CN104342491A - Novel non-invasive blood type gene detection kit - Google Patents
Novel non-invasive blood type gene detection kit Download PDFInfo
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- CN104342491A CN104342491A CN201410019867.1A CN201410019867A CN104342491A CN 104342491 A CN104342491 A CN 104342491A CN 201410019867 A CN201410019867 A CN 201410019867A CN 104342491 A CN104342491 A CN 104342491A
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Abstract
The invention relates to the technical field of medical and biological detection, relates to application of gene detection in human blood type non-invasive detection, and in particular relates to a novel non-invasive kit for detecting ABO blood type of an individual by non-blood biological sample genome DNA through a PCR-RFLP method. The kit disclosed by the invention, by combining the non-invasive sampling method and PCR-RFLP method for the first time, can obtain mass non-blood biological samples at a large scale non-invasively and stably, so as to rapidly and accurately detect ABO blood type of the individual. The kit disclosed by the invention is simple and convenient to operate, safe, rapid, low in cost and capable of realizing mass operation; and the kit has remarkable advantages in aspects of blood type screening in a large population as well as clinical blood type detection for people difficult to draw blood samples and some difficult and miscellaneous specimens.
Description
Technical field
The present invention relates to technical field of biomedical detection, relate to the application of gene test in human blood type Noninvasive detects, specifically, relate to and utilize non-blood biological sample genomic dna, by PCR-RFLP method, detect the Novel noninvasive test kit of individual abo blood group.
Background technology
Blood group is the method to blood classification, typically refers to erythrocytic somatotype, and its foundation is whether erythrocyte surface exists some heritable antigenic substance.Up to the present, have been found that and be that the blood group system that international Blood Transfusion Association is admitted has 30 kinds, wherein ABO is most important blood group system, and its application extends to many aspects [1,2] such as biology, genetics, medical jurisprudence and anthropology from clinical blood transfusion.
Serological method is adopted to the somatotype of blood group always, this method is simple and quick, but as the research tool of abo blood group, serology makes to carry out more deep research and is restricted, it can not carry out the somatotype of abo blood group gene hypotype, therefore in clinical blood transfusion, there is certain limitation.The method can not explain that the identical and genotype of phenotype does not clinically conform to the case [3] of genetic development.
Current genotyping technique mainly contains following several: 1. PCR-DNA sequencing 2. PCR-RFLP (digestion with restriction enzyme) method 3. PCR-SSP method.PCR-DNA sequencing cost is higher, and detection time is longer; PCR-SSP method needs to design multipair primer, often pair of primer increases respectively, if for the identification of abo blood group genotype, need the multitube that increases, pcr amplification product also needs porous electrophoresis detection, and will internal reference be set, very complicated is compared in experiment, is easily subject to the impact of many factors, result stable not [4].PCR-RFLP method, in conjunction with PCR specific amplification and restriction enzymatic endonuclease digestion, can carry out polymorphism to 1-2 bar PCR primer, cost is low, easy and simple to handle, and result is stablized.
But at present, apply the research that DNA typing technology carries out abo blood group somatotype in clinical and scientific research, in acquisition DNA sample source, mostly adopt venous blood sample.But, in large-scale crowd sampling, often there is the problem of blood sample sampling difficulty, during children and some difficult samples samplings especially for bloodletting difficulty.Further, also there is the problem stored with transport difficult in blood sample.And compared with venous blood, collutory, hair and nail etc. belong to non-invasive sample, have a lot of advantage, its feature is conflicted little for the person of being sampled is psychological, sample easily stores and transports, and therefore in large-scale crowd sampling, has clear superiority.Current research shows, collutory, hair and nail have certain operability and using value [5,6] as DNA extracting sample.
The present invention utilizes the non-blood biological sample genomic dnas such as collutory, hair and nail, by PCR-RFLP method, accurately can complete the detection of individual abo blood group.
Reference:
Zhao Qin, Wang Shujun (1997) abo blood group is in medicolegal application. Handan Medical college journal, 1:19.
The distribution of Peng Deren (1991) Chinese han population abo blood group. China's blood transfusion magazine, 4 (1): 20-23.
The application of Xu Hua, Zhang Yangpei (2004) PCR in the difficult bracket for blood grouping of ABO. institute of Military Medical Science Institute prints, and 28 (1): 85-87.
The gene type of Shi Ning (1997) ABO blood group system. foreign medical science: blood transfusion and hematology fascicle, 20 (5): 286-289.
Zhang Hong, Chen Hongfan, An Yu, Wang Hongyan, Duan Wenyuan. the discussion of (2009) margo liber unguis and hair center DNA method for extracting. China follows card youngster medical science, and 4 (5): 431-435.
Yan Wei, Jiang Xianhua, Lv Shihui (1994) saliva and the DNA analysis containing saliva sample. Chinese law medical journal, 9 (2): 65-67.
Summary of the invention
The object of the present invention is to provide one to utilize non-blood biological sample genomic dna, by PCR-RFLP method, detect the test kit of individual abo blood group.
For achieving the above object, this test kit extracts genomic dna from the non-blood biological samples such as collutory, hair and nail, pcr amplification is carried out to three multiple equipotential single copy genes of the ABO blood group system in the genomic dna extracted, and utilize digestion with restriction enzyme amplified fragments, then use the abo blood group of agarose gel electrophoresis identification and detection sample.
Described non-blood biological sample can be cotton swab containing human oral epithelial cells or collutory, the hair of people, nail, seminal stain equal samples;
Affiliated genome DNA extraction method can be the methods such as saturated sodium-chloride method, paramagnetic particle method, glass bead-extraction, post partition method, and more excellent is saturated sodium-chloride method;
Described pcr amplified fragment is utilize primer amplification to obtain, and be arranged in the section of ABO allelotrope the 6th, the 7th exon containing nucleotide difference of karyomit(e) 9q34, more excellent is following primer sequence (SEQ No.1-SEQ No.4);
SEQ NO.1：5'-CCTGTCCCTTTGTTCTCCAA-3’
SEQ NO.2：5’-AATGTCCACAGTCACTCGCC-3’
SEQ NO.3：5’-CGCATGGAGATGATCAGTG-3’
SEQ NO.4：5’-CACAAGTACTCGGGGGAGAG-3’
Described restriction enzyme is can otherness Nucleotide in specific recognition amplified fragments, and enzyme cuts the DNA restriction enzyme of this amplification sheet degree, and more excellent is Kpn I and Alu I;
Present invention also offers the method using mentioned reagent box to detect for Noninvasive blood group gene, the method comprises the following steps:
A () utilizes the reagent in this test kit (comprise but be not limited only to: STE damping fluid, 10% sodium laurylsulfonate, 10mg/mL Proteinase K etc.), carry out cracking to non-blood biological samples such as collutory, hair and nails;
B () utilizes saturated sodium-chloride method, add Virahol mixing after, ice for, centrifugal, washing, dissolve, obtain the genomic dna after separation and purification.
C () utilizes this seminal plasma fructose detection kit (comprise but be not limited only to: DNA synthetic enzyme, SEQ No.1-SEQ No.4PCR amplimer, PCR damping fluid, MgC12 solution, dNTP mixed solution etc.), carry out pcr amplification to the section containing nucleotide difference in ABO allelotrope the 6th, the 7th exon of 9q34 in genomic dna, pcr amplified fragment size is as shown in table 1;
Table 1ABO blood group genotype detects design of primers information
D () is carried out enzyme respectively to the fragment application Kpn1 enzyme of PCR acquisition in (c) and Alu I enzyme and is cut, then identify with agarose gel electrophoresis, blood type analysis judges that (when Kpn I can cut 261 site, result is expressed as M, can not cut and be expressed as m with reference to table 2; When Alu I can cut 703 site, result is expressed as N, can not cut and be expressed as n).
Table 2PCR product enzyme is cut qualification result and is judged for blood type analysis
The present invention, first in conjunction with the Noninvasive method of sampling and PCR-RFLP method, without wound, extensive, a large amount of non-blood biological sample of stable acquisition, can detect individual abo blood group fast, accurately.The test kit that the present invention relates to is easy and simple to handle, safe, quick, cost is low, can mass-producing operation, in the examination of large-scale crowd blood group, and the crowd of bloodletting difficulty and the clinical Blood grouping application aspect of some difficult samples have significant advantage.
Accompanying drawing explanation
Fig. 1: the not homoallelic cDNA difference schematic diagram of abo blood group;
The detected result in Fig. 2 .BO Blood grouping PCR primer restriction enzyme digestion and electrophoresis photo a. the 6th exon 2 61 site: can determine that No. 1 and No. 5 is OO type blood; No. 2 is AO or BO; No. 3 and No. 4 do not have gene O, are the one in AA, AB or BB.B. the detected result in the 7th exon 7 03 site: in conjunction with 261 site results, can determine that No. 1 and No. 5 is OO type blood; No. 2 BO types; No. 3 is AB type; No. 4 is BB type.Positive control is the PCR primer that non-enzyme is cut.
Embodiment
Below in conjunction with drawings and Examples, the invention will be further described, but enforcement of the present invention is not limited only to this.
Embodiment 1: prepare test kit of the present invention
Test kit of the present invention is composed as follows:
1. genomic nucleic acids extraction agent:
STE damping fluid (in every 100ml, adds the Tris-Hcl of 1ml1mol/L, the sodium-chlor of 2ml5mol/L, the EDTA of 200ul0.Smol/L), 10% sodium laurylsulfonate, 10mg/mL Proteinase K, 1mol/L-1DTT (the equal available from Sigma of above-mentioned medicine);
2.PCR amplifing reagent:
Pcr amplification primer (SEQ No.1-SEQ No.4, Sangon Biotech (Shanghai) Co., Ltd. synthesizes), 2 × Taqman Universal PCR Master Mix (TA KARA)
3. restriction analysis reagent:
Kpn I and Alu I restriction enzyme and damping fluid (purchased from NEB company) thereof.
Embodiment 2: the detection of test kit of the present invention
1. the collection of non-blood biological sample and pre-treatment:
The collection of 1.1 sample of hair and pre-treatment:
Gather self-sow and the boiling hot dye of 10 adult healthy volunteers, be with the black hair of root of hair some.10 minutes are respectively soaked with distilled water and dehydrated alcohol.Cut the segment of 0.3 ~ 0.5cm containing hair follicle as root of hair sample from every root root of hair, every 3 is 1 part, totally 10 parts.
The collection of 1.2 nail samples and pre-treatment:
Gather the margo liber unguis of 10 adult healthy volunteers, totally 10 parts.With distilled water immersion 10 minutes, carefully scrub 1 time with fine, soft fur brush, distilled water vibration rinsing 2 times, each 5 minutes.Shred with scissors after drying.
The collection of 1.3 collutory samples and pre-treatment:
Gather the collutory of 10 adult healthy volunteers, gargle with 15mL sterile saline, collect collutory, centrifugal 5 minutes of 5000 revs/min of rotating speeds, abandon supernatant ,-20 DEG C store for future use.
2. extracting genome DNA:
Adopt in the present embodiment but be not limited only to saturated sodium-chloride method extracting genomic dna.
1. the nail sample shredded is placed in 2mL centrifuge tube, adds the STE damping fluid 410 μ L of configuration, 10% sodium laurylsulfonate (SDS) 90 μ L, 10mg/mL Proteinase K 40 μ L; 2. root of hair sample is placed in 2mL centrifuge tube, adds STE damping fluid 410 μ L, 10%SDS10 μ L, 10mg/mL Proteinase K 10 μ L; 3. the collutory sample after centrifugal is placed in 2mL centrifuge tube, adds STE damping fluid 410 μ L, 10%SDS20 μ L, 10mg/mL Proteinase K 8 μ L, 1mol/L-1DTT8 μ L; Equal 56 DEG C of water-baths digestion is spent the night.Above-mentioned sample is with saturated sodium-chloride method extracting DNA after digestion, and add 400 μ L Virahols and put upside down jog 2-3 minute repeatedly, 4 DEG C, 12000r/ minute centrifugal 15 minutes, abandons supernatant; Add 1ml75% washing with alcohol precipitation, jog 2-3 minute repeatedly, 4 DEG C, 12000r/ minute centrifugal 10 minutes, and repeated washing once; Precipitate 4 DEG C and naturally evaporate into drying, add the mixing of 10-30 μ LTE damping fluid.
The section pcr amplification step of 3.ABO allelotrope the 6th, the 7th exon otherness:
PCR amplification system and reaction conditions as follows:
Utilize SEQ No.1 and SEQ No.2 for the amplified fragments that upstream and downstream primer obtains be 552bp; Utilize SEQ No.3 and SEQ No.4 for the amplified fragments that upstream and downstream primer obtains be 413bp;
4. restriction enzyme is cut and electrophoretic analysis:
SEQ No.1 and SEQ No.2 increase, and to cut system as follows for the restriction enzyme of 552bp fragment:
SEQ No.3 and SEQ No.4 increase, and to cut system as follows for the restriction enzyme of 413bp fragment:
Enzyme is cut and is all reacted more than 3 hours in 37 DEG C of water-baths, then electroresis appraisal in 1.5% agarose gel electrophoresis, and by table 2, sample blood group genotype is analyzed (Fig. 2), and compare in the serotype identification results of the identification of blood of same sample; The blood group genotype of result non-blood sample and the serotype identification results of blood check sample completely the same.
Claims (6)
1., for detecting a nothing wound test kit for individual abo blood group, it is characterized in that utilizing non-blood biological sample genomic dna, by PCR-RFLP method, detecting individual abo blood group; Its feature is specifically related to extract genomic dna from non-blood biological sample, pcr amplification is carried out to three multiple equipotential single copy genes of the ABO blood group system in the genomic dna extracted, and utilize digestion with restriction enzyme amplified fragments, then use the abo blood group of agarose gel electrophoresis identification and detection sample.This test kit comprises:
1.1 genomic nucleic acids extraction agents;
1.2PCR amplifing reagent;
1.3 restriction analysis reagent.
2. non-blood biological sample according to claim 1, is characterized in that, include but are not limited to the cotton swab containing human oral epithelial cells or collutory, the non-blood biological sample such as hair, nail, seminal stain of people.
3. genome DNA extraction method according to claim 1, is characterized in that, include but are not limited to the methods such as saturated sodium-chloride method, paramagnetic particle method, glass bead-extraction, post partition method, more excellent is saturated sodium-chloride method.
4. pcr amplified fragment according to claim 1 is utilize primer amplification to obtain, be arranged in the section of ABO allelotrope the 6th, the 7th exon containing nucleotide difference of karyomit(e) 9q34, more excellent is following primer sequence, and has the sequence of 80% and above similarity with following sequence:
SEQ NO.1：5'-CCTGTCCCTTTGTTCTCCAA-3’
SEQ NO.2：5’-AATGTCCACAGTCACTCGCC-3’
SEQ NO.3：5’-CGCATGGAGATGATCAGTG-3’
SEQ NO.4：5’-CACAAGTACTCGGGGGAGAG-3’ 。
5. restriction enzyme according to claim 1 is can amplified fragments in specific recognition claim 4, and the DNA restriction enzyme that enzyme is cut, more excellent is Kpn I and Alu I, and has the restriction enzyme in same identification site with Kpn I and Alu I.
6., for the method that Noninvasive blood group gene detects, it is characterized in that, described method comprises:
A () application rights requires the arbitrary described test kit of 2-7, carry out pre-treatment, the extraction of genomic nucleic acids, pcr amplification to detection experimenter non-blood sample, enzyme is cut and identified with agarose gel electrophoresis;
B () analyzes blood group genotype:
According to the restriction enzyme in claim 5, the blood group genotype that effect judges given the test agent is cut to the enzyme of amplified fragments in claim 4;
If Kpn I can ABO allelotrope the 6th exon 2 61 site of complete degestion karyomit(e) 9q34, Alu I can not enzyme when cutting ABO allelotrope the 7th exon 7 03 site, and the blood group genotype of given the test agent is OO, and Blood group phenotype is O;
If Kpn I can not cut ABO allelotrope the 6th exon 2 61 site of karyomit(e) 9q34 by enzyme, Alu I can not enzyme when cutting ABO allelotrope the 7th exon 7 03 site, and the blood group genotype of given the test agent is AA, and Blood group phenotype is A;
If Kpn I can not cut ABO allelotrope the 6th exon 2 61 site of karyomit(e) 9q34 by enzyme, during Alu I complete degestion ABO allelotrope the 7th exon 7 03 site, the blood group genotype of given the test agent is BB, and Blood group phenotype is B;
If Kpn I can not cut ABO allelotrope the 6th exon 2 61 site of karyomit(e) 9q34 by enzyme, during partially digested ABO allelotrope the 7th exon 7 03 site of Alu I, the blood group genotype of given the test agent is AB, and Blood group phenotype is AB;
If ABO allelotrope the 6th exon 2 61 site of the partially digested karyomit(e) 9q34 of Kpn I, Alu I can not enzyme when cutting ABO allelotrope the 7th exon 7 03 site, and the blood group genotype of given the test agent is AO, and Blood group phenotype is A;
If ABO allelotrope the 6th exon 2 61 site of the partially digested karyomit(e) 9q34 of Kpn I, during partially digested ABO allelotrope the 7th exon 7 03 site of Alu I, the blood group genotype of given the test agent is BO, and Blood group phenotype is B.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113789374A (en) * | 2021-09-24 | 2021-12-14 | 广西医科大学第一附属医院 | Taqman hydrolysis probe for gene level ABO chimera and detection method |
Citations (3)
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| CN101065499A (en) * | 2004-09-22 | 2007-10-31 | 英国西英格兰大学，布里斯托尔 | RHD and ABO genotyping by multiplex PCR |
| CN101403007A (en) * | 2008-11-03 | 2009-04-08 | 深圳市血液中心 | Detection agent for ABO blood type gene and shaping method |
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- 2014-01-16 CN CN201410019867.1A patent/CN104342491A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101065499A (en) * | 2004-09-22 | 2007-10-31 | 英国西英格兰大学，布里斯托尔 | RHD and ABO genotyping by multiplex PCR |
| CN1900314A (en) * | 2006-07-19 | 2007-01-24 | 辽宁省刑事科学技术研究所 | Fluorescence labelling ABO gene typing method and its reagent kit |
| CN101403007A (en) * | 2008-11-03 | 2009-04-08 | 深圳市血液中心 | Detection agent for ABO blood type gene and shaping method |
Non-Patent Citations (1)
| Title |
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| 肖文昕 等: "利用漱口水、毛发和指甲进行非侵入性的ABO血型的基因检测", 《现代人类学通讯》, vol. 7, 31 December 2013 (2013-12-31) * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113789374A (en) * | 2021-09-24 | 2021-12-14 | 广西医科大学第一附属医院 | Taqman hydrolysis probe for gene level ABO chimera and detection method |
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Application publication date: 20150211 |