CN112391463B

CN112391463B - Special primer for detecting SNP (single nucleotide polymorphism) site of invasive aspergillosis risk after allogeneic hematopoietic stem cell transplantation and application of special primer

Info

Publication number: CN112391463B
Application number: CN202011458525.1A
Authority: CN
Inventors: 孙于谦
Original assignee: Peking University Peoples Hospital
Current assignee: Peking University Peoples Hospital
Priority date: 2020-12-10
Filing date: 2020-12-10
Publication date: 2022-10-11
Anticipated expiration: 2040-12-10
Also published as: CN112391463A

Abstract

The invention belongs to the technical field of biology, and provides a special primer for detecting SNP sites of invasive aspergillosis risk after allogeneic hematopoietic stem cell transplantation, which comprises a primer group 1 and a primer group 2, wherein the primer group 1 is a specific PCR primer consisting of 3 pairs of primers, and the primer group 2 is a single-base extension primer consisting of 3 primers. The invention also provides a preparation of the SNP locus for detecting the risk of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation, and an SNP locus information method for detecting the risk of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation. By adopting the special primer provided by the invention, the association of the gene polymorphism of 3 SNP sites related to the occurrence risk of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation and the occurrence risk of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation can be efficiently and simultaneously completed through one-time detection, thereby greatly reducing the detection time and saving the cost.

Description

Special primer for detecting SNP (single nucleotide polymorphism) site of invasive aspergillosis risk after allogeneic hematopoietic stem cell transplantation and application of special primer

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a special primer for detecting an SNP locus of invasive aspergillosis risk after allogeneic hematopoietic stem cell transplantation and application thereof.

Background

Invasive Aspergillosis (IA) is a common and important complication after allogeneic Hematopoietic Stem Cell Transplantation (HSCT) and has the following characteristics: the incidence is high: the incidence rate after the allogeneic transplantation is about 5-10 percent, and even can be as high as 20 percent after the matched-type incompatibility/haplotype hematopoietic stem cell transplantation; poor prognosis: the mortality rate of the patients reaches 30-90%, and researches show that the long-term survival rates of the patients with the combined invasive aspergillosis and the patients without the combined invasive aspergillosis are 24.4% and 71.2% respectively; 3. the treatment time is long and the treatment cost is high.

In view of the above, the prevention of IA is very important in HSCT clinical practice. The preventive strategy in current clinical practice is still rough and does not allow for stratified prevention depending on the individual's risk of developing IA: under-prevention may be a problem in patients at high risk for developing IA (high risk), and over-prevention may be a problem in low risk patients; in addition, the fungus medicine has long treatment period, high cost and organ toxicity, and brings heavy economic burden and organ risk. Therefore, the corresponding stratification prevention according to the patient IA risk is an important research topic in the field of HSCT. The premise of reasonable layered prevention is that the IA high risk group can be accurately predicted.

It is well known that polymorphisms in genetic loci (primarily in the form of SNPs) can cause alterations in DNA structure and gene function, and are the genetic basis for differences in biological traits among different individuals. For example, polymorphism at SNP site results in different individuals with different susceptibility to complex diseases and different treatment susceptibility to different drugs. Therefore, the early discovery and early warning of diseases can be realized by detecting the single nucleotide polymorphism of the individual gene; for the sick individual, help the doctor to screen out the effective treatment scheme, make the most correct treatment decision to the patient, has already saved the invalid medical cost of patient, and improved the therapeutic effect.

However, the traditional methods for detecting the genotype of the SNP site of a patient mainly include sanger sequencing, fluorescent quantitative PCR and the like. Although the method may provide better value clinically, the method has the problems of time consumption, high economic cost, high time cost and the like, and cannot be popularized and used clinically on a large scale.

According to the latest research results, the matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) technology (MASSARRAY platform) can simultaneously detect a plurality of gene polymorphism sites, has the advantages of strong compatibility, high flux, high precision and high cost performance, and can finish the detection of the three sites at one time. However, there has been no report of the correlation between the use of MALDI-TOF-MS for detecting the risk of occurrence of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a special primer, a preparation and a detection method for detecting the SNP locus of the risk of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation, and application of the primer group, the kit and the detection method in preparing products for detecting the risk of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation; the special primer can accurately predict which patients belong to invasive aspergillosis high risk group after allogeneic hematopoietic stem cell transplantation.

In a first aspect, the invention provides a special primer for detecting SNP sites of invasive aspergillosis risk after allogeneic hematopoietic stem cell transplantation, which comprises a primer group 1 and a primer group 2, wherein:

the primer group 1 is a specific PCR primer and comprises a primer pair 1, a primer pair 2 and a primer pair 3;

the primer pair 1 consists of a single-stranded DNA molecule shown in a sequence 1 and a single-stranded DNA molecule shown in a sequence 2;

the primer pair 2 consists of a single-stranded DNA molecule shown in a sequence 3 and a single-stranded DNA molecule shown in a sequence 4;

the primer pair 3 consists of a single-stranded DNA molecule shown in a sequence 5 and a single-stranded DNA molecule shown in a sequence 6;

the primer group 2 is a single-base extension primer, and comprises a single-base extension primer 1, a single-base extension primer 2 and a single-base extension primer 3;

the single-base extension primer 1 is a single-stranded DNA molecule shown in a sequence 7;

the single-base extension primer 2 is a single-stranded DNA molecule shown in a sequence 8;

the single-base extension primer 3 is a single-stranded DNA molecule shown in a sequence 9.

In a second aspect, the invention provides a preparation for detecting SNP sites of risk of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation, which comprises a first reagent and a second reagent; wherein:

the first reagent comprises a primer pair 1-a primer pair 3 in the primer group 1 according to the first aspect of the invention, a PCR buffer solution, mgCl ₂ Dntps and DNA polymerase;

the second reagent comprises the single base extension primer 1-3 in the primer set 2 according to the first aspect of the invention, and single base extension buffer, single base extension enzyme, and single base extension termination Mix.

In a third aspect, the invention provides an application of a special primer for detecting the SNP locus of the risk of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation provided by the first aspect of the invention or a preparation for detecting the SNP locus of the risk of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation provided by the second aspect of the invention in preparation of products for detecting the risk of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation.

In a fourth aspect, the present invention provides a method for detecting SNP sites at risk of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation, comprising the steps of:

1) Carrying out PCR amplification by using the primer pair 1-the primer pair 3 in the primer group 1 according to the first aspect of the invention and using the genomic DNA of a sample to be detected as a template to obtain a PCR amplification product;

2) Carrying out alkaline phosphatase digestion on the PCR amplification product to obtain a digestion product;

3) Carrying out single base extension reaction on the digestion product by using a single base extension primer 1-a single base extension primer 3 in a primer group 2 to obtain a single base extension reaction product;

4) And purifying the single base extension reaction product, and then carrying out matrix-assisted laser desorption ionization time-of-flight mass spectrometry detection to obtain SNP site information of invasive aspergillosis risk after allogeneic hematopoietic stem cell transplantation in a sample to be detected.

By using the special primer for detecting the SNP loci of the risk of the invasive aspergillosis after the allogeneic hematopoietic stem cell transplantation, the relevance of the gene polymorphism of 3 SNP loci (namely rs4257674 (CXCL 10), rs3921 (CXCL 10) and rs1800629 (TNF)) related to the incidence risk of the invasive aspergillosis after the allogeneic hematopoietic stem cell transplantation and the incidence risk of the invasive aspergillosis after the allogeneic hematopoietic stem cell transplantation can be efficiently and simultaneously completed through one-time detection. The primer designed according to the adopted SNP locus has high specificity and good accuracy; by adopting the special primer and the method provided by the invention, the detection time is greatly reduced, the cost is saved, the special primer and the method have very high application value for guiding the association of the gene polymorphism of 3 SNP sites related to the occurrence risk of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation and the occurrence risk of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation, and are suitable for popularization and application.

Drawings

FIG. 1 shows the result of detection of site rs4257674 in example 2.

FIG. 2 shows the result of detection of the rs3921 site in example 2.

Fig. 3 shows the detection result of the rs1800629 locus in example 2.

FIG. 4 shows the result of detection of site rs4257674 in example 4.

FIG. 5 shows the result of detection of the rs3921 site in example 4.

Fig. 6 shows the detection result of the rs1800629 locus in example 4.

Detailed Description

In order to make the technical solution, objects and advantages of the present invention clearer, the present invention is further described in detail by the following specific examples. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.

In a first aspect, the invention provides a special primer for detecting SNP sites of invasive aspergillosis risks after allogeneic hematopoietic stem cell transplantation, which comprises a primer group 1 and a primer group 2; wherein:

the primer pair 3 consists of a single-stranded DNA molecule shown in a sequence 5 and a single-stranded DNA molecule shown in a sequence 6.

The primer group 2 is a single base extension primer (UEP) which consists of a series of single base primers with gradually increased molecular weights and gradually increased base numbers, and is respectively a single base extension primer 1, a single base extension primer 2 and a single base extension primer 3. Each primer of the series only extends one base, and the molecular weight and the base number of 3 extension products are gradually increased.

The primers (single-stranded DNA molecules represented by the sequences 1 to 6) in the primer set 1 to the primer set 3 were mixed at an equimolar ratio to constitute an amplification primer Mix.

The single-base extension primers 1 to 3 (single-stranded DNA molecules represented by SEQ ID Nos. 7 to 9) in the primer set 2 were mixed at an equimolar ratio to form a single-base extension primer Mix.

Wherein the SNP sites comprise: rs4257674, rs3921 and rs1800629.

The distribution of the rs4257674 genotype in people is AA/AG/GG, which respectively corresponds to high risk, medium risk and low risk; the distribution of the rs3921 genotype in the population is CC/CG/GG, which respectively corresponds to high risk, medium risk and low risk; the distribution of the rs1800629 genotype in the population is AA/AG/GG, which corresponds to high risk, medium risk and low risk, respectively.

In a second aspect, the invention provides a preparation for detecting SNP sites of invasive aspergillosis risks after allogeneic hematopoietic stem cell transplantation, which comprises a first reagent and a second reagent; wherein:

the first reagent comprises the primer pair 1-primer pair 6 in the primer set 1 according to the first aspect of the invention, PCR buffer solution, mgCl ₂ dNTPs and DNA polymerase.

The second reagent comprises the single base extension primer 1-the single base extension primer 3 in the primer set 2 according to the first aspect of the invention, and a single base extension buffer, a single base extension enzyme, and a single base extension termination Mix.

Wherein the SNP site is as defined above.

In the reagent I, the concentration of 6 primers in the primer pairs 1-3 in the reagent I is 0.5 mu M. The PCR buffer solution can be obtained commercially, and the components in the PCR buffer solution are common PCR buffer components. Said MgCl ₂ The concentration in reagent one was 2mM. The concentration of the dNTP in the first reagent is 0.5mM. The DNA polymerase can be Hotstar Taq DNA polymerase, and the concentration in reagent one is 0.2U/. Mu.L.

In the second reagent, the concentrations of the single-base extended primer 1 to the single-base extended primer 3 in the second reagent are all 0.5. Mu.M. The single-base extension buffer, single-base extension enzyme, and single-base extension termination Mix are commercially available, and are all commercially available, for example, from Agena BIOSCIENCE.

In the second aspect of the present invention, the reagent I and the reagent II are used in combination to form a multiple nested PCR reaction system.

In a third aspect, the invention provides the application of the special primer for detecting the SNP locus of the risk of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation provided by the first aspect of the invention or the preparation for detecting the SNP locus of the risk of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation provided by the second aspect of the invention in the preparation of products for detecting the risk of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation.

1) Carrying out PCR amplification by using the primer pair 1-the primer pair 3 in the primer group 1 and using the genome DNA of a sample to be detected as a template to obtain a PCR amplification product;

4) And purifying the single base extension reaction product, and then carrying out matrix assisted laser desorption ionization time-of-flight mass spectrometry to obtain SNP site information of invasive aspergillosis risk after allogeneic hematopoietic stem cell transplantation in a sample to be detected.

Wherein, in the step 1), the PCR amplification procedure is as follows:

first 94 ℃ for 15min (pre-denaturation); then, 45 cycles of 94 ℃, 20s,56 ℃, 30s,72 ℃ and 60s are carried out; then 72 ℃ for 3min.

In step 2), the alkaline phosphatase digestion conditions are as follows: at 37 ℃ for 40min; storing at 85 deg.C for 5min and 4 deg.C;

in step 3), the procedure of the single base extension reaction is as follows: 30s at 94 ℃; firstly, carrying out 40 cycles of 94 ℃ for 5s, then carrying out 5s at 52 ℃ and 5s at 80 ℃; then 3min at 72 ℃; finally, storing at 4 ℃.

In step 4), the purification may be resin purification.

Wherein the SNP site is as described above.

Currently, in patients receiving HSCT, the incidence of AI is closely related to genetic background of the donor in addition to the hypoimmunity of the patient. Among them, the cytokine-related gene of the donor is closely related to the incidence of AI and plays an important role in the anti-Aspergillus immune process.

The CXCL10 gene encodes a member of the Chemokine family (Chemokine family), a chemotactic cytokine that controls cell migration and cell localization throughout development, homeostasis, and inflammatory processes. The immune system, which is dependent on the cooperative migration of cells, is particularly dependent on chemokines for its function. Chemokines not only direct immune effector cells to the site of infection or inflammation, but also coordinate interactions between immune cells. In this manner, chemokines can facilitate the interaction between the innate and adaptive immune systems, thereby shaping and providing the necessary environment for the development of an optimal adaptive immune response. The chemotactic factor coded by the CXCL10 gene can stimulate the migration of monocytes, NK cells and T cells to the infection or inflammation part and participate in the inflammatory reaction.

The TNF (TNF-a) gene encodes a multifunctional pro-inflammatory cell, belonging to the tumor necrosis factor, produced mainly by activated macrophages. It binds to receptors and participates in the regulation of a wide range of biological processes and the development of a variety of diseases.

Particularly, in Han people in China, the relevance of the polymorphism of CXCL10 genes and TNF (TNF-a) genes and the occurrence risk of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation does not exist at present, and other related researches and data do not exist, so that the invention has important significance.

The invention combines effective indexes of IA occurrence risk through early warning or prediction, and the scientific and clinical significance of the invention comprises the following steps: (1) The method provides guidance for selecting bone marrow donors in clinical work, reduces the incidence rate of Invasive Aspergillosis (IA) of patients after clinical allogeneic Hematopoietic Stem Cell Transplantation (HSCT), and has important clinical significance for preventing IA after HSCT; meanwhile, the method also conforms to the concept of 'preventing diseases'. (2) Accurate prediction of IA occurrence risk is helpful for guiding the hierarchical management of patients after HSCT, on one hand, more active and targeted prevention strategies are carried out on high-risk patients, and on the other hand, unnecessary prevention is reduced for low-risk patients, so that economic cost and organ damage risk are reduced.

Various reagents, materials and the like used in the following examples are commercially available products unless otherwise specified; unless otherwise specified, all the tests and detection methods used in the following examples are conventional in the art and can be obtained from textbooks, tool books or academic journals.

Example 1

This example is intended to illustrate the primers specifically used for detecting SNP sites at risk of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation, which are provided by the present invention.

Specific PCR primers and UEP primers are designed aiming at rs4257674, rs3921 and rs1800629 sites, and the generation risk gene detection of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation is realized by utilizing a matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) technology.

Specific PCR primers include:

sequence 1, rs1800629 PCR primer 1:

5′-ACGTTGGATGGATTTGTGTGTAGGACCCTG-3′；

sequence 2, rs1800629 PCR primer 2:

5′-ACGTTGGATGGGTCCCCAAAAGAAATGGAG-3′；

sequence 3, rs4257674 PCR primer 1:

5′-ACGTTGGATGGTATCATGTGCTTTCTACTG-3′；

sequence 4, rs4257674 PCR primer 2:

5′-ACGTTGGATGACTGGATGATGAATCGTGGC-3′；

sequence 5, rs3921 PCR primer 1:

5′-ACGTTGGATGTCCTACAGGAGTAGTAGCAG-3′；

sequence 6, rs3921 PCR primer 2:

5′-ACGTTGGATGCATGGAGTATATGTCAAGCC-3′。

the UEP primers comprise:

sequence 7, rs1800629 single base extension primer:

5′-GAGGCTGAACCCCGTCC-3′；

sequence 8, rs4257674 single base extension primer:

5′-GTGCTTTCTACTGCTAGCTACTA-3′；

sequence 9, rs3921 single base extension primer:

5′-GGAGCAGCTGATTTGGTGACCAT-3′。

example 2

This example illustrates the method for detecting SNP sites at risk of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation, which is provided by the present invention.

1. Extraction of template DNA

Genomic DNA was extracted from 800 ex vivo peripheral blood samples and diluted to 20-30 ng/. Mu.L.

2. PCR reaction

The reaction was prepared in 5 μ L PCR 96-well plates:

1. Mu.L of template DNA, 1. Mu.L of amplification primer Mix, 10 XPCR Buffer (containing 15mM Mg) ²⁺ )0.5μL，MgCl ₂ (25 mM) 0.4. Mu.L (MgCl in the reaction System) ₂ The final concentration of (2 mM), 0.1. Mu.L of dNTP (25 mM) (the final concentration of dNTP in the reaction system is 0.5 mM), 0.2. Mu.L of Hotstar Taq (5U/. Mu.L) (the final concentration of Hotstar Taq in the reaction system is 0.2U/. Mu.L), and 5. Mu.L of the reaction system was supplemented with MBG water. The 384 well plates were sealed with a sealing membrane.

The amplification primers Mix consist of primers shown in sequences 1-6 and water, wherein the final concentration of each primer is 0.5. Mu.M.

And carrying out PCR reaction on the reaction system to obtain a PCR amplification product.

The PCR reaction program was set up as follows:

3. alkaline phosphatase treatment (SAP digestion reaction)

Adding the following components into the PCR amplification product obtained in the step 2 to obtain an SAP digestion reaction system:

SAP Buffer 0.17. Mu.L, SAP Enzyme (Agena BIOSCIENCE, product ref:100021, lot.

Carrying out digestion reaction on the digestion reaction system according to the following digestion reaction procedures to obtain a digestion reaction product: at 37 ℃ for 40min; 5min at 85 ℃;4 ℃ and infinity.

4. Single base extension reaction

Adding the following components into the digestion reaction product obtained in the step 3: 0.2. Mu.L of iPLEX Buffer plus (Agena BIOSCIENCE, ref:01431, lot.

The primer Mix consists of primers shown in sequences 7-9 and water, and the concentration of each primer is 0.5. Mu.M.

And carrying out single base extension reaction on the single base extension reaction system to obtain a single base extension reaction product.

The above single-base extension reaction procedure was as follows:

5. resin purification

Performing resin purification on the single base extension reaction product obtained in the step 4, wherein the resin purification is as follows:

1) And (3) centrifuging the 384 reaction plates containing the single-base extension products obtained in the step (4) at 3000rpm for 2min, adding 16 mu L of MBG water into each hole, sealing the reaction plates by using a sealing film, and performing instant centrifugation at 3000 rpm.

2) A clean A4 paper is taken, a resin plate (specification is 6 mg) is placed on the paper, a proper amount of resin (Agena BIOSCIENCE, product ref:08040, lot.

3) The centrifuged 384 reaction plate was inverted on the resin plate with resin laid, with one-to-one correspondence between the wells of the two plates. The plates were inverted with the resin plate on top and the 384 reaction plate on the bottom. The back of the resin plate was gently tapped evenly to drop the resin into a 384 reaction plate containing the single base extension product.

4) The 384 reaction plate containing the single base extension product and the resin is sealed with a sealing film, and the shaker is rotated vertically at a low speed for 30min to bring the resin into full contact with the reactants.

5) And centrifuging the 384 reaction plates after the reaction is finished at 3000rpm for 5min to enable the resin to sink to the bottom of the tube, wherein the supernatant is the purified product.

6. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) detection

The purified product obtained in the step 5 is transferred to a chip (Agena BIOSCIENCE, product ref:01509, lot.

The results are shown in FIGS. 1-3, and the genotypes of the locus rs4257674, rs3921 and rs1800629 are obtained. As can be seen from the figures, the detection rate of rs4257674 is 99.6%, the detection rate of rs3921 is 99.7%, the detection rate of rs1800629 is 99.7%, and the clustering effect is good, so that the special primer and the method can be used for information detection of the SNP sites.

7. And (4) interpretation of results: and (3) accurately predicting and early warning the occurrence risk of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation by using a mass spectrum detection result and referring to the following table 1.

TABLE 1 information on 3 SNP sites of peripheral blood samples for phenotypic analysis

Note that: in the above table, if the clinical significance of the detection results of different SNP sites of the same individual is determined to be inconsistent, the clinician will refer to different individuals with different contribution degrees to the occurrence risk of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation for different detection sites, and select the most suitable bone marrow donor for the patient.

Example 3

This example is used to illustrate the method for detecting SNP loci of risk of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation and the application of the specific primers.

1. Extraction of template DNA

Genomic DNA of peripheral blood of 8 subjects was extracted and diluted to 25 ng/. Mu.L in a uniform manner. The 8 subjects were all bone marrow donors for allogeneic hematopoietic stem cell transplantation.

2. And (3) PCR reaction: see example 2, step 2.

3. Alkaline phosphatase treatment (SAP digestion reaction): see example 2, step 3.

4. Single base extension reaction: see example 2, step 4.

5. Resin purification: see example 2, step 5.

6. And (3) matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) detection: see example 1, step 6.

Through detection, in 8 samples examined in this example, the detectable rate of each site is 100%.

The results are shown in table 2 below.

Table 2: SNP site detection results of 8 peripheral blood test samples

Sample 1

Sample 2

Sample 3

Sample 4

Sample 5

Sample 6

Sample 7

Sample 8

rs4257674

GG

AA

AG

GG

AG

rs3921

GG

CC

CG

GG

CG

rs1800629

GG

AA

AG

GG

AG

According to the test results in table 2, it can be seen that:

bone marrow recipients of

samples

1 and 5 were not susceptible to invasive aspergillosis after allogeneic hematopoietic stem cell transplantation; after the bone marrow recipients of

samples

4, 6, 7 and 8 are transplanted with allogeneic hematopoietic stem cells, invasive aspergillosis has certain immunity; bone marrow recipients of

samples

2 and 3 were susceptible to invasive aspergillosis following allogeneic hematopoietic stem cell transplantation.

Clinical verification shows that the occurrence risk of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation of the samples 1-8 is consistent with the result obtained by the special primer and the method for detecting the SNP locus related to the occurrence risk of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation provided by the invention (the specific result is not repeated), which indicates that the occurrence risk of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation can be well predicted by the special primer and the method for detecting the SNP locus related to the occurrence risk of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation provided by the invention.

Example 4

The same procedure as in example 2 was repeated, except that 800 ex vivo peripheral blood samples in example 2 were replaced with 384 bone marrow samples in this example in the extraction of the template DNA in step 1.

The results are shown in FIGS. 4-6, and the genotypes of the locus rs4257674, rs3921 and rs1800629 are obtained. As can be seen from the figures, the detection rate of each site is more than 98%, which indicates that the special primer and the method of the invention can be used for information detection of the SNP sites.

Example 5

The procedure was carried out in the same manner as in example 2 except that, in the extraction of the template DNA in step 1, the peripheral blood of 8 subjects in example 3 was replaced with the bone marrow of 8 allogeneic hematopoietic stem cell transplantation bone marrow donors in this example, and the bone marrow of these 8 subjects was used as the specimens 9 to 16.

The results are shown in table 3 below.

Table 3: SNP site detection results of 8 bone marrow samples

Sample 9

Sample 10

Sample 11

Sample 12

Sample 13

Sample 14

Sample 15

Sample 16

rs4257674

AG

GG

AG

GG

AG

rs3921

CG

GG

CG

GG

CG

rs1800629

AG

GG

AG

GG

AG

From the test results in table 3, it can be seen that:

bone marrow recipients of

samples

11, 12, 14 were less susceptible to invasive aspergillosis following allogeneic hematopoietic stem cell transplantation;

samples

9, 10, 13, 15, and 16 had some immunity to invasive aspergillosis after allogeneic hematopoietic stem cell transplantation in bone marrow recipients.

Clinical verification shows that the occurrence risk of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation of samples 9-16 is consistent with the result obtained by the special primer and method for detecting the SNP locus related to the occurrence risk of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation (the specific result is not repeated), which indicate that the occurrence risk of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation can be well predicted by the special primer and method for detecting the SNP locus related to the occurrence risk of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation.

Comparative example 1

The design of the primer is the key of the invention, the combination rate of the primer and the template directly influences the PCR amplification efficiency, thereby influencing the sensitivity and the detection rate of the detection result, especially the design requirement of the UEP primer is very high, and the 3 UEP primers must be increased in gradient in length design so as to generate molecular weight difference and realize mass spectrum detection except for the requirement of complete base complementary match between the sequence and the sequence in front of the detection site. Non-specific binding of the primer to the template results in non-specific amplification products, which also interfere with the experimental results.

For each SNP site, the inventors designed 3 sets of PCR primers and UEP primers in parallel, namely primer set 1, primer set 2 and primer set 3 (primers used in examples 1-5 above). The detection effect of the primer set 3 used in example 2 was the best when 800 samples of the extracorporeal peripheral blood sample used in example 2 were detected with 3 sets of primers according to the method of example 2, by comparison: high detection rate, high sensitivity, good clustering effect and high specificity.

TABLE 4 primer set 1

TABLE 5 primer set 2

Primer set 3 is shown in the sequence 1-9.

The detection rate of the sites of the primer group 1 and the primer group 2 is low, and the clustering effect is poor.

When the primer group 1 is used, the detection rate of each site in 800 samples to be detected is as follows:

the detection rate of rs4257674 is 87.5%, the clustering effect is poor, and the clustering is dispersive; the detection rate of rs3921 is 95.0%, and the clustering effect is good; the detection rate of rs1800629 is 87.5%, the clustering effect is poor, and the clustering is dispersive.

When the primer group 2 is used, the detection rate of each site in 800 samples to be detected is as follows:

the detection rate of rs4257674 is 87.8%, the clustering effect is poor, and the clustering effect is dispersive; the detection rate of rs3921 is 98.1%, and the clustering effect is good; the detection rate of rs1800629 is 81.3%, the clustering effect is poor, and the clustering is dispersive.

Comparative example 2

The procedure was as in example 2 except that a primer for the SNP site RS5743836 responsible for cytomegalovirus infection after transplantation was additionally added to all the primers used in example 2.

Wherein, the primer sequence of the SNP site RS12979860 related to cytomegalovirus infection after transplantation is as follows:

rs5743836-PCR primer 1:

5′-ACGTTGGATGTGAGGGTCTTATGGCCTATG-3′；

rs5743836-PCR primer 2:

5′-ACGTTGGATGACATTGGCTTTCCTGGCAAC-3′；

rs5743836-UEP primer:

5′-GAGACTTGGGGGAGTTT-3′。

as a result, it was found that, under the condition of keeping all the operation parameters of example 2 unchanged, even if a primer set related to the post-transplantation cytomegalovirus infection related SNP site rs12979860 other than the above 3 sites is added, the compatibility of the reaction system and the reaction conditions of example 2 is destroyed, so that the detection rate of the above 3 SNP sites is reduced, and the clustering effect is deteriorated. Specifically, the detection rates of the respective sites were as follows: the detectable rate of rs4257674 is 58.2%, the detectable rate of rs3921 is 88.4% and the detectable rate of rs1800629 is 90.3%.

Comparative example 3

When the reaction systems of examples 1 and 2 were investigated, the following parallel reaction conditions were investigated:

the other operating parameters and materials of the two optimized conditions (optimized condition 1 and optimized condition 2) in this comparative example were the same as in example 2, except for the following parameters.

Optimization Condition 1

And (3) PCR reaction: the final concentration of each primer was 0.5. Mu.M, mgCl ₂ The final concentration of (2 mM), dNTP (0.5 mM), and Hotstar Taq (0.1U/. Mu.L). The PCR reaction program was set up as follows:

SAP enzymatic digestion: digestion reaction procedure: 45min at 37 ℃; 5min at 85 ℃;4 ℃ and infinity

Single base extension reaction:

optimization Condition 2

SAP enzymatic digestion: digestion reaction procedure: at 37 ℃ for 45min; 5min at 85 ℃;4 ℃ and infinity

Single base extension reaction:

as a result, it was found that, in 800 samples examined using the optimized condition 1, the detection rate of each site was as follows:

the detection rate of rs4257674 is 93.2%, and the clustering effect is good; the detection rate of rs3921 is 87.5%, the clustering effect is poor, and the clustering is dispersive; the detection rate of rs1800629 is 87.0%, the clustering effect is poor, and the clustering is scattered.

Using the optimized condition 2, the detection rate of each site in 800 samples tested is as follows:

the detection rate of rs4257674 is 91.8%, and the clustering effect is good; the detection rate of rs3921 is 89.9%, the clustering effect is poor, and the clustering is scattered; the detection rate of rs1800629 is 87.0%, the clustering effect is poor, and the clustering is scattered.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Beijing university Hospital

<120> special primer for detecting SNP site of invasive aspergillosis risk after allogeneic hematopoietic stem cell transplantation and application

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acgttggatg gtatcatgtg ctttctactg 30

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Claims

1. A special primer for detecting SNP loci of invasive aspergillosis risks after allogeneic hematopoietic stem cell transplantation is characterized in that:

the special primer consists of a primer group 1 and a primer group 2, wherein:

the primer group 1 is a specific PCR primer and consists of the following primers: primer pair 1, primer pair 2, primer pair 3;

the primer group 2 is a single-base extension primer and consists of the following primers: a single-base extension primer 1, a single-base extension primer 2, and a single-base extension primer 3;

the single-base extension primer 1 is a single-stranded DNA molecule shown as a sequence 7;

the single-base extension primer 3 is a single-stranded DNA molecule shown in a sequence 9;

the SNP loci are as follows: rs4257674, rs3921, rs1800629.

2. The special primer according to claim 1, wherein:

the primers in the primer pair 1-the primer pair 3 in the primer group 1 are mixed in an equimolar ratio to form an amplification primer Mix;

the single-base extension primers 1 to 3 in the primer set 2 are mixed at an equimolar ratio to constitute a single-base extension primer Mix.

3. A preparation for detecting SNP loci of invasive aspergillosis risk after allogeneic hematopoietic stem cell transplantation, which is characterized in that:

the primer in the preparation consists of the special primer in claim 1.

4. The formulation of claim 3, wherein:

the preparation comprises a first reagent and a second reagent; wherein:

the reagent comprises the primer group 1 of claim 1, PCR buffer solution, mgCl ₂ Dntps and DNA polymerase;

the reagent two comprises the primer group 2 of claim 1, a single base extension buffer, a single base extension enzyme, and a single base extension termination Mix.

5. The formulation of claim 4, wherein:

in the reagent I, the concentrations of 6 primers in the primer pairs 1-3 in the reagent I are all 0.5 mu M;

said MgCl ₂ The concentration in the first reagent is 2mM;

the concentration of the dNTP in the reagent I is 0.5mM;

the concentration of the DNA polymerase in the first reagent is 0.2U/. Mu.L;

in the second reagent, the concentrations of the single-base extended primer 1 to the single-base extended primer 3 in the second reagent were all 0.5. Mu.M.

6. The formulation of claim 5, wherein:

the SNP loci are as follows: rs4257674, rs3921 and rs1800629.

7. Use of the primers as defined in any one of claims 1 to 2 or the formulation as defined in any one of claims 3 to 4 for the preparation of a product for detecting the risk of invasive aspergillosis after allogeneic hematopoietic stem cell transplantation.