in comprehensive non-deletion type ground, 3 non-deletion type α it is poor normal
Exist as long as just representing the genes of α 2 with reference to one site in the site positive, if a kind of non-deletion type β poor allele normal ginseng
It is the positive according to site, that is, proves that β genes are present, must consider.
Compared with prior art, the method have the characteristics that：
1st, using kit of the present invention 2 pipes can be realized to alpha Thalassemia --SEA、--THAI、-α2.4、-α21.9、-
α4.2、-α3.7、αCSα、αQSα、αWestmeadα and Hb Q-Thailand, and beta Thalassemia HPFH-SEA,Gγ+(Aγδβ
)0、β-90、β-29、β-28、βCap+40-43、βInt M、βInt CD、βCD14-15、βCD17、βCD26、β27/28、βIVS-I-1、βIVS-I-5、βCD37、
βCD41-42、βCD43、βCD71–72、βCD95And βIVS-II-654Adopt genotype to be detected, without extra agarose gel electrophoresis, hybridization
Operation, it is to avoid PCR primer pollution.
2nd, detection of the kit of the present invention to detection site has sensitivity, stability and the accuracy of height, with
And higher specificity；Primer therein has good specificity and the repeatability tested；
3rd, the use of fluorescence labeling, requires to reduce to the DNA concentration of tested sample, can be using the hair, oral cavity for having hair follicle
Swab etc. carries out DNA extractions and completes detection；
4th, kit of the present invention can carry out the detection of 96 samples simultaneously, and automatic detection platform lowers artificial participate in
Degree, reduces human operational error；
5th, kit of the present invention has detection quick (extracting result acquisition from DNA only needs 4-5 hour), standard
Really, easy to operate, detection range is wide, it is low-cost the characteristics of；
6th, the detection range of kit of the present invention is adapted to the detection machine of with detecting Chinese population poor allelic variation
Structure is used.
Brief description of the drawings
Fig. 1 is the testing result parting figure of 2# samples in the embodiment of the present invention 1.
Embodiment
With reference to specific embodiment, the present invention is described in further detail, to more fully understand present disclosure, but
The present invention is not limited to following examples.
Embodiment 1：Using testing result of the kit of the present invention in known pattern sheet
1. the composition of kit
(1) primer, including：
1.1) detection α poor mutation primer：α-globin gene cluster (NG_000006.1) sequence according to disclosed in NCBI
Design α poor mutation detection primer, wherein --SEA、--THAI、-α2.4、-α21.9Primer ,-α are designed using Gap-PCR methods4.2With-
α3.7Then completed according to the relative quantification of Y1 sections and Y2 sections, αCSα、αQSα、αWestmeadα and Hb Q-Thailand use AS-
PCR methods design primer；
1.2) detection β poor mutation primer：Beta-globin gene cluster (NG_000007.3) sequence according to disclosed in NCBI
Be designed, wherein HPFH-SEA andGγ+(Aγδβ)0Primer, β are designed using Gap-PCR methods-90、β-29、β-28、βCap+40-43、
βInt M、βInt CD、βCD14-15、βCD17、βCD26、β27/28、βIVS-I-1、βIVS-I-5、βCD37、βCD41-42、βCD43、βCD71–72、βCD95With
βIVS-II-654Primer, wherein α are designed using AS-PCR methodsCSα、αQSα、αWestmeadα、β-28、βCD17、βCD26、β27/28、βIVS-I-1、
βCD41-42、βCD43、βCD71–72、βIVS-II-654It is designed with normal control SNP detection primers.
1.3) the AMXY primers of sex are detected.
2 detection pipe packing of above-mentioned all primers point, 2 detection pipes are designated as tube1 and tube2 respectively, wherein：
The Primer and sequence that tube1 detection pipes are included are as described in Table 5：
Table 5：
In above-mentioned table 5, " M " represents mutational site, and " N " represents normal control site, and FAM refers to hydroxyl fluorescein, and ROX is
Refer to carboxy-X-rhodamine, HEX refers to chlordene -6- methylfluoresceins.In table 5, Beta1-FAM-F, Beta2-FAM-R, Beta3-
FAM-R be non-deletion type β poor amplified allele common indicium primer end, mutation end included for amplification 18 kinds it is non-lack
Mistake type β poor allele test side；CQW-co-ROX-R be non-deletion type α poor amplified allele consensus primer
End, test side detection be 3 kinds of non-deletion types poor allele normal control point；AMXY primer pairs are to detect drawing for sex
Thing pair, Y1-Y2 primer pairs are to detect Y1 and Y2 copy numbers.Numerical value shown in table is the amplified fragments size of correspondence primer.
The detection of Y1 and Y2 copy numbers is used to point out-α4.2With-α3.7Presence, with Y1 detection peak value and Y2 detection peak
Value is compared, and specific determination methods are：Work as Y1=Y2, and non-deletion type α poor control point detection product peak when, then sample to be tested
Do not include-α3.7Or-α4.2；If Y1=2Y2, and non-deletion type α poor control point detection product peak when, then the base of sample to be tested
Because type contains-α4.2, without other α missings；If 2Y1=Y2, and non-deletion type α poor control point detection product peak when, then treat test sample
This genotype contains-α3.7, without other α missings；The detection peak of remaining genotype with referring to deletion form poor allele remakes
It is specific to judge.
The sequence of the foregoing Y1 sections being related to is：5’-GTGATTTCTCAGGCTGTTTTCTCCT
CAGTACCATCCCCCCAAAAAACATCACTTTTCATGCACAGGGATGCACC
CACTGGCACTCCTGCACCTCCCACCCTTCCCCAGAAGTCCACCCCTTCCTTCCTCACCCTGCAGGAGCTGGCCAGCC
TCATCACCCCAACATCTCCCCACCTCCATTCTCCAACCACAGGGCCCTTGTCTCCTCTGTCCTTTCCCCTCCCCGAG
CCAAGCCTCCTCCCTCCTCCACCTCCTCCACCTAATACATATCCTTAAGTCTCACCTCCTCCAGGAAGCCCTCAGAC
TAACCCTGGTCACCTTGAATGCCTCGTCCACACCTCCAGACTTCCTCAGGGCCTGTGATGAGGTCTGCACCTCTGTG
TGTACTTGTGTGATGGTTAGAGGACTGCCTACCTCCCAGAGGAGGTTGAATGCTCCAGCCGGTTCCAGCTATTGCTT
TGTTTACCTGTTTAACCAGTATTTACCTAGCAAGTCTTCCATCAGATAG-3’(SEQ ID NO：30)；The Y1 sections
Characteristic sequence y1 is：5’-CAGGGATGCACCCACTGGCACTCCTGCACCTCCCACCCTTCCCCAGAAGTCCACCCCTTCC
TTCCTCACCC-3’(SEQ ID NO：31).The sequence for the Y2 sections being related to is：5’-CACCGAGCCTGGCCAAACCATCACT
TTTCATGAGCAGGGATGCACCCACTGGCACTCCTGCACCTCCCACCCTCCCCCTCGCCAAGTCCACCCCTTCCTTCC
TCACCCCACATCCCCTCACCTACATTCTGCAACCACAGGGGCCTTCTCTCCCCTGTCCTTTCCCTACCCAGAGCCAA
GTTTGTTTATCTGTTTACAACCAGTATTTACCTAGCAAGTCTTCCATCAGATAG-3’(SEQ ID NO：32)；The Y2
The characteristic sequence y2 of section is：5’-CAGGGATGCACCCACTGGCACTCCTGCACCTCCCACCCTCCCCCTCGCCAAGTCC
ACCCCTTCCTTCCTCACCC-3’(SEQ ID NO：33).
The Primer and sequence that Tube2 detection pipes are included are as described in Table 6：
Table 6：
In table 6, " M " represents mutational site, and " N " represents normal control site, and FAM refers to hydroxyl fluorescein, and HEX refers to six
Chloro- 6- methylfluoresceins, TAMRA refers to carboxyl tetramethylrhodamine.In table 6, Beta1-TAMRA-F, Beta2-TAMRA-R,
Beta3-TAMRA-R be non-deletion type β poor amplified allele common indicium primer end, mutation end included for amplification
9 kinds of non-deletion type β the reference normal locations of poor allele test side, wherein 43/41-42N-F detections for CD43M and
CD41-42M shared reference point；CQW-co-HEX-R be non-deletion type α poor amplified allele consensus primer end, inspection
Survey end detection is the poor allele in 3 kinds of non-deletion types ground；The detection pipe is with also including detection deletion form α poor --SEA、--THAI、-
α2.4With-α21.9Primer pair, and detection deletion form β poor HPFH-SEA andGγ+(Aγδβ)0Primer pair.Number shown in table
It is worth for the amplified fragments size of correspondence primer.
Final composition concentration of the primer that the primer and Tube2 detection pipes that Tube1 detection pipes are included are included in kit
Respectively as shown in table 7 below and table 8：
Table 7：
Table 8：
(2) other components：
GoldStar Taq DNA Polymerase and buffer solution supporting GoldStar Taq DNA Polymerase
Health is purchased from for century with dNTPs, MgCl2Purchased from Life Technology.
The PCR reaction systems of Tube1 detection pipes and Tube2 detection pipes are prepared by table 9 below and table 10 respectively：
Table 9：
Table 10：
2. implementation
PCR reaction instruments are Bio-Rad real time thermocyclers CFX96.PCR response procedures are：PCR response procedures
For, 95 DEG C 10 minutes, 94 DEG C 30 seconds, 62.5 DEG C 30 seconds and 72 DEG C 30 seconds, 28-35 circulation, 62 DEG C 60 minutes extend.
Sample process：General DNA kits extract DNA, are diluted to 20~50ng/ μ L with distilled water standby.
Pattern detection：By known type sample to be checked (20 parts, numbering is 1#, 2#......20# respectively) by Tube1 bodies
System and Tube2 systems are respectively configured, are placed in PCR instrument and run PCR response procedures.Taken respectively after PCR program end of runs
Tube1 detection pipes PCR primer and each 0.4 μ l of Tube2 detection pipe PCR primers, mix the molecular weight internal reference of sequencing system, and mixing is surveyed
Sequence loading carrier deionized formamide (HiDi) 10 μ l, carry out loading sequencing, using fragment analysis side after mixing using sequenator
Method testing result.
3. samples sources：Conventional Gap-PCR technologies are hung oneself in all sample standard deviations source or sequencing technologies determine the DNA of genotype
Sample.
4. data analysis and result judgement：Read using GeneMapper softwares and analysis result, comprehensive detection result is sentenced
Genotype is read, it is as a result as follows：
1# samples are male α α/α α, βCD17/βN；
2# samples are women α α/α α, β-28/β-28, its testing result parting figure is as shown in Figure 1；
3# samples are male α α/α α, β-29/βN；
4# samples are male-α3.7/αα,βCD14-15/βN；
5# samples are male-α3.7/αCSα,βN/βN；
6# samples are women-α4.2/αWestmeadα,βN/βN；
7# samples are women α α/α α, β27/28/βN；
8# samples are women α α/α α, βCD37/βN；
9# samples are male α α/α α, βCD41-42/βN；
10# samples are women α α/α α, β-28/βCD71–72；
11# samples are male α α/α α, βIVS-II-654/βIVS-II-654；
12# samples are male α α/α α, β-90/βN；
13# samples are male α α/α α, βCap+40-43/βN；
14# samples are male α α/α α, βCD26/βN；
15# samples are male αWestmeadα/αα,βCD95/βN；
16# samples are women α α/α α,Gγ+(Aγδβ)0/βN；
17# samples are women α α/α α, HPFH-SEA/ βN；
18# samples are male α α/α α, βInt CD/βN；
19# samples are women α α/α α, βInt M/βN；
20# samples are women α α/α α, βIVS-I-5/βN。
From result, kit of the present invention can distinguish various Common genes types；And the base of all samples of detection
Because type is identical with the genotype that it is determined through conventional Gap-PCR, PCR-RDB technology, illustrate kit of the present invention to
There is good accuracy during the detection of major gene pattern sheet.
SEQUENCE LISTING
<110>Huang, moral is precious
<120>A kind of kit of quick detection thalassemia common mutations gene
<130> 2017
<160> 62
<170> PatentIn version 3.5
<210> 1
<211> 23
<212> DNA
<213>Artificial sequence
<400> 1
tcctaagcca gtgccagaag agc 23
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<212> DNA
<213>Artificial sequence
<400> 2
cctgagactt ccacactgat gc 22
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<211> 25
<212> DNA
<213>Artificial sequence
<400> 3
agttggactt agggaacaaa ggaac 25
<210> 4
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<212> DNA
<213>Artificial sequence
<400> 4
acctccattg ttggcacatt cc 22
<210> 5
<211> 24
<212> DNA
<213>Artificial sequence
<400> 5
ccctgggctc tgtaaagaat agtg 24
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<212> DNA
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<400> 6
atcagagctt aaactgggaa gctg 24
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ggactcaaag aacctctggg act 23
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<212> DNA
<213>Artificial sequence
<400> 8
aaaaaaacaa aaatcctcag gagtcagttg cact 34
<210> 9
<211> 23
<212> DNA
<213>Artificial sequence
<400> 9
gtagattggc caacccaagg gta 23
<210> 10
<211> 30
<212> DNA
<213>Artificial sequence
<400> 10
caaaacaata gatggctctg ccctgactac 30
<210> 11
<211> 21
<212> DNA
<213>Artificial sequence
<400> 11
gatggctctg ccctgccttt c 21
<210> 12
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<212> DNA
<213>Artificial sequence
<400> 12
cctcaggagt cagatgcatc c 21
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<211> 27
<212> DNA
<213>Artificial sequence
<400> 13
aaaaacaact tcatccacgt tcgccta 27
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<211> 25
<212> DNA
<213>Artificial sequence
<400> 14
aaaagatgca ccatggtgtc tgagg 25
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<212> DNA
<213>Artificial sequence
<400> 15
ccacgttcac cttgctctac ca 22
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<211> 20
<212> DNA
<213>Artificial sequence
<400> 16
accaacctgc acagggtctt 20
<210> 17
<211> 24
<212> DNA
<213>Artificial sequence
<400> 17
ctgtcttgta accttgatgc caaa 24
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<212> DNA
<213>Artificial sequence
<400> 18
aacttaaacc tgtcttgtaa ccttgacag 29
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<211> 25
<212> DNA
<213>Artificial sequence
<400> 19
ttttgatacc aacctgacca ggagc 25
<210> 20
<211> 24
<212> DNA
<213>Artificial sequence
<400> 20
caagaaagtg ctcggtgcct gtaa 24
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<212> DNA
<213>Artificial sequence
<400> 21
gtgagctgca ctgtgacgaa g 21
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<212> DNA
<213>Artificial sequence
<400> 22
ccttggaccc agaggttcgt tt 22
<210> 23
<211> 38
<212> DNA
<213>Artificial sequence
<400> 23
caaaaaaaaa acaaaactac ccttggaccc agatgttg 38
<210> 24
<211> 24
<212> DNA
<213>Artificial sequence
<400> 24
cagtgataat ttctgggttg aggt 24
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<212> DNA
<213>Artificial sequence
<400> 25
agttcacctc tgcggtgcac 20
<210> 26
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<212> DNA
<213>Artificial sequence
<400> 26
cgtgctgacc tccaaatact gtt 23
<210> 27
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<212> DNA
<213>Artificial sequence
<400> 27
ctgcggtgca agcctacct 19
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<212> DNA
<213>Artificial sequence
<400> 28
cagggatgca cccactggca 20
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<212> DNA
<213>Artificial sequence
<400> 29
ctcgacacgc atctgctcag gggtgaggaa ggaaggggtg 40
<210> 30
<211> 508
<212> DNA
<213>Artificial sequence
<400> 30
gtgatttctc aggctgtttt ctcctcagta ccatcccccc aaaaaacatc acttttcatg 60
cacagggatg cacccactgg cactcctgca cctcccaccc ttccccagaa gtccacccct 120
tccttcctca ccctgcagga gctggccagc ctcatcaccc caacatctcc ccacctccat 180
tctccaacca cagggccctt gtctcctctg tcctttcccc tccccgagcc aagcctcctc 240
cctcctccac ctcctccacc taatacatat ccttaagtct cacctcctcc aggaagccct 300
cagactaacc ctggtcacct tgaatgcctc gtccacacct ccagacttcc tcagggcctg 360
tgatgaggtc tgcacctctg tgtgtacttg tgtgatggtt agaggactgc ctacctccca 420
gaggaggttg aatgctccag ccggttccag ctattgcttt gtttacctgt ttaaccagta 480
tttacctagc aagtcttcca tcagatag 508
<210> 31
<211> 71
<212> DNA
<213>Artificial sequence
<400> 31
cagggatgca cccactggca ctcctgcacc tcccaccctt ccccagaagt ccaccccttc 60
cttcctcacc c 71
<210> 32
<211> 233
<212> DNA
<213>Artificial sequence
<400> 32
caccgagcct ggccaaacca tcacttttca tgagcaggga tgcacccact ggcactcctg 60
cacctcccac cctccccctc gccaagtcca ccccttcctt cctcacccca catcccctca 120
cctacattct gcaaccacag gggccttctc tcccctgtcc tttccctacc cagagccaag 180
tttgtttatc tgtttacaac cagtatttac ctagcaagtc ttccatcaga tag 233
<210> 33
<211> 74
<212> DNA
<213>Artificial sequence
<400> 33
cagggatgca cccactggca ctcctgcacc tcccaccctc cccctcgcca agtccacccc 60
ttccttcctc accc 74
<210> 34
<211> 23
<212> DNA
<213>Artificial sequence
<400> 34
tcctaagcca gtgccagaag agc 23
<210> 35
<211> 22
<212> DNA
<213>Artificial sequence
<400> 35
cctgagactt ccacactgat gc 22
<210> 36
<211> 25
<212> DNA
<213>Artificial sequence
<400> 36
agttggactt agggaacaaa ggaac 25
<210> 37
<211> 22
<212> DNA
<213>Artificial sequence
<400> 37
acctccattg ttggcacatt cc 22
<210> 38
<211> 22
<212> DNA
<213>Artificial sequence
<400> 38
tagatggctc tgccctgaca tt 22
<210> 39
<211> 22
<212> DNA
<213>Artificial sequence
<400> 39
caacttcatc cacgttcacc at 22
<210> 40
<211> 20
<212> DNA
<213>Artificial sequence
<400> 40
accaacctgc tcagggactc 20
<210> 41
<211> 24
<212> DNA
<213>Artificial sequence
<400> 41
ctgtcttgta accttgatac catc 24
<210> 42
<211> 25
<212> DNA
<213>Artificial sequence
<400> 42
aaaatgatac caacctgccc agcgc 25
<210> 43
<211> 24
<212> DNA
<213>Artificial sequence
<400> 43
caagaaagtg ctcggtgcct tgag 24
<210> 44
<211> 22
<212> DNA
<213>Artificial sequence
<400> 44
ccttggaccc agaggttcct tg 22
<210> 45
<211> 44
<212> DNA
<213>Artificial sequence
<400> 45
caaaaaacaa aaaaaaaaaa cagtgataag ttctgggtta aggc 44
<210> 46
<211> 21
<212> DNA
<213>Artificial sequence
<400> 46
gagttcaccc ctgcggttca g 21
<210> 47
<211> 23
<212> DNA
<213>Artificial sequence
<400> 47
cgtgctgacc tccaaataca gtc 23
<210> 48
<211> 19
<212> DNA
<213>Artificial sequence
<400> 48
ctgcggtgca cggctcacc 19
<210> 49
<211> 24
<212> DNA
<213>Artificial sequence
<400> 49
cttggatctg cacctctggg tagg 24
<210> 50
<211> 28
<212> DNA
<213>Artificial sequence
<400> 50
gaataaagcg agaggaatca cattcctc 28
<210> 51
<211> 21
<212> DNA
<213>Artificial sequence
<400> 51
agaagctgag tgatgggtcc g 21
<210> 52
<211> 24
<212> DNA
<213>Artificial sequence
<400> 52
tggacttaag tgatcctcct gccc 24
<210> 53
<211> 26
<212> DNA
<213>Artificial sequence
<400> 53
aaaaccagcc tcatggtagc agaatc 26
<210> 54
<211> 20
<212> DNA
<213>Artificial sequence
<400> 54
tggtatctgc agcagttgcc 20
<210> 55
<211> 22
<212> DNA
<213>Artificial sequence
<400> 55
ccagaaattg cctcatgtct ct 22
<210> 56
<211> 31
<212> DNA
<213>Artificial sequence
<400> 56
cacatataaa atgctgctaa tgcttcatta c 31
<210> 57
<211> 20
<212> DNA
<213>Artificial sequence
<400> 57
accacgacct ctaggccagt 20
<210> 58
<211> 20
<212> DNA
<213>Artificial sequence
<400> 58
caaccagccc tctgctgtac 20
<210> 59
<211> 23
<212> DNA
<213>Artificial sequence
<400> 59
ggagcttttc cttccctgga acg 23
<210> 60
<211> 22
<212> DNA
<213>Artificial sequence
<400> 60
tgtggttgga gaatggaggt gg 22
<210> 61
<211> 19
<212> DNA
<213>Artificial sequence
<400> 61
aacgccgtgg cgcacttgc 19
<210> 62
<211> 26
<212> DNA
<213>Artificial sequence
<400> 62
caaaacagca ggcagtggct taggag 26
Claims (2)
1. a kind of kit of quick detection thalassemia common mutations gene, it is characterised in that：Including for deletion form
Poor gene --SEA、--THAI、-α2.4、-α21.9, HPFH-SEA andGγ+(Aγδβ)0The primer of design, for the poor base in deletion form ground
Because of-α4.2With-α3.7The primer designed using Y1 sections and Y2 sections relative quantitative assay, for the poor allele in non-deletion type ground
αCSα、αQSα、αWestmeadα、Q-Thailand、β-90、β-29、β-28、βCap+40-43、βIntM、βIntCD、βCD14-15、βCD17、βCD26、
β27/28、βIVS-I-1、βIVS-I-5、βCD37、βCD41-42、βCD43、βCD71–72、βCD95、βIVS-II-654The primer of design, for non-deletion type
The poor allele α in groundCSα、αQSα、αWestmeadα、β-28、βCD17、βCD26、β27/28、βIVS-I-1、βCD41-42、βCD43、βCD71–72、
βIVS-II-654The normal control primer of design, and detect the AMXY primers of sex, 2 detection pipes packing of these primers point, 2
Detection pipe is designated as tube1 and tube2 respectively, wherein：
The Primer and sequence that tube1 detection pipes are included are as follows：
Beta1-FAM-F：5’-6FAM-TCCTAAGCCAGTGCCAGAAGAGC-3’；
Beta2-FAM-R：5’-6FAM-CCTGAGACTTCCACACTGATGC-3’；
Beta3-FAM-R：5’-6FAM-AGTTGGACTTAGGGAACAAAGGAAC-3’；
CQW-co-ROX-R：5’-ROX-ACCTCCATTGTTGGCACATTCC-3’；
AMXY-6FAM-F：5’-6-FAM-CCCTGGGCTCTGTAAAGAATAGTG-3’；
AMXY–R：5’-ATCAGAGCTTAAACTGGGAAGCTG-3’；
CD37M-R：5’-GGACTCAAAGAACCTCTGGGACT-3’；
IntM-R：5’-AAAAAAACAAAAATCCTCAGGAGTCAGTTGCACT-3’；
-90M-R：5’-GTAGATTGGCCAACCCAAGGGTA-3’；
-28M-R：5’-CAAAACAATAGATGGCTCTGCCCTGACTAC-3’；
-29M-R：5’-GATGGCTCTGCCCTGCCTTTC-3’；
IntCD-R：5’-CCTCAGGAGTCAGATGCATCC-3’；
CD17M-R：5’-AAAAACAACTTCATCCACGTTCGCCTA-3’；
Cap+40-43M-R：5’-AAAAGATGCACCATGGTGTCTGAGG-3’；
CD14-15(+G)M-R：5’-CCACGTTCACCTTGCTCTACCA-3’；
CD26M-R：5’-ACCAACCTGCACAGGGTCTT-3’；
IVS-Ⅰ-1M-R：5’-CTGTCTTGTAACCTTGATGCCAAA-3’；
IVS-Ⅰ-5M-R：5’-AACTTAAACCTGTCTTGTAACCTTGACAG-3’；
CD27/28(+C)M-R：5’-TTTTGATACCAACCTGACCAGGAGC-3’；
CD71-72(+A)M-F：5’-CAAGAAAGTGCTCGGTGCCTGTAA-3’；
CD95M-F：5’-GTGAGCTGCACTGTGACGAAG-3’；
CD43M-F：5’-CCTTGGACCCAGAGGTTCGTTT-3’；
CD41-42M-F：5’-CAAAAAAAAAACAAAACTACCCTTGGACCCAGATGTTG-3’；
654M-F：5’-CAGTGATAATTTCTGGGTTGAGGT-3’；
WS-N-F：5’-AGTTCACCTCTGCGGTGCAC-3’；
CS-N-F：5’-CGTGCTGACCTCCAAATACTGTT-3’；
QS-N-F：5’-CTGCGGTGCAAGCCTACCT-3’；
Y1-Y2-HEX-F：5’-HEX-CAGGGATGCACCCACTGGCA-3’；
Y1-Y2-R：5’-CTCGACACGCATCTGCTCAGGGGTGAGGAAGGAAGGGGTG-3’；
The Primer and sequence that Tube2 detection pipes are included are as follows：
Beta1-TAMRA-F：5’-TAMRA-TCCTAAGCCAGTGCCAGAAGAGC-3’；
Beta2-TAMRA-R：5’-TAMRA-CCTGAGACTTCCACACTGATGC-3’；
Beta3-TAMRA-R：5’-TAMRA-AGTTGGACTTAGGGAACAAAGGAAC-3’；
CQW-co-HEX-R：5’-HEX-ACCTCCATTGTTGGCACATTCC-3’；
-28N-R：5’-TAGATGGCTCTGCCCTGACATT-3’；
CD17N-R：5’-CAACTTCATCCACGTTCACCAT-3’；
CD26N-R：5’-ACCAACCTGCTCAGGGACTC-3’；
IVS-Ⅰ-1N-R：5’-CTGTCTTGTAACCTTGATACCATC-3’；
CD27/28N-R：5’-AAAATGATACCAACCTGCCCAGCGC-3’；
CD71-72N-F：5’-CAAGAAAGTGCTCGGTGCCTTGAG-3’；
43/41-42N-F：5’-CCTTGGACCCAGAGGTTCCTTG-3’；
654N-F：5’-CAAAAAACAAAAAAAAAAAACAGTGATAAGTTCTGGGTTAAGGC-3’；
WS-M-F：5’-GAGTTCACCCCTGCGGTTCAG-3’；
CS-M-F：5’-CGTGCTGACCTCCAAATACAGTC-3’；
QS-M-F：5’-CTGCGGTGCACGGCTCACC-3’；
A1-74M-HEX-F：5’-HEX-AACGCCGTGGCGCACTTGC-3’；
A1-QT-R：5’-CAAAACAGCAGGCAGTGGCTTAGGAG-3’；
THAI-HEX-R：5’-HEX-CTTGGATCTGCACCTCTGGGTAGG-3’；
THAI-F：5’-GAATAAAGCGAGAGGAATCACATTCCTC-3’；
SEA-HEX-F：5’-HEX-AGAAGCTGAGTGATGGGTCCG-3’；
SEA-R：5’-TGGACTTAAGTGATCCTCCTGCCC-3’；
HPFH-F：5’-AAAACCAGCCTCATGGTAGCAGAATC-3’；
HPFH-M-FAM-R：5’-6FAM-TGGTATCTGCAGCAGTTGCC-3’；
DBT-M-FAM-F：5’-6FAM-CCAGAAATTGCCTCATGTCTCT-3’；
DBT-R：5’-CACATATAAAATGCTGCTAATGCTTCATTAC-3’；
2.4-HEX-F：5’-HEX-ACCACGACCTCTAGGCCAGT-3’；
2.4R：5’-CAACCAGCCCTCTGCTGTAC-3’；
21.9-F：5’-GGAGCTTTTCCTTCCCTGGAACG-3’；
21.9-HEX-R：5’-HEX-TGTGGTTGGAGAATGGAGGTGG-3’；
In above-mentioned primer, " M " represents mutational site, and " N " represents normal control site, and FAM refers to hydroxyl fluorescein, and ROX refers to
Carboxy-X-rhodamine, HEX refers to chlordene -6- methylfluoresceins, and TAMRA refers to carboxyl tetramethylrhodamine.
2. the kit of quick detection thalassemia common mutations gene according to claim 1, it is characterised in that：Should
Kit also includes buffer solution, enzyme liquid, Mg2+And dNTP.
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| CN105176995A (en) * | 2015-10-26 | 2015-12-23 | 钦州市妇幼保健院 | Hydrolysis probe method based kit capable of simultaneously detecting deletion -alpha<21.9> and alpha2 variation of thalassemia |
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| CN106868187A (en) * | 2015-11-04 | 2017-06-20 | 深圳市瀚海基因生物科技有限公司 | Multiple PCR primer, kit and purposes |
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