CN111088349B - KIR3DL1 genotyping primer group and application thereof - Google Patents
KIR3DL1 genotyping primer group and application thereof Download PDFInfo
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Abstract
The invention provides a KIR3DL1 genotyping primer group and application thereof. The KIR3DL1 genotyping primer group comprises a high-expression type KIR3DL1 h allele specific primer pair; a low expression KIR3DL1 allele-specific primer pair and a universal KIR3DL1 gene-specific primer pair. Based on 147 KIR3DL1 alleles published by the latest international Database IPD-KIR Database, a pair of specific primers was designed for all high-expression KIR3DL1 h, low-expression KIR3DL1 l alleles and all KIR3DL1 alleles, respectively, so that they can be completely distinguished from all alleles by fewer assays. Meanwhile, the specific amplified fragments of the specific primers are smaller than 1kb, the extension time is only about 1min in the reaction, the time consumption is shorter, and the method is more suitable for clinical detection of 'quick and good' requirements.
Description
Technical Field
The invention relates to the technical field of genotyping detection, in particular to a method for detecting the genotyping of a human bodyKIR3DL1Genotyping primer set and application thereof.
Background
Natural Killer (NK) cells are an important component of the innate immune system and are the first defense barrier of the body in the early stages of viral infection and tumorigenesis. NK cells can receive HLA class I molecule-mediated "education" from normal cells during development through their surface-inhibitory killer cell immunoglobulin-like receptor (KIR) to gain functional maturation. The KIR gene family includes 14 functional genes and 2 pseudogenes. Located in the 19 th chromosome short arm (19q13.4) LRC regionKIR3DL1The gene is one of the members of the KIR gene family. KIR3DL1 coordinates with HLA-A or HLA-B molecule (HLA-Bw 4) carrying Bw4 domain, delivering a strong inhibitory signal, normally inhibiting NK cell activity.
KIR3DL1The gene has abundant allelic polymorphism, and the expression level and the function of different alleles are different. By 11 months of 2018, 147 IPD-KIR Database was publishedKIR3DL1Alleles encoding 91 protein molecules. Depending on the level of protein expression,KIR3DL1alleles are classified as highly expressedKIR3DL1*h(e.g.:KIR3DL1* 001. 002,/008,/009,/015 and/or/020), under-expressedKIR3DL1*l(e.g.:KIR3DL1*005 and 007) and notExpression typeKIR3DL1*null(e.g.:KIR3DL1*004 and 019). Have been found in ligands by researchersHLA-B*57High expression in the presence of genesKIR3DL1*hAlleles are effective in controlling serum HIV viral load and in delaying the progression of AIDS.
Currently, inKIR3DL1In the genotyping process of genes, multiple primers are often required to be designed for multiple alleles of the same expression type to completely distinguish the alleles, and the operation is complex in the actual use process. Therefore, it is necessary to provide a simple operationKIR3DL1Genotyping primer sets.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the invention proposes a method ofKIR3DL1Genotyping primer set and application thereof.
In a first aspect, one embodiment of the present invention provides a method ofKIR3DL1Genotyping primer sets comprising the following primer pairs:
high expression typeKIR3DL1*hAllele-specific primer pairs:
KIR3DL1*h-PCR-SSP-F：5′-AACAAAAAAAGTAAGTCTCACGG-3′（SEQ ID No.1），
KIR3DL1*h-PCR-SSP-R：5′-GAGGAGCMCCTACCTCGCTGTTG-3′（SEQ ID No.2）；
low expression typeKIR3DL1*lAllele-specific primer pairs:
KIR3DL1*l-PCR-SSP-F：5′-AACAAAAAAAGTAAGTCTCACGC-3′（SEQ ID No.3），
KIR3DL1*l-PCR-SSP-R：5′-GATGCCCTAAGATGMAGACTCAC-3′（SEQ ID No.4）；
universal typeKIR3DL1Gene-specific primer pairs:
KIR3DL1-PCR-SSP-F：5′-CAGCTCAGGTATGAGGGGAGCT-3′（SEQ ID No.5），
KIR3DL1-PCR-SSP-R：5′-GGCTGAGAGAGAAGGTTTCTCATAT-3′（SEQ ID No.6）。
wherein, the high expression typeKIR3DL1*hUpstream primer of allele specific primer pairKIR3DL1*h-PCR-SSP-F) located atKIR3DL1Genome nt12956-nt12978, downstream primerKIR3DL1*h-PCR-SSP-R) located atKIR3DL1Genome nt13471-nt13493, and the length of the amplified target fragment is 538bp; low expression typeKIR3DL1*lUpstream primer of allele specific primer pairKIR3DL1*l-PCR-SSP-F) located atKIR3DL1Genome nt12956-nt12978, downstream primerKIR3DL1*l-PCR-SSP-R) located atKIR3DL1Genome nt13877-nt13899, and amplifying target fragment length 944bp; universal typeKIR3DL1Upstream primer of gene specific primer pairKIR3DL1-PCR-SSP-F) located atKIR3DL1Genome nt4996-nt5017, downstream primerKIR3DL1-PCR-SSP-R) located atKIR3DL1Genomic nt5069-nt5093, and the length of the amplified target fragment is 98bp.
Embodiments of the inventionKIR3DL1The genotyping kit has at least the following beneficial effects:
147 published based on latest International Database IPD-KIR DatabaseKIR3DL1Alleles for all high expression levelsKIR3DL1*hLow expression typeKIR3DL1*lAlleles and allKIR3DL1Alleles each were designed with a pair of specific primers, and a more comprehensive detection was performed with fewer primers to completely distinguish all alleles. Meanwhile, the specific amplified fragments of the specific primers are smaller than 1kb, the extension time is only about 1min in the reaction, the time consumption is shorter, and the method is more suitable for clinical detection of 'quick and good' requirements.
In a second aspect, an embodiment of the present invention provides a method as described aboveKIR3DL1Preparation of genotyping primer setKIR3DL1Use of genotyping agents. Preparing typing reagent based on the primer group to obtainKIR3DL1The genotyping reagent can rapidly and accurately finish the genotyping work of the gene, and save the detection time.
In a third aspect, one embodiment of the present invention provides a method ofKIR3DL1Genotyping kit comprising the above-describedKIR3DL1Genotyping primer sets. Based on the foregoingKIR3DL1Genotyping primer setObtained byKIR3DL1Compared with the genotyping kit designed only aiming at specific alleles with different expression types in the prior art, the genotyping kit can more accurately detect possible genotyping and cover more comprehensively. Meanwhile, the primer groups can react under the same reaction system and reaction parameters, so that synchronous amplification is realized, and the aims of time and labor saving are achieved.
According to some embodiments of the inventionKIR3DL1The genotyping kit also comprises dNTPs, DNA polymerase and PCR buffer solution. The DNA polymerase may be DNA polymerase having a certain thermostability and fidelity, which is optional in the art, for example, taq DNA polymerase. The PCR buffer may be added with Mg 2+ Buffers containing no Mg 2+ Buffer of ions. When the buffer does not contain Mg 2+ When ions are reacted, additional Mg is required to be added 2+ Ions to ensure that amplification proceeds.
In a fourth aspect, an embodiment of the present invention provides a method as described aboveKIR3DL1Genotyping primer set, or the aboveKIR3DL1Genotyping kit for non-diagnostic purposesKIR3DL1Application in genotyping. Wherein the non-diagnostic purposeKIR3DL1Genotyping may be performed by, but is not limited to, such as pairingKIR3DL1Basic or application studies performed by genetic population of genes and the like.
In a fifth aspect, one embodiment of the present invention provides a method ofKIR3DL1A rapid genotyping method for a gene, the rapid genotyping method comprising the steps of:
s1, extracting DNA of a sample to be detected;
s2, using the DNA of the sample to be detected as a template, and using the aboveKIR3DL1The genotyping primer group is used as an amplification primer for PCR amplification;
s3, determining a typing result according to the amplification product;
the rapid typing method is suitable for non-diagnostic purposes.
By the method ofKIR3DL1Gene performanceTyping, wherein three pairs of primers are respectively aimed at all high expression typesKIR3DL1*hLow expression typeKIR3DL1*lAlleles and allKIR3DL1Alleles are designed to allow a more comprehensive assay to be accomplished with fewer reactions than in the prior art. Meanwhile, the primer groups can react under the same reaction system and reaction parameters, so that synchronous amplification is realized. In addition, the specific amplified fragments are smaller than 1kb, the extension time is only about 1min in the reaction, the time consumption is shorter, and the method is more suitable for clinical detection of the requirement of 'quick and good'.
According to the rapid typing method of some embodiments of the present invention, the method for determining the typing result in S3 is: detecting the amplified product by gel electrophoresis, judging the specific PCR product band, and determining the genotype of the DNA of the sample to be detected. The specific method comprises the following steps: when the amplified target fragment with the length of 98bp appears, the DNA of the sample to be detected comprisesKIR3DL1A gene; when the amplified target fragment with the length of 538bp appears, the sample to be detected carries a high expression typeKIR3DL1*hAn allele; when the amplified target fragment with the length of 944bp appears, the sample to be detected carries a low expression typeKIR3DL1*lAlleles. The primer group used in the method is designed based on PCR-SSP technology, so that when the typing result is judged according to the amplified product, the typing result can be dyed and then electrophoresed in agarose gel, and the existence and the size of each specific PCR product strip are observed by contrasting with a molecular marker, thereby judging the existence of the detected object of the DNA to be detectedKIR3DL1Genotype.
According to some embodiments of the invention, the procedure for PCR amplification in S2 is: 95 ℃ for 4min;95 ℃,30s,68 ℃,30s,72 ℃,1min,35 cycles; 72℃for 10min. By setting the above-mentioned cycle parameters, the PCR amplification process can be performed more efficiently.
According to some embodiments of the present invention, the reaction system of the PCR in S2 is as follows:
10×PCR Buffer1.0μL；
2.5mM dNTP 0.8. Mu.L;
5mM Mg 2+ 3.0μL；
10. Mu.M of each of the PCR primers was 0.4. Mu.L;
30-100 ng/. Mu.L of DNA to be detected and 1.0 mu.L of DNA to be detected;
5U/. Mu.L Taq DNA polymerase 0.1. Mu.L;
ddH 2 o was added to 10.0. Mu.L.
The PCR amplification is carried out by the reaction system, so that the reaction efficiency of the PCR can be effectively maintained, the amplification reaction can be carried out in a relatively efficient manner, and the quantity of amplified products is increased.
Drawings
FIG. 1 is a high/low expression pattern of 8 Chinese Han healthy individuals based on PCR-SSP technique according to one embodiment of the inventionKIR3DL1Agarose gel electrophoresis effect diagram of rapid genotyping of genes.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.
Example 1
This example shows the PCR-SSP technology based high/low expression of 8 Chinese Han healthy individualsKIR3DL1The genes were typed rapidly. The parting method comprises the following steps:
(1) Peripheral blood of 8 Chinese Han healthy individuals is extracted in an anticoagulant tube, and genomic DNA is extracted as DNA to be detected.
(2) To be used forKIR3DL1The genotyping primer group carries out specific PCR amplification reaction on 8 cases of DNA to be detected, and the adopted primer groupKIR3DL1The genotyping primer sets are shown in the following table:
TABLE 1 primer set information
Wherein, the PCR amplification reaction system comprises:
all PCR amplification reactions can be synchronously amplified under the same cycle parameters, and the cycle parameters adopted in the amplification process are as follows:
six PCR primer sequences were commissioned for synthesis by Invitrogen corporation. The amplification enzyme adopts Takara TaqTM high-fidelity enzyme produced by Takara Bio-engineering (Dalian) limited company, so that the accuracy of the sequence is ensured.
(3) Taking 2 mu L of each PCR amplified product, staining by a nucleic acid dye, electrophoresis in 2% agarose gel, contrasting with TakaraDL2000DNA molecular marker, and observing the existence and the size of each specific PCR product band in a gel imaging system to judge whether the detected object is DNA to be detectedKIR3DL1The genotype is as follows:
when the amplified target fragment with the length of 98bp appears, the sample to be detected comprisesKIR3DL1A gene;
when the amplified target fragment with the length of 538bp appears, the sample to be detected carries a high expression typeKIR3DL1* h allele;
when the amplified target fragment with the length of 944bp appears, the sample to be detected carries a low expression typeKIR3DL1* l allele.
As a result, FIG. 1 shows that FIG. 1 is a high/low expression pattern of 8 Chinese Han healthy individuals based on PCR-SSP technique according to one embodiment of the present inventionKIR3DL1Agarose gel electrophoresis effect diagram of rapid genotyping of genes. It can be seen that sample numbers 1, 2 and 4 carry high expression levelsKIR3DL1*hAllele, sample No. 3 carries low expressionKIR3DL1*lAllele, sample nos. 5, 6, 7 and 8 carry high expressionKIR3DL1*hAnd low expression typeKIR3DL1*lAlleles. M in the figure is DL2000DNA Marker purchased from Takara company.
Example 2
Genotyping result comparison
Identification of 200 healthy individuals of Chinese Han nationality by the first-generation sequencing technique and the method of example 1, respectivelyKIR3DL1Genotype. Wherein, identification is based on a first generation sequencing technologyKIR3DL1The genotyping method refers to the Chinese patent application CN107058544A "14 functional killer cell immunoglobulin-like receptor KIRs gene synchronous sequencing typing method".
The results of the identification are shown in Table 2, from which it can be seen that the present example providesKIR3DL1The typing result of the rapid genotyping method is completely the same as that obtained by adopting the first generation sequencing technology, and has very good consistency, which shows that the primer group provided by the embodiment has the following characteristicsKIR3DL1The typing of genes has high accuracy.
TABLE 2 comparison of the typing results of different genotyping techniques
As can be seen from the above examples, the typing primer set provided in the examples of the present invention is based on 147 published primer setsKIR3DL1Alleles were designed for all high expression levelsKIR3DL1*hAlleles and all underexpressionKIR3DL1*lAlleles and allKIR3DL1Alleles are each designed with a pair of specific primers to accomplish a more comprehensive detection with fewer primers. Compared with the first generation sequencing technology, the rapid parting method formed by the method does not need to be provided with an expensive first generation sequencer and a matched reagent, and parting work can be completed only by a conventional PCR instrument, an agarose gel electrophoresis instrument and a matched imaging system, so that the cost is low, the operation is simple and rapid, the result is easy to interpret, and the clinical detection requirement is met. Meanwhile, the adopted primer reaction system and reaction parameters can be the same, so that synchronous amplification can be realized, and time and labor are saved.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
SEQUENCE LISTING
<110> Shenzhen Baoan district women and child health care hospital
<120> KIR3DL1 genotyping primer group and application thereof
<130> 1
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<170> PatentIn version 3.5
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aacaaaaaaa gtaagtctca cgg 23
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gaggagcmcc tacctcgctg ttg 23
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aacaaaaaaa gtaagtctca cgc 23
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gatgccctaa gatgmagact cac 23
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Claims (2)
1. The method comprises the following steps ofKIR3DL1The rapid genotyping method of the gene is characterized by comprising the following steps of:
s1, extracting DNA of a sample to be detected;
s2, using the DNA as a templateKIR3DL1The genotyping primer group is used as an amplifying primer for PCR amplification, and the PCR amplification process comprises the following steps: 95 ℃ for 4min;95 ℃,30s,68 ℃,30s,72 ℃,1min,35 cycles; 72 ℃ for 10min;
s3, determining a parting result according to the amplification product, wherein the method for determining the parting result comprises the following steps: detecting the amplified product by gel electrophoresis, judging the specific PCR product band, and determining the genotype of the DNA;
the rapid typing method is suitable for non-diagnostic purposes;
the saidKIR3DL1The genotyping primer set comprises the following primer pairs:
high expression typeKIR3DL1*hAllele-specific primer pairs:
KIR3DL1*h-PCR-SSP-F：5′-AACAAAAAAAGTAAGTCTCACGG-3′，
KIR3DL1*h-PCR-SSP-R：5′-GAGGAGCMCCTACCTCGCTGTTG-3′；
low expression typeKIR3DL1*lAllele-specific primer pairs:
KIR3DL1*l-PCR-SSP-F：5′-AACAAAAAAAGTAAGTCTCACGC-3′，
KIR3DL1*l-PCR-SSP-R：5′-GATGCCCTAAGATGMAGACTCAC-3′；
universal typeKIR3DL1Gene specificitySex primer pair:
KIR3DL1-PCR-SSP-F：5′-CAGCTCAGGTATGAGGGGAGCT-3′，
KIR3DL1-PCR-SSP-R：5′-GGCTGAGAGAGAAGGTTTCTCATAT-3′。
2. the rapid typing method according to claim 1, wherein the reaction system of the PCR in S2 is as follows:
10×PCR Buffer 1.0μL；
2.5mM dNTP 0.8. Mu.L;
5mM Mg 2+ 3.0μL；
10. Mu.M of each of the PCR primers was 0.4. Mu.L;
30-100 ng/. Mu.L of DNA to be detected and 1.0 mu.L of DNA to be detected;
5U/. Mu.L Taq DNA polymerase 0.1. Mu.L;
ddH 2 o was added to 10.0. Mu.L.
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