CN104046695B

CN104046695B - The quickly detection allelic primer of HLA-B*27, test kit and method

Info

Publication number: CN104046695B
Application number: CN201410292976.0A
Authority: CN
Inventors: 潘捷; 郑仲征; 孟伟
Original assignee: Di Shuobeiken Bio Tech Ltd Shanghai
Current assignee: Shanghai Tissuebank Biotechnology Co ltd; Shanghai Tissuebank Medical Laboratory Co ltd; Shenzhen Tissuebank Precision Medicine Co ltd
Priority date: 2014-06-25
Filing date: 2014-06-25
Publication date: 2016-06-22
Anticipated expiration: 2034-06-25
Also published as: CN104046695A

Abstract

The invention belongs to gene engineering technology field, disclose a kind of quick primer of detection HLA-B*27 gene, the test kit containing this primer and the method adopting this primer and test kit quickly to detect HLA-B*27 gene。Present invention uses the specific primer of HLA-B*27 gene and the modular design of employing multi-primers, completely covers the SNP site of HLA-B*27 gene, enhance specific combination, be more effectively prevented from false-positive generation。In addition, the present invention is by specific primer, dNTPs, PCR buffer and dyestuff are pre-mixed, it is greatly saved operating time and workload, have quick, easy, accurately, feature intuitively, examination and the typing assay of whole gene can be completed in 3 hours, solve HLA-B*27 gene for the problem to ankylosing spondylitis auxiliary diagnosis。

Description

The quickly detection allelic primer of HLA-B*27, test kit and method

Technical field

The present invention relates to gene engineering technology field, be specifically related to quickly the detection allelic primer of HLA-B*27, test kit and method。

Background technology

HLA antigen is major histocompatibility complex (MajorHistocompatibilityComplex, MHC) expression product, in immune system, being mutually distinguishable between primary responsibility cell is reacted with induction of immunity, has the function regulating immunne response。Recent study HLA and disease associated data show, the incidence rate of some disease is relevant with some special types other HLA recall rate, these patients are that pathogeny is failed to understand and with immunologic dysfunction and the disease having genetic predisposition mostly, therefore, analyze HLA antigen presentation situation and not only facilitate understanding pathogeny, all significant for the diagnosis of disease, prevention and Index for diagnosis。

HLA-B*27 gene belongs to I type mhc gene, substantially expresses in body on all cells having core, and especially lymphocytic surface has abundant content。Before more than 20 years, people just have been found that the expression of HLA-B*27 antigen and ankylosing spondylitis have high correlation, its HLA-B*27 antigen presentation of ankylosing spondylitis patient more than 90% is positive, in general population, only 5-10% is positive, and ankylosing spondylitis is similar to numerous disease and be difficult to make a definite diagnosis due to symptom, therefore important in inhibiting in the detection of HLA-B*27 diagnosis in disease。In the disease of this class of spondyloarthropathy except ankylosing spondylitis, also having other diseases many and the expression of HLA-B*27 antigen to have dependency more or less, therefore the detection of HLA-B*27 is a very valuable index in the diagnosis of these diseases。

The allelic detection method of current HLA-B*27 mainly has the methods such as serological typing, direct Sequencing and primer specificity amplification。The method of serological typing has had a strong impact on the reliability of HLA genotyping result owing to affinity of antibody is more weak, titer is relatively low, be easily generated cross reaction。The method of direct Sequencing can directly obtain accurate sequence information, but the complex steps of detection, somewhat expensive, detection cycle longer (more than 1 day), be not suitable for the needs of disease auxiliary quick diagnosis。PCR-SSP method is then utilize the primer complementary with HLA allelic sequences, sample to be tested DNA is carried out specific PCR amplification, has cost low, the characteristic that the time is short。Although, a kind of method that generally speaking PCR-SSP is relatively easy convenience, specificity is high, and existing PCR-based-SSP method carries out the test kit of single special primer detection HLA-B*27 at present, but the gene type that test kit on the market is contained at present is less, many emerging HLA-B*27 gene tests do not go out, and can not meet far away quick, easy, high specific to drug safety and detect the demand of HLA-B*27 gene。

Summary of the invention

The present invention is directed to the drawbacks described above existed in prior art, based on up-to-date HLA data base, consider the factors such as the position of different primers design, sequence, the allele quantity that contains, and apply Single-tube multiplex-PCR reaction system, namely the other detection of genotype is achieved by a reaction, thus provide a kind of quickly, simplicity, high specific, the easy standardization ground PCR-SSP method of qualitative detection HLA-B*27 gene。

For this, one aspect of the present invention provides the primer of a kind of quick detection HLA-B*27 gene, and it comprises the detection primer B27F01 shown in SEQIDNO.1-5, B27R01, B27R02, B27R03 and B27R04。

In a preferred embodiment of the present invention, this primer further comprises internal reference primer NCXF and the NCXR shown in SEQIDNO.6 and SEQIDNO.7。

Another aspect of the present invention provides the test kit of a kind of quick detection HLA-B*27 gene, it comprises PCR reaction mixture and Taq enzyme, described PCR reaction mixture comprises the primer quickly detecting HLA-B*27 gene as above, and the concentration of described Taq enzyme is preferably 5U/ μ L

In present invention embodiment more preferably, in this test kit quickly the detection primer B27F01 of HLA-B*27 gene, B27R01, B27R02, B27R03 and B27R04 concentration be 0.5 μM。

In another preferred embodiment of the present invention, the PCR reaction mixture of this test kit comprises internal reference primer as above further。

In present invention embodiment more preferably, in this test kit, the concentration of internal reference primer NCXF and NCXR is 0.2 μM。

In the another preferred embodiment of the present invention, comprising dNTPs, dyestuff and PCR buffer in the PCR reaction mixture of this test kit further, dyestuff is cresol red more preferably。

In present invention embodiment more preferably, in this test kit, dNTPs concentration is 0.2mM, and dye strength is 0.01%, and PCR buffer consists of: Tris-HCL concentration 10mM, potassium chloride concentration 50mM, density of magnesium chloride 1.5mM, gelatin concentration 0.001%。

Another aspect of the present invention provides a kind of method of quick detection HLA-B*27 gene, and it comprises the steps of

1, the DNA solution of detected sample is extracted；

2, adopting primer as above or adopt test kit as above, the DNA solution extracted in step 1, as template, carries out pcr amplification；

3, pcr amplification product is after electrophoresis, carries out result interpretation, when internal reference band and HLA-B*27 gene band occur simultaneously, it was shown that this gene is HLA-B*27 gene masculine, and when only there is internal reference band, then shows that this gene is HLA-B*27 gene negative。

Further aspect of the present invention provides the application in quickly detection HLA-B*27 gene of primer as above or test kit as above。

Seen from the above description, compared with prior art, the HLA-B*27 gene screening test kit of PCR-based-SSP technological development of the present invention, owing to employing specific primer, and adopts the modular design of multi-primers, completely covers the SNP site of HLA-B*27 gene, enhance specific combination, be more effectively prevented from false-positive generation, have quick, easy, accurately, feature intuitively, and experimental cost is low, it is only necessary to needed for trace sample can complete experiment detection。Meanwhile, the detection kit of the present invention can have nil case according to pcr amplification rear electrophoresis band very intuitively, namely can determine whether the allelotype of HLA-B*27, thus completing the rapid screening of HLA-B*27。

Additionally, the present invention is by primer, dNTPs, PCR buffer and dyestuff are pre-mixed, and can be greatly saved operating time and the workload of experimenter。Further, allelic multipair primer is mixed by the present invention, completes to expand simultaneously, achieve a PCR reaction and can complete allelic typing, meet high-throughout experiment demand, immediately arrive at typing and screening results, there is sensitive, stable, feature accurately and efficiently。When having qualified DNA sample, the present invention can complete whole gene screening and typing assay in 3 hours, solves HLA-B*27 gene for the problem to ankylosing spondylitis auxiliary diagnosis。

Accompanying drawing explanation

Fig. 1: HLA-B*27 gene masculine sample gel electrophoresis figure。

Fig. 2: HLA-B*27 gene chance sample gel electrophoresis figure。

Detailed description of the invention

The present invention is described in further detail by the examples below, it is intended to is used for illustrating rather than the restriction present invention。It should be pointed out that, to those skilled in the art, under the premise without departing from the principles of the invention, it is also possible to the present invention carries out some improvement and modification, these improve and modify and fall into too within protection scope of the present invention。

Embodiment 1: quickly detect the design of primers of HLA-B*27 gene

The allelic Data Source of all HLA that PCR primer design is wanted is in IMGT/HLADatabase (Release3.15.0,2014-01-17), and concrete network address is: http://www.ebi.nc.uk/ipd/imgt/hla/。Design of primers adopts manual method, and the primer of design is compared in IMGT data base, confirms that this primer can specific binding HLA-B*27 allele。In design of primers process, it is important to make the primer of design under specific PCR buffer system environment, it is possible to amplification HLA-B*27 gene specifically, namely this primer is a kind of " having sequence-specific SSP "。

In order to adopt specific PCR-SSP that HLA-B*27 gene is carried out examination, upstream at HLA-B*27 gene SNP site, design specific upstream primer B27F01 (SEQIDNO.1), 4 primer B27R01 (SEQIDNO.2), B27R02 (SEQIDNO.3), B27R03 (SEQIDNO.4) and B27R04 (SEQIDNO.5) is designed, to prevent the missing inspection of HLA-B*27 gene in all SNP site covering HLA-B*27 gene。

Meanwhile, the specific designs introducing base mismatch is held in the 3 ' of downstream primer, to strengthen the specificity of primer amplification。

In order to ensure being normally carried out of reaction system, have also been devised a pair upstream and downstream primer NCXF (SEQIDNO.6) and NCXR (SEQIDNO.7), as internal reference primer。

The primer situation of specific design is as shown in table 1。

Table 1. quickly detects the primer of HLA-B*27 gene

Primer numbers	Serial number	Primer sequence
			B27F01	SEQ ID NO.1	GCT ACG TGG ACG ACA CCC T
B27R01	SEQ ID NO.2	CTC GGT CAG TCT GTG CCT C
			B27R02	SEQ ID NO.3	TCT CGG TAA GTC TGT GCC GT
B27R03	SEQ ID NO.4	CTC GGT CAG TCT GTG TGT TGG
			B27R04	SEQ ID NO.5	CTC GGT CAG TCT GTG CCT GGG CCT TG

NCXF	SEQ ID NO.6	AGC GAG CAT CCC CCA AAG TT
			NCXR	SEQ ID NO.7	GGG CAC GAA GGC TCA TCA TT

When adopting above-mentioned primer pair HLA-B*27 allele to detect, PCR target fragment is sized to 150bp。Meanwhile, the product that internal reference primer carries out pcr amplification is adopted to be sized to 285bp。

Embodiment 2: preparation quickly detects the test kit of HLA-B*27 gene

1, the synthesis of primer

Primer is synthesized by Shanghai Ying Jun Bioisystech Co., Ltd, and primer sequence is respectively shown in SEQIDNO.1-7, and particular sequence is as shown in table 1。

2, the preparation of PCR reaction mixture

By the primer shown in SEQIDNO.1-7, dNTPs, dyestuff cresol red, PCR buffer mixes。The concentration of detection primer SEQIDNO.1-5 is 0.5 μM, the concentration of internal reference primer SEQIDNO.6-7 respectively 0.2 μM, and dyestuff cresol red concentration is 0.01%, and dNTPs concentration is 0.2mM。PCR buffer is: Tris-HCL concentration is 10mM, and potassium chloride concentration is 50mM, and density of magnesium chloride is 1.5mM, and gelatin concentration is 0.001%。To pack after above-mentioned PCR reaction mixture and Taq enzyme subpackage, form test kit。

Embodiment 3: the sizing detection of HLA-B*27 gene in sample

Choose the DNA sample of 16 parts of known HLA-B*27 gene masculines, randomly select the DNA sample of 32 parts of known HLA-B*27 genes。It is separately added in PCR pipe, in often pipe, adds Taq enzyme simultaneously。Reaction mixture is mixed after completing by application of sample, of short duration centrifugal, carries out PCR reaction。

The condition of PCR reaction is: 94 DEG C of 5min；30 circulations, 94 DEG C of 1min, 65 DEG C of 2min, 72 DEG C of 1min are run successively again according to following procedure；Last 72 DEG C of 10min, are cooled to 15 DEG C, can carry out detected through gel electrophoresis。

Use 0.5 × tbe buffer liquid, configure 2% agarose gel。Taking the direct point sample of 3ulPCR product to gel pore, electrophoresis 20 minutes, then Taking Pictures recording under ultraviolet light, concrete gel electrophoresis figure is referring to attached Fig. 1 and 2。

Result interpretation: when internal reference band normally occurs, the result of detection HLA-B*27 gene has two kinds of possibilities。One is internal reference band and HLA-B*27 gene band occurs simultaneously, it was shown that this gene is HLA-B*27 gene masculine。Another kind is internal reference band only occur, then show that this gene is HLA-B*27 gene negative。

Claims

1. a combination of primers for quick detection HLA-B*27 gene, internal reference primer NCXF and the NCXR shown in its detection primer B27F01 shown in SEQIDNO.1-5, B27R01, B27R02, B27R03 and B27R04 and SEQIDNO.6 and SEQIDNO.7 forms。

2. a test kit for quick detection HLA-B*27 gene, it comprises PCR reaction mixture and Taq enzyme, and described PCR reaction mixture comprises the combination of primers quickly detecting HLA-B*27 gene described in claim 1, and the concentration of described Taq enzyme is 5U/ μ L。

3. test kit according to claim 2, wherein quickly detection the primer B27F01 of HLA-B*27 gene, B27R01, B27R02, B27R03 and B27R04 concentration be 0.5 μM。

4. the test kit according to Claims 2 or 3, wherein the concentration of internal reference primer NCXF and NCXR is 0.2 μM。

5. the test kit according to Claims 2 or 3, wherein comprises dNTPs, dyestuff and PCR buffer further in PCR reaction mixture, dyestuff is cresol red。

6. test kit according to claim 5, wherein dNTPs concentration is 0.2mM, and dye strength is 0.01%, and PCR buffer consists of: Tris-HCL concentration 10mM, potassium chloride concentration 50mM, density of magnesium chloride 1.5mM, gelatin concentration 0.001%。

7. the combination of primers quickly detecting HLA-B*27 gene described in claim 1 quickly detects the application in the test kit of HLA-B*27 gene in preparation。