CN104109716A

CN104109716A - Human HLA-B27 gene typing kit

Info

Publication number: CN104109716A
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: 2014-10-22
Anticipated expiration: 2034-07-15
Also published as: CN104109716B

Abstract

The invention provides a human HLA-B27 gene typing kit. The human HLA-B27 gene typing kit comprises a PCR amplification primer, DNA polymerase and a reaction buffer solution, wherein the DNA polymerase is a hot initiated Taq polymerase which is modified by polyethylene glycol (PEG) and can tolerate PCR reaction inhibitors in blood. The invention also provides a nucleotide sequence of the PCR amplification primer. The human HLA-B27 gene typing kit has the technical effects that the adopted DNA polymerase is the hot initiated Taq polymerase which is modified by PEG and can tolerate PCR reaction inhibitors, PCR amplification on whole blood of human (nucleic acid extraction does not need to be carried out) can be realized, PCR amplification is directly carried out by virtue of blood, and DNA extraction does not need to be carried out; the human HLA-B27 gene typing kit can realize HLA-B27 negative and positive identification on a sample; meanwhile, a gene subtype result of an HLA-B27 positive sample can be obtained, and 24 HLA-B27 gene subtypes and other gene subtypes of 13 HLAB sites can be detected by every eight holes (eight linked pipes).

Description

A kind of human HLA-B27 gene type test kit

Technical field

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

Background technology

Human leucocyte antigen (Human Leukocyte Antigen, HLA) is positioned at 21.31st district of No. 6 the short arm of a chromosome, is the region that in current known human chromosomal, Gene Density is the highest, polymorphism is the abundantest, is 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, and known have 105 kinds of hypotypes, a called after HLA-B*27:01～HLA-B*27:105.

Ankylosing spondylitis (Ankylosing Spondylitis, AS) be a kind of male adults that is mainly in, and take axis joint chronic inflammatory diseases and be main agnogenic autoimmune disorder, clinical manifestation with flank pain, stiff discomfort for common sympton.The total incidence of China AS is 0.3%, approximately 4,500,000 examples, and the male sex is more than women, and men and women's PMR is about 10:1.Research report, expression and the AS of HLA-B27 antigen are high correlation, and this is unique related antigen that has clinical value at present, in hitherto known HLA and disease associated, is the most by force with most typical.Clinical studies show, AS patient's HLA-B27 positive rate is up to 96%, and only 5%～10% 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, suffering from struvite lumbago and backache and do not observing classical radiographic feature and when not clear few sacroiliitis or Enthesopathy, the detection of HLA-B27 will avoid clinicist to diagnose unclear predicament 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, from crowd, distributes, and 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 hereditary ancestors of B27, and other genes are all by its evolution.Studies show that with the AS relevant gene hypotype of falling ill and mainly contain 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 more AS of research, HLA-B27 and many other diseases also exist certain dependency.Research discovery, more teenager's rheumatic arthritis patient HLA-B27 antigen is positive, and in the patient of the HLA-B27 positive, B*2705 accounts for 60% left and right, and all the other are B*2704, B*2702 and B*2707.In acute uveitis patient, also found that HLA-B*27 is 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 are significant to clinical diagnosis, disease discriminating and medical research.

On domestic market, conventional HLA-B27 detection method 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, whether can only detect the existence of HLA-B27 antigen, cannot detect its allelotrope, cannot carry out HLA-B27 gene type.

MLCT method is microlymphocytotoxicity test, claims again complement-dependent cellulotoxic experiment.Basic skills is to get HLA blood grouping serum, adds examinee's lymphocyte, can make to carry with the lymphocyte of blood grouping serum type same antigen dead in the presence of complement, and inverted microscope is observed and counted, and common dead cell is greater than 40% and is judged to the HLA-B27 positive.MLCT method cost is low, easily universal, but operation steps is many, has subjectivity during result interpretation, and serum, complement and separated lymphocytic quality all can affect the accuracy of result.

ELISA method is enzyme linked immunosorbent assay, conventionally adopts double antibody sandwich method to measure HLA-B27 level in sample.Basic skills is the coated microwell plate of HLA-B27 antibody with purifying, makes insolubilized antibody; In the micropore of coated monoclonal antibody, add successively HLA-B27, then with the HLA-B27 antibodies of HRP mark, form antibody-antigen-hrp-antibody complex, after thorough washing, add substrate TMB colour developing; By microplate reader, under 450nm wavelength, measure absorbancy (OD value), by HLA-B27 concentration in typical curve calculation sample.ELISA method cost is low, and result data is objective, but complicated operation affects reaction factor more, result poor accuracy, and the coated difficulty of solid phase carrier reaches consistent between each individuality, and therefore each mensuration all needs to do typical curve.

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

PCR-SSP method is the PCR reaction of sequence specific primer guiding, it is one of method detecting from gene aspect at present HLA-B27, its basic skills is from blood, to extract genomic dna, design HLA-B27 sequence specific primers, by specific PCR reaction system each allelotype DNA fragment specific that increases, produce corresponding specific amplification products band, and detect PCR product with agarose gel electrophoresis.The required instrument of 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 increased detection time and the factor that affects result.

Although above-mentioned 4 kinds of methods are being played the part of 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 be played 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 gene type test kit is provided.

Human HLA-B27 gene type test 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 reaction inhibition in the tolerated blood of modifying through polyoxyethylene glycol (PEG), and the nucleotide sequence of described pcr amplification primer is as follows:

Also comprise a pair of 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 gene type test kit, the composition of described reaction buffer and each component concentration 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 dimethyl sulfoxide (DMSO) (DMSO) 5% (v/v), methane amide 2.5% (v/v) and the trimethyl-glycine 5% (v/v) of the agent of PCR increased response and stablizer.

Described human HLA-B27 gene type test kit, also comprises DNA Loading dye.

The present invention develops and a kind ofly from people's whole blood sample, directly carries out PCR (polymerase chain reaction) amplification whether to detect the sample HLA-B27 antigen gene positive and to carry out method and the test kit thereof of HLA-B27 subtype gene somatotype simultaneously, not only result is accurately and reliably for this method, and early stage is without blood sample is carried out to nucleic acid extraction operation, 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 containing PCR reaction inhibition in the tolerated blood of modifying through polyoxyethylene glycol (PEG) 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, with guarantee experimentation stable and experimental result accurately.

The present invention has following technique effect:

1) archaeal dna polymerase is the warm start Taq polysaccharase of PCR reaction inhibition in the tolerated blood of modifying through polyoxyethylene glycol (PEG), can realize the pcr amplification to human whole blood (without carrying out nucleic acid extraction), directly through blood, carry out pcr amplification, without carrying out DNA extraction;

2) the HLA-B27 yin and yang attribute of realizing sample is identified;

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

Accompanying drawing explanation

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

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

Embodiment

Embodiment 1

1 raw material and equipment:

1.1 test kit inclusion:

1) 8 hole 200ul PCR Sptting plates;

2) 13 pairs of specificity upstream and downstream amplimers, by particular arrangement freeze-drying, in PCR Sptting plate bottom, it is set internal reference primer that each PCR Sptting plate bottom is also added with a pair of, 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 in PCR Sptting plate bottom.

The distribution in PCR Sptting plate of the 13 pairs of specificity amplification primers is as follows:

In each hole, also add a pair of 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’

3) PCR reaction reagent

Archaeal dna polymerase: the warm start Taq polysaccharase of PCR reaction inhibition in the tolerated blood of modifying through polyoxyethylene glycol (PEG).

Reaction buffer: composition and each component concentration 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 dimethyl sulfoxide (DMSO) (DMSO) 5% (v/v), methane amide 2.5% (v/v) and the trimethyl-glycine 5% (v/v) of the agent of PCR increased response and stablizer.

DNA?Loading?dye。

The source of 1.2 samples

Fresh or the freezing preservation of collecting with EDTA anticoagulant tube is without the whole blood sample of multigelation.

1.3 required experimental installations

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

2 gene type processes

The configuration of 2.1 PCR reaction systems

The somatotype that once only carries out a sample of take is example, and 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

Frozen in advance in each PCR reaction orifice plate have 0.4 μ l10uM internal reference primer, and the 8th hole contains 0.4 μ l10uM B27 universal primer, the 13 pairs of Auele Specific Primers each PCR reaction orifice plate distribute and consumption as follows:

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

2.2 PCR 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 2% sepharose, with the voltage electrophoresis 5-7 minute of 200V, reading result.As shown in Figure 1, interpretation form is as shown in table 3 for result.

According to the HLA-B27 gene hypotype sequence of announcing 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 exons, search homologous sequence, for each homologous sequence, design 13 pairs of primers; By product difference in size, the large and not high primer of the frequency of occurrences is arranged in same hole, as the 6th and No. 7 holes; In the 8th hole, contain HLA-B27 universal primer, design HLA-B27 subtype gene interpretation form (in Table 3); According to gel electrophoresis, read the positive hole number of sample, the homologous sequence that known sample comprises, thus release sample HLA-B27 gene hypotype; In addition, also can detect 13 kinds of other gene hypotypes of HLA B site.

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 the hole positive prompts 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 configure PCR reaction system: 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 title	Primer consumption
			The 1st hole	Primer 1	0.4
The 2nd hole	Primer 2	0.4
			The 3rd hole	Primer 3	0.4
The 4th hole	Primer 4	0.4

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

With the PCR response procedures the same with the direct amplification of whole blood, increase (response procedures is as shown in table 2), after having increased, carry out electrophoretic analysis, electrophoresis result as shown in Figure 2.Compare with the experimental result of whole blood amplification: 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 approximately 1.5 hours, experimental result is accurate, and HLA-B27 gene yin and yang attribute that can not only discriminating experiment sample by test kit of the present invention, and the gene hypotype that can inform HLA-B27, prompting onset risk and better auxiliary doctor diagnose.

Claims

1. human HLA-B27 gene type test 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 reaction inhibition in the tolerated blood of modifying through polyoxyethylene glycol (PEG), and the nucleotide sequence of described pcr amplification primer is as follows:

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

2. human HLA-B27 gene type test kit according to claim 1, is characterized in that, the composition of described reaction buffer and each component concentration 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 dimethyl sulfoxide (DMSO) (DMSO) 5% (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 gene type test kit according to claim 1, is characterized in that, also comprises DNA Loading dye in described test kit.