CN104109716B

CN104109716B - A kind of human HLA-B27 genotyping kit

Info

Publication number: CN104109716B
Application number: CN201410337737.2A
Authority: CN
Inventors: 陈炤源; 王浩
Original assignee: JIANGSU WEIHE BIOTECHNOLOGY Co Ltd
Current assignee: JIANGSU WEIHE BIOTECHNOLOGY Co Ltd
Priority date: 2014-07-15
Filing date: 2014-07-15
Publication date: 2015-12-02
Anticipated expiration: 2034-07-15
Also published as: CN104109716A

Abstract

The invention provides a kind of human HLA-B27 genotyping kit, comprise pcr amplification primer, archaeal dna polymerase and reaction buffer, described archaeal dna polymerase is the warm start Taq polysaccharase of PCR response inhabitation thing in the tolerated blood modified through polyoxyethylene glycol (PEG), additionally provides the nucleotide sequence of pcr amplification primer.The present invention has following technique effect: archaeal dna polymerase used is the warm start Taq polysaccharase of PCR response inhabitation thing in the tolerated blood modified through polyoxyethylene glycol (PEG), the pcr amplification to human whole blood (without the need to carrying out nucleic acid extraction) can be realized, directly carry out pcr amplification through blood, without the need to carrying out DNA extraction; This test kit can realize the HLA-B27 yin and yang attribute qualification of sample; The gene hypotype result of HLA-B27 positive sample can be drawn simultaneously, can every 8 holes (8 union) detect 24 kinds of HLA-B27 gene hypotypes and 13 kinds of HLA? other gene hypotype of B site.

Description

A kind of human HLA-B27 genotyping kit

Technical field

The present invention relates to a kind of human HLA-B27 genotyping kit, belong to gene engineering technology field.

Background technology

Human leucocyte antigen (HumanLeukocyteAntigen, HLA) 21.31st district of No. 6 the short arm of a chromosome are positioned at, be the region that in known at present human chromosomal, Gene Density is the highest, polymorphism is the abundantest, be divided into HLA-I, II and III genoid.HLA-B27 is classical HLA-class Ⅰmolecule, is expressed in all karyocytes and platelet surface, has height polymorphism, there will be a known 105 kinds of hypotypes, called after HLA-B*27:01 ~ HLA-B*27:105.

Ankylosing spondylitis (AnkylosingSpondylitis, AS) be that one is mainly in male adults, and based on the agnogenic autoimmune disorder of axis joint chronic inflammatory diseases, clinical manifestation is most common sympton with flank pain, stiff discomfort.The total incidence of China AS is 0.3%, and about 4,500,000 examples, the male sex is more than women, and men and women's PMR is about 10:1.Research report, the expression of HLA-B27 antigen and AS be high correlation, and this is the related antigen uniquely having at present clinical value, are the most by force with most typical in the associating of hitherto known HLA and disease.Clinical studies show, the HLA-B27 positive rate of AS patient is up to 96%, and 5% ~ 10% be only positive in general population, the Relative Risk that AS occurs HLA-B27 antigen carrier reaches 70%.The symptom of AS is similar to numerous disease and be difficult to make a definite diagnosis, when suffering from struvite lumbago and backache and do not observe classical radiographic feature and not clear few sacroiliitis or Enthesopathy, the detection of HLA-B27 diagnoses unclear predicament by avoiding clinicist to conditions of patients.As can be seen here, the detection of HLA-B27 important in inhibiting in the diagnosis of disease about spine.

The allelotrope of known HLA-B*27 reaches 105 kinds more than, and from Crowds Distribute, B*2705 is more common in white people and American Indian, B*2704 is common in Aisa people, B*2702 is more common in Mediterranean Sea crowd, and B*2706 sees South East Asia crowd, and B*2709 sees Italian southern Sardinia crowd.Wherein, B*2705 gene distribution is the widest, almost sees all ethnic groups, it is generally acknowledged that B*2705 is the genetic ancestry of B27, and other genes are all evolved by it.Research shows and AS falls ill, and relevant gene hypotype mainly contains B*2702, B*2703, B*2704, B*2705, B*2706, B*2707, B*2708, B*2710, B*2714, B*2715 and B*2719, wherein the strongest with the cognation of B*2704 and AS, be secondly B*2705, B*2702 and B*2707.

Except the AS that research is more, HLA-B27 and many other diseases also also exist certain dependency.Research finds, more juvenile rheumatoid arthritis patient HLA-B27 antigen positive, and in the patient of the HLA-B27 positive, B*2705 accounts for about 60%, and all the other are B*2704, B*2702 and B*2707.In acute uveitis patient, have also discovered the HLA-B*27 positive, and be mainly HLA-B*2704 and B*2705 genotype.In addition, also there is certain dependency in HLA-B27 and behcets disease, psoriatic, arthritis type enteritis, leukemia and heart disease.

As can be seen here, the allelic detection of HLA-B*27 and further somatotype clinical diagnosis, disease are differentiated and medical research significant.

HLA-B27 detection method conventional on domestic market mainly contains MLCT method, ELISA method, FCM method and PCR-SSP method.Wherein, MLCT method, ELISA method, FCM method are all the serological methods based on antigen antibody reaction, and the existence that can only detect HLA-B27 antigen whether, cannot detect its allelotrope, cannot carry out HLA-B27 gene type.

MLCT method and microlymphocytotoxicity test, test also known as complement dependent cytotoxicity.Basic skills gets HLA blood grouping serum, adds examinee's lymphocyte, and can make to carry the cell death with blood grouping serum type same antigen in the presence of complement, inverted microscope is observed and counted, and usual dead cell is greater than 40% and is judged to the HLA-B27 positive.MLCT method cost is low, easily popularizes, but operation steps is many, there is subjectivity during result interpretation, serum, complement and be separated lymphocytic quality, all can affect the accuracy of result.

ELISA method and enzyme linked immunosorbent assay, adopt double antibody sandwich method to measure HLA-B27 level in sample usually.Basic skills uses the HLA-B27 antibody bag of purifying by microwell plate, makes insolubilized antibody; In the micropore that Sheet is anti-, add HLA-B27 successively, then with the HLA-B27 antibodies that HRP mark, form antibody-antigene-hrp-antibody complex, after thoroughly washing, add substrate TMB develop the color; Under 450nm wavelength, absorbancy (OD value) is measured, by HLA-B27 concentration in typical curve calculation sample by microplate reader.ELISA method cost is low, and result data is objective, but complicated operation, affects reaction factor more, result poor accuracy, and the bag of solid phase carrier be killed in a disaster and reach between each individuality consistent, therefore each mensuration all needs to make typical curve.

FCM method and flow cytometry, be utilize flow cytometer to carry out the detection means of quantitative analysis and sorting to cell or other biological particle, can be used for the detection of HLA-B27.Basic skills is made by sample unicellularly to select smelting, with fluorescent mark specificity HLA-B27 antibodies, uses the HLA-B27 automatic detection analysis software set to measure.FCM method detection speed is fast, accuracy is better, but its resolving power is low, operative technique requires high, greatly, high to sample requirement, the quality of selected antibody directly affects result for instrument calibration and maintenance difficulties, and the repeatability of result is not good enough, therefore the application in HLA-B27 gene test is not yet widely used.

The PCR reaction that PCR-SSP method and sequence specific primers guide, it is one of current method from gene aspect detection HLA-B27, its basic skills extracts genomic dna from blood, design HLA-B27 sequence specific primers, to be increased each allelotype DNA fragment specific by specific PCR reaction system, produce corresponding specific amplification products band, and detect PCR primer with agarose gel electrophoresis.Instrument needed for PCR-SSP method is simple, and project is easily carried out, the easy interpretation of result, and accuracy is better, but whole blood DNA extraction operation steps is many, length consuming time, and adds detection time and the factor affecting result.

Although above-mentioned 4 kinds of methods play key player on clinical HLA-B27 detects, and respectively have its defect, a kind of easy and simple to handle, quick, and the exploitation of the high detection method of accuracy will play key clinical effect.

Summary of the invention

The object of the invention is to overcome the deficiencies in the prior art part, a kind of human HLA-B27 genotyping kit is provided.

Human HLA-B27 genotyping kit of the present invention, comprise pcr amplification primer, archaeal dna polymerase and reaction buffer, described archaeal dna polymerase is the warm start Taq polysaccharase of PCR response inhabitation thing in the tolerated blood modified through polyoxyethylene glycol (PEG), and the nucleotide sequence of described pcr amplification primer is as follows:

Also comprise a pair internal reference primer, in order to the product of the 600bp that increases, internal reference primer sequence is as follows:

5’-TGCATCTGGACATGCTTGCT-3’
	5’-TGGCTGGAGGAGACTCCAAA-3’

In described human HLA-B27 genotyping kit, composition and each component concentration of described reaction buffer are as follows: 0.18mM deoxynucleotide (dNTP), 1.8mM magnesium chloride (MgCl ₂), 60.3mM Repone K (KCl), 18.9mM Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl), glycerine (glycerol) 0.6% (v/v), as the dimethyl sulfoxide (DMSO) (DMSO) 5% (v/v) of the agent of PCR increased response and stablizer, methane amide 2.5% (v/v) and trimethyl-glycine 5% (v/v).

Described human HLA-B27 genotyping kit, also comprises DNALoadingdye.

The present invention develops that a kind of from people's whole blood sample, directly carry out PCR (polymerase chain reaction) amplification whether positive and carry out method and the test kit thereof of HLA-B27 subtype gene somatotype to detect sample HLA-B27 antigen gene simultaneously, not only result is accurately and reliably for this method, and early stage is without the need to carrying out nucleic acid extraction operation to blood sample, reduce laboratory pollution risk, reduce experimental cost and reduce the experimental implementation time.

The present invention is directed to the different subtype gene of HLA-B27, design multipair specific gene amplification primer, and freeze-drying is in the bottom of PCR Sptting plate, and, the invention provides a set of special PCR reaction system, the warm start Taq polysaccharase of PCR response inhabitation thing in the tolerated blood modified through polyoxyethylene glycol (PEG) is contained in this system, and in reaction buffer, add dimethyl sulfoxide (DMSO) (DMSO), methane amide and trimethyl-glycine as the agent of PCR increased response and stablizer, to ensure the accurate of the stable of experimentation and experimental result.

The present invention has following technique effect:

1) archaeal dna polymerase is the warm start Taq polysaccharase of PCR response inhabitation thing in the tolerated blood modified through polyoxyethylene glycol (PEG), the pcr amplification to human whole blood (without the need to carrying out nucleic acid extraction) can be realized, directly carry out pcr amplification through blood, without the need to carrying out DNA extraction;

2) the HLA-B27 yin and yang attribute qualification of sample is realized;

3) can draw the gene hypotype result of HLA-B27 positive sample, every 8 holes (8 union) can detect 24 kinds of HLA-B27 gene hypotypes and 13 kinds of other gene hypotypes of HLAB site simultaneously.

Accompanying drawing explanation

Fig. 1 is the electrophorogram that the PCR-SSP being template with EDTA anti-freezing human blood carries out HLA-B27 gene type;

Fig. 2 is take DNA as the electrophorogram that the PCR-SSP of template carries out HLA-B27 gene type.

Embodiment

Embodiment 1

1 raw material and equipment:

1.1 kit contents:

1) 8 hole 200ulPCR Sptting plates;

2) 13 pairs of specificity upstream and downstream amplimers, by particular arrangement freeze-drying bottom PCR Sptting plate, also be added with a pair bottom each PCR Sptting plate for set internal reference primer, and last reacting hole contains B27 universal primer to judge the yin and yang attribute of sample; The equal freeze-drying of all primers is bottom PCR Sptting plate.

The 13 pairs of distributions of specificity amplification primer in PCR Sptting plate are as follows:

Also add a pair internal reference primer in each hole, in order to the product of the 600bp that increases, internal reference primer sequence is as follows:

5’-TGCATCTGGACATGCTTGCT-3’
	5’-TGGCTGGAGGAGACTCCAAA-3’

3) PCR reaction reagent

Archaeal dna polymerase: the warm start Taq polysaccharase of PCR response inhabitation thing in the tolerated blood that polyoxyethylene glycol (PEG) is modified.

Reaction buffer: composition and each component concentration as follows: 0.18mM deoxynucleotide (dNTP), 1.8mM magnesium chloride (MgCl ₂), 60.3mM Repone K (KCl), 18.9mM Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl), glycerine (glycerol) 0.6% (v/v), as the dimethyl sulfoxide (DMSO) (DMSO) 5% (v/v) of the agent of PCR increased response and stablizer, methane amide 2.5% (v/v) and trimethyl-glycine 5% (v/v).

DNALoadingdye。

The source of 1.2 samples

Fresh or the freezen protective collected with EDTA anticoagulant tube is without the whole blood sample of multigelation.

Experimental installation needed for 1.3

The pipettor of gene-amplificative instrament, electrophoresis apparatus and supporting electrophoresis chamber, different range, small desk whizzer (containing 8 union horizontal heads).

2 genotyping process

The configuration of 2.1PCR reaction system

Once only to carry out the somatotype of a sample, reaction system is as shown in table 1:

Table 1:PCR reaction system

	PCR mixing mother liquor (microlitre)	Every hole content (microlitre)
			Archaeal dna polymerase	0.54	0.06
Reaction buffer	72	8
			DNA Loading dye	18	2
Aseptic deionized water	54	6
			Whole blood (replacement DNA profiling)	36	4
Summation	180.54	20.06

Each PCR reacts frozen in advance in orifice plate 0.4 μ l10uM internal reference primer, and 0.4 μ l10uMB27 universal primer is contained in the 8th hole, the 13 pairs of Auele Specific Primers each PCR react orifice plate distribution and consumption as follows:

Mixed by PCR mixing mother liquor (1 person-portion), then each reacting hole of 8 unions divides the mixed solution getting 20ul, covers 8 union lid or films, puts into gene-amplificative instrament.

2.2PCR response procedures

PCR response procedures is as shown in table 2:

Table 2:PCR response procedures

2.3 interpretation

By PCR reaction product, be loaded onto on the sepharose of 2%, with the electrophoresis 5-7 minute of 200V, read result.As shown in Figure 1, interpretation form is as shown in table 3 for result.

According to the HLA-B27 gene hypotype sequence announced in GENBANK database, select 24 kinds of HLA-B27 gene hypotypes that the frequency of occurrences is relatively high in crowd; In each subtype gene the 2nd, 3 and 4 exon, search homologous sequence, design 13 pairs of primers for each homologous sequence; Product difference in size is comparatively large and the primer that the frequency of occurrences is not high is arranged in same hole, as the 6th and No. 7 holes; Containing HLA-B27 universal primer in 8th hole, design HLA-B27 subtype gene interpretation form (see table 3); Read the positive hole number of sample according to gel electrophoresis, i.e. the homologous sequence that comprises of known sample, thus release sample HLA-B27 gene hypotype; In addition, 13 kinds of other gene hypotypes of HLAB site can also be detected.

Table 3: interpretation form

The interpretation form of synopsis 3, interpretation is as follows:

1) positive hole: 1,5 and 8;

2) in conjunction with interpretation table:

B*2704/25

1

5

+

No. 8 hole positives prompt for HLA-B27 positive, and sample hypotype is B*2704/25.

Meanwhile, by above-mentioned blood sample, carry out extracting genome DNA, measuring DNA concentration is 10ng/ul.Then PCR reaction system is configured: PCR reaction system is as shown in table 4:

Table 4:PCR reaction system

	PCR mixing mother liquor (microlitre)	Every hole content (microlitre)
			Reaction buffer	72	8
DNA Loading dye	18	2
			Taq enzyme (5U/ microlitre)	0.54	0.06
DNA (10-80ng/ microlitre)	18	2
			Summation	108.54	12.06

Reacting hole	Primer	Primer consumption
			1st hole	Primer 1	0.4
2nd hole	Primer 2	0.4
			3rd hole	Primer 3	0.4
4th hole	Primer 4	0.4

Mixed by PCR mixing mother liquor (1 person-portion), then each reacting hole of 8 unions divides the mixed solution getting 12ul, covers 8 union lid or films, puts into gene-amplificative instrament.

To carry out increase (response procedures is as shown in table 2) with the PCR response procedures that whole blood directly increases the same, after having increased, carry out electrophoretic analysis, electrophoresis result as shown in Figure 2.The experimental result comparison of increasing with whole blood: positive Kong Yiwei 1,5 and 8, result interpretation is HLA-B*2704/25, identical with whole blood amplification.

Conclusion: whole experiment flow only needs about 1.5 hours, experimental result is accurate, and can not only the HLA-B27 gene yin and yang attribute of discriminating experiment sample by test kit of the present invention, and the gene hypotype of HLA-B27 can be informed, prompting onset risk and better assist physician are diagnosed.

Claims

1. a human HLA-B27 genotyping kit, comprise pcr amplification primer, archaeal dna polymerase and reaction buffer, it is characterized in that, described archaeal dna polymerase is the warm start Taq polysaccharase of PCR response inhabitation thing in the tolerated blood of polyoxyethylene glycol PEG modification, and the nucleotide sequence of described pcr amplification primer is as follows:

5’-TGCATCTGGACATGCTTGCT-3’ 5’-TGGCTGGAGGAGACTCCAAA-3’。

2. human HLA-B27 genotyping kit according to claim 1, is characterized in that, composition and each component concentration of described reaction buffer are as follows: 0.18mM deoxynucleotide dNTP, 1.8mM magnesium chloride Mg Cl ₂, 60.3mM potassium chloride (KCl), 18.9mM Tri(Hydroxymethyl) Amino Methane Hydrochloride Tris-HCl, glycerine glycerol0.6%v/v, as dimethyl sulfoxide (DMSO) DMSO5%v/v, methane amide 2.5%v/v and the trimethyl-glycine 5%v/v of the agent of PCR increased response and stablizer.

3. human HLA-B27 genotyping kit according to claim 1, is characterized in that, also comprise DNALoadingdye in described test kit.