Multiplex digital PCR (polymerase chain reaction) probe primer group and kit for Diego blood type detection

Abstract

The invention discloses a multiplex digital PCR (Polymerase Chain Reaction) probe primer group and a kit for Diego blood type detection. The multiplex digital PCR probe primer group for Diego blood type detection comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1, a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2, a Dia probe with a nucleotide sequence as shown in SEQ ID NO.3 and a Dib probe with a nucleotide sequence as shown in SEQ ID NO.4. One end of the Dia probe and one end of the Dib probe are labeled with fluorophores, and the other end of the Dia probe and the other end of the Dib probe are labeled with quenching groups. And a fluorophore labeled by the Dia probe is different from a fluorophore labeled by the Dib probe. According to the method for detecting the Diego blood type and the population distribution frequency based on the multiple digital PCR technology, accurate detection of the Diego blood type of an individual sample is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a multiplex digital PCR probe primer set and a kit for Diego blood group detection. Background Art

[0002] The Diego blood group system is one of the human blood group systems, named DI, 010 by the International Society of Blood Transfusion (ISBT). At present, two pairs of allelic antigens have been found in the Diego blood group system: Di a and Di b as well as Wr a and Wr b , plus 17 low-frequency antigens. Among them, the most clinically significant ones are Di a and Di b , both of which can cause hemolytic transfusion reactions and hemolytic disease of the newborn. The Di a antigen was first discovered in the 1950s, and more than a decade later, its corresponding antigen Di b was also discovered. Di a and Di b are the main Diego blood group antigens. Di a is very rare in people of European or African descent, but according to existing literature reports, its prevalence is about 5% in people of Chinese or Japanese descent, and even higher in the indigenous people of North and South America, reaching 40 - 54%. Di b is a high-frequency antigen expressed in almost all populations, but there are reports of negative Di b phenotypes everywhere. The Di a and Di b antigens are not expressed on the surfaces of other blood cells except on the surfaces of red blood cells. Research literature and clinical data both show that the Di b antigen is a high-frequency antigen in the distribution of the local population, and anti-Di b is a very rare irregular antibody against a high-frequency antigen.

[0003] Currently, the methods for identifying the Diego blood group are divided into serological detection and gene detection. However, since it is difficult to obtain red blood cells negative for the Di b antigen and there is no commercially available anti-Di b antibody for identifying the antigen, it is very difficult to identify the anti-Di b antibody by serological methods. This highlights the important role of genotype methods represented by PCR technology in the identification of the Diego blood group, which can obtain the gene distribution frequency of the Diego blood group in the region and is of great significance for understanding the population distribution frequency of blood groups in the region.

[0004] Population genetics is the discipline that studies genes, gene frequencies, and genotype frequencies in populations and their changes. Population genetics reveals the genetic relationship between gene frequencies and genotype frequencies in randomly mating populations through the Hardy-Weinberg principle. However, in practice, gene frequencies are affected by various factors such as mutation, natural selection, genetic drift, immigration, and emigration. Blood group genes are one of the key markers in population genetics, and detecting the distribution frequency of blood group genes is an important issue in population genetics. There is a strong connection between blood group genes and the expression of blood group antigens. Due to genetic drift and founder effects, blood group gene frequencies vary in different regions, and clarifying blood group genotype typing is of great significance in research such as clinical blood transfusion, organ transplantation, human kinship, and ethnic migration. Therefore, it is of great significance to design a simple, economical, and accurate method for detecting gene frequencies and genotype frequencies.

[0005] However, the conventional PCR method is time-consuming, costly, and requires a relatively high technical level of operators, making it difficult to achieve high-throughput and low-cost detection. Digital PCR technology is a highly sensitive nucleic acid absolute quantification analysis technology that performs PCR amplification by dividing the reaction system into a large number of independent reaction units and calculates nucleic acid copy number quantification analysis based on Poisson distribution and positive proportion. This technology is mostly applied to the detection of tumor markers, microorganisms, and pathogens, and it has significant advantages in the detection of trace specimens and rare mutations. Measuring virus load by digital PCR can not only shorten the window period of disease detection but also provide guidance for the development and prognosis of diseases. However, single-channel digital PCR technology can only detect a single gene fragment of a single sample at a time. In actual work, multiple genes of a single sample often need to be detected simultaneously, and single-channel digital PCR technology greatly increases the workflow and the use of experimental consumables. Summary of the Invention

[0006] To address the deficiencies in the prior art, the present invention aims to provide a multiplex digital PCR probe primer set and a kit for Diego blood group detection. The present invention introduces multiplex digital PCR technology, in which in a non-competitive biphasic reaction, different scatter clusters overlap in a two-dimensional space to form a scatter plot with more than 2D. Each scatter cluster represents a different target, and through model interpretation, multiple targets can be quantified simultaneously.

[0007] The specific technical solution of the present invention is as follows:

[0008] The present invention provides a multiplex digital PCR probe primer set for Diego blood group detection, which includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1, a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2, a Dia probe with a nucleotide sequence as shown in SEQ ID NO.3, and a Dib probe with a nucleotide sequence as shown in SEQ ID NO.4. One end of the Dia probe and the Dib probe is labeled with a fluorescent group, and the other end is labeled with a quenching group, and the fluorescent groups labeled on the Dia probe and the Dib probe are different from each other.

[0009] Further, the fluorescent group is selected from FAM or VIC;

[0010] The quenching group is selected from MGB.

[0011] Further, the fluorescent group labeled on the Dia probe is FAM, and the fluorescent group labeled on the Dib probe is VIC.

[0012] Further, the 5'-end of the Dia probe and the Dib probe is labeled with a fluorescent group, and the 3'-end is labeled with a quenching group.

[0013] The present invention also provides a kit for Diego blood group detection, which includes the multiplex digital PCR probe primer set.

[0014] Further, the kit also includes a blood genomic DNA extraction reagent, a digital PCR premix, and ultrapure water.

[0015] Further, the kit also includes a positive control and a negative control.

[0016] The present invention also provides the application of the multiplex digital PCR probe primer set or the kit in the preparation of a Diego blood group detection kit.

[0017] The present invention provides a method for using the kit, which includes the following steps:

[0018] (1) Extract genomic DNA from peripheral blood;

[0019] (2) Prepare a digital PCR reaction system: 10 μL of digital PCR premix, 0.3 μL each of 10 μM upstream primer and downstream primer, 0.2 μL each of 10 μM Dia probe and Dib probe, 2 μL of ultrapure water, and 7 μL of DNA sample;

[0020] (3) Perform dual-channel digital PCR amplification, and the amplification process is as follows: Set the heating rate to 2 °C / s per step for thermal cycling, and run at 95 °C for 10 minutes; then denature at 94 °C for 30 seconds, anneal and extend at 60 °C for 30 seconds, for 40 cycles; inactivate the enzyme at 98 °C for 10 minutes; finally, hold at 4 °C;

[0021] (4) Perform model interpretation to determine the Diego blood type.

[0022] The beneficial effects of the present invention are as follows:

[0023] The present invention provides a multiplex digital PCR probe primer set for Diego blood type detection, thereby realizing the detection of Diego blood type based on the multiplex digital PCR method and the large-scale detection of the population distribution frequency. The method for detecting Diego blood type and population distribution frequency based on the multiplex digital PCR technology of the present invention can accurately detect the Diego blood type of individual samples and obtain the distribution frequency of Diego blood type in the local population, which is of great significance for understanding the gene distribution polymorphism and genetic heterogeneity in the local area. Compared with the prior art: 1) The present invention changes the previous dual digital PCR that can only detect single-point mutations to detecting multiple mutation sites in a single well and single detection, greatly saving the detection time and cost. 2) For the first time, digital PCR is used to simultaneously detect two or more mutation sites, achieving multiplex detection that has not been achieved before. Description of the Drawings

[0024] Figure 1 For Diego probe specificity verification; among them, Figure 1 A. Di(a+b-) sample, Figure 1 B. Di(a-b+) sample;

[0025] Figure 2 For Diego blood type digital PCR dual-channel typing; among them, Figure 2 A. Di(a+b+) sample; Figure 2 B. Negative control;

[0026] Figure 3 For the screening results of the Diego blood type population frequency in a 1000-person blood pool; among them, Figure 3 A. Results of detecting 20 groups of blood pools with the Dia probe, Figure 3 B. Results of detecting 20 groups of blood pools with the Dib probe. Detailed Embodiments

[0027] To understand the present invention more clearly, the present invention will be further described below with reference to the following embodiments and accompanying drawings. The embodiments are for explanation only and do not limit the present invention in any way. In the embodiments, all original reagent materials are commercially available, and the experimental methods without specific conditions are conventional methods and conventional conditions well-known in the art, or the conditions recommended by the instrument manufacturer.

[0028] Example 1: Establishment of a method for multiplex digital PCR detection of Diego blood group

[0029] 1. Recruitment of experimental subjects

[0030] A total of 1,000 unpaid blood donors in Northwest China were recruited for this experiment. The genomic DNA of the patients was extracted from EDTA-K2 anticoagulated peripheral blood using a blood genomic DNA extraction centrifugal column kit. Donor samples with Di(a+b-), Di(a-b+), and Di(a+b+) phenotypes screened in the laboratory in the early stage were used for the verification of the multiplex digital PCR method.

[0031] 2. DNA extraction

[0032] (1) Treatment of blood samples

[0033] a. When extracting 200 μL of blood sample, the next experiment can be carried out directly.

[0034] b. When extracting less than 200 μL of blood sample, buffer can be added to make up the volume to 200 μL, and then the next experiment can be carried out. Note: Steps a and b are applicable to the extraction of most 100 - 200 μL blood samples. However, for some blood samples with high protein, carbohydrate, or lipid content or poor sample storage conditions, the OD260 / OD230 ratio may be low. If it is necessary to increase the OD260 / OD230 ratio, 1 - 2.5 times the volume of cell lysis solution CL can be added to the sample for treatment, and the specific steps are the same as c.

[0035] c. When extracting more than 200 μL of blood sample, cell lysis solution treatment is required. The specific steps are as follows: Add 1 - 2.5 times the volume of cell lysis solution to the sample, invert and mix well, centrifuge at 10,000 rpm (~11,500 g) for 1 min, aspirate the supernatant, and leave the nuclear precipitate (if the lysis is not complete, 1 - 2.5 times the volume of cell lysis solution can be added to repeat the lysis once). Add 200 μL of buffer to the nuclear precipitate, shake until thoroughly mixed, and then carry out the next experiment.

[0036] d. When the blood sample is a blood clot, the liquefaction column CX1 (TIANGEN, RK165) can be selected to liquefy the blood clot. The specific steps are as follows:

[0037] d1) Transfer the blood clot to the liquefaction column CX1, centrifuge at 12,000 rpm (~11,500 g) for 1 min, and collect the filtrate (if the blood clot is large, it can be centrifuged through the column in batches multiple times to collect the filtrate).

[0038] d2) Add 100 μL - 1 mL of the filtrate to the cell lysate with a volume 1 - 2.5 times that of the blood sample, invert and mix well, centrifuge at 10,000 rpm (~11,500 xg) for 1 min, aspirate the supernatant, and leave the nuclear precipitate (if the lysis is incomplete, 1 - 2.5 times the volume of the cell lysate can be added to repeat the lysis once). Add 200 μL of buffer to the collected nuclear precipitate, shake until thoroughly mixed, and then proceed to the next experiment.

[0039] (2) Add a premixed solution of 200 μL of buffer GB and 20 μL of Proteinase K to the 200 μL of the sample obtained from the above treatment, invert and mix well thoroughly, incubate at 56 °C for 10 min, and invert and mix several times during this period. The solution should become clear (if the solution does not become completely clear, extend the lysis time until the solution is clear).

[0040] (3) After standing at room temperature for 2 - 5 min, add 350 μL of buffer BD, invert and mix well thoroughly. At this time, flocculent precipitates may appear.

[0041] (4) Add the solution and the flocculent precipitate obtained in the previous step to an adsorption column CG2 (the adsorption column CG2 is placed in a collection tube), centrifuge at 12,000 rpm (~13,400 g) for 30 sec, pour out the waste liquid in the collection tube, and place the adsorption column CG2 in the collection tube.

[0042] (5) Add 500 μL of buffer to the adsorption column CG2, centrifuge at 12,000 rpm (~13,400 g) for 30 sec, pour out the waste liquid in the collection tube, and place the adsorption column CG2 in the collection tube.

[0043] (6) Add 600 μL of the washing solution (containing absolute ethanol) to the adsorption column CG2, centrifuge at 12,000 rpm (~13,400 g) for 30 sec, pour out the waste liquid in the collection tube, and place the adsorption column CG2 in the collection tube.

[0044] (7) Repeat operation step 6.

[0045] Note: If the blood sample has been treated with the cell lysate, step (7) can be omitted.

[0046] (8) Centrifuge at 12,000 rpm (~13,400 g) for 2 min, pour out the waste liquid. Place the adsorption column CG2 at room temperature for 2 min to thoroughly dry the residual washing solution in the adsorption material.

[0047] (9) Transfer the adsorption column CG2 into a 1.5 mL centrifuge tube. Drop 50 - 200 μL of elution buffer onto the middle of the adsorption membrane in a suspended manner. Let it stand at room temperature for 2 min, then centrifuge at 12,000 rpm (~13,400 g) for 2 min, and collect the solution into the centrifuge tube.

[0048] Note: The volume of the elution buffer should not be less than 50 μL. Too small a volume will affect the recovery efficiency. To increase the yield of genomic DNA, the solution obtained by centrifugation can be added back to the adsorption column CG2, let it stand at room temperature for 2 min, and then centrifuge at 12,000 rpm (~13,400 g) for 2 min. The pH of the eluent has a great impact on the elution efficiency. If ddH2O is used as the eluent, its pH value should be ensured to be within the range of 7.0 - 8.5. A pH value lower than 7.0 will reduce the elution efficiency; and the DNA product should be stored at -20 °C to prevent DNA degradation.

[0049] 3. Primers and Probes

[0050] Use NCBI (National Center for Biotechnology Information) to retrieve the Diego (Dia / Dib) blood group gene sequence. According to the gene sequence, design forward (Diego FP) and reverse primers (Diego FP), as well as probes (Dia-probe and Dib-probe) that can distinguish single nucleotide polymorphisms in these regions. Use Primer 5 software to design and synthesize the primers and probes. All primers and probes are synthesized by IDT company, and the working concentration of the primers and probes is 10 μM. Among them, the 5' end of the Dia-probe is labeled with the fluorescent group 6-FAM, and the 3' end is labeled with MGB; the 5' end of the Dib-probe is labeled with the fluorescent group 6-VIC, and the 3' end is labeled with MGB. The primers and probes used for digital PCR to detect blood types are shown in Table 1.

[0051] Table 1 Primers and Probes for Diego Blood Group Genotyping

[0052]

[0053] 4. Workflow and Detection Components of Digital PCR

[0054] The digital PCR reaction system is: 10 μL of digital PCR premix, 0.3 μL of 10 μM primer, 0.2 μL of 10 μM probe, 2 μL of ultrapure water, and 7 μL of DNA sample, with a final reaction volume of 20 μL.

[0055] The process of digital PCR amplification is as follows: set the thermal cycle with a heating rate of 2°C / s per step, run at 95°C for 10 minutes; then denaturation at 94°C for 30 seconds, annealing and extension at 60°C for 30 seconds, 40 cycles; enzyme inactivation at 98°C for 10 minutes; and finally maintain at 4°C.

[0056] 5. Data Analysis Methods

[0057] All digital PCR data were generated using dPCRAnalyzer software from Beijing Xinyi Company. Single-channel and dual-channel experiments were analyzed using the same software. SPSS (25.0) statistical analysis software was used to establish a database for data processing, and one-way ANOVA was used for inter-group analysis, and Dunnett's post-test was used to evaluate significant differences. The comparison of rates was performed using the χ2 test, and P < 0.05 was considered statistically significant.

[0058] 6. Experimental Results

[0059] (1) Specificity verification of digital PCR

[0060] Blood donors with Di(a-b+) and Di(a+b-) phenotypes were screened to evaluate the specificity of the probe. The annealing temperature and probe sequence were optimized to make the negative and positive droplets of the fluorescent droplet clusters generated by the digital PCR instrument clearly separated and without cross-reaction. The results showed that the Di(a+b-) sample only bound to the corresponding probe of Dia, and a positive signal was observed in the FAM channel ( Figure 1 A); Di(a-b+) samples only bind to the Dib corresponding probe, and a positive signal is observed in the VIC channel ( Figure 1 B). No cross-reactivity was detected between the two probe sets and non-correlated samples, demonstrating that the probes are highly specific.

[0061] (2) Using dual-channel digital PCR for Diego blood type genotyping

[0062] The two sets of probes Dia and Dib and the Di(a+b+) sample were simultaneously put into the reaction system for double PCR detection. The amplified products of the two templates could specifically bind to their corresponding probes and were detected by FAM and VIC fluorescence, respectively. There was no positive band in the NC group, indicating that the two templates and the two probes did not interfere with each other during double PCR. Diego samples with different blood types are conducive to locating different droplet clusters. In multiple experiments, the position of the target droplet cluster remained unchanged.

[0063] (3) Using dual-channel digital PCR to screen Diego blood type population frequency

[0064] Using dual-channel digital PCR, 50 blood samples randomly selected from 1000 blood donors were tested in 20 blood pools. Two pairs of probes were used to identify the Dia antigen and Dib antigen in the Diego blood group ( Figure 3 ), and compared with the distribution of Dia and Dib antigen positives in the population in previous literature. The frequency ratios of Di(a+b-), Di(a+b+), and Di(a-b+) phenotypes detected by digital PCR were: 0.281%: 6.972%: 92.747%. The Dia antigen and Dib antigen were 7.253% and 99.719% for Dia and Dib respectively, as shown in Table 2. The experimental results showed that the results of this experiment were consistent with the distribution of 5% positive Dia antigen and 99% positive Dib antigen in the population, and the results among the 20 groups were similar with good repeatability. It was confirmed that the method was successfully established and the results were accurate and reliable.

[0065] Table 2 Frequencies of Diego blood group detected by digital PCR in 20 blood pools

[0066]

[0067] Obviously, the above embodiments are merely examples for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. The obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A multiplex digital PCR probe primer set for Diego blood group detection, characterized in that, It includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1, a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2, a Dia probe with a nucleotide sequence as shown in SEQ ID NO.3, and a Dib probe with a nucleotide sequence as shown in SEQ ID NO.

4. One end of the Dia probe and the Dib probe is labeled with a fluorophore, and the other end is labeled with a quencher group, and the fluorophore labeled on the Dia probe is different from the fluorophore labeled on the Dib probe.

2. The multiplex digital PCR probe primer set according to claim 1, wherein The fluorophore is selected from FAM or VIC; The quencher group is selected from MGB.

3. The multiplex digital PCR probe primer set according to claim 1, characterized in that, The fluorophore labeled on the Dia probe is FAM, and the fluorophore labeled on the Dib probe is VIC.

4. The multiplex digital PCR probe primer set according to claim 1, wherein The 5'-end of the Dia probe and the Dib probe is labeled with a fluorophore, and the 3'-end is labeled with a quencher group.

5. A kit for Diego blood group detection, characterized in that, It includes the multiplex digital PCR probe primer set according to any one of claims 1-4.

6. The kit according to claim 5, wherein The kit further includes a blood genomic DNA extraction reagent, a digital PCR premix, and ultrapure water.

7. The kit according to claim 5, wherein The kit further includes a positive control and a negative control.

8. Use of the multiplex digital PCR probe primer set according to claim 1 or the kit according to claim 5 in the preparation of a Diego blood group detection kit.

9. A method for using the kit according to claim 5, characterized in that, It includes the following steps: (1) Extract genomic DNA from peripheral blood; (2) Prepare a digital PCR reaction system: 10 μL of digital PCR premix, 0.3 μL each of 10 μM upstream primer and downstream primer, 0.2 μL each of 10 μM Dia probe and Dib probe, 2 μL of ultrapure water, and 7 μL of DNA sample; (3) Perform dual-channel digital PCR amplification. The amplification process is as follows: Set the heating rate to 2 °C / s per step for thermal cycling, and run at 95 °C for 10 minutes; then denature at 94 °C for 30 seconds, anneal and extend at 60 °C for 30 seconds for 40 cycles; inactivate the enzyme at 98 °C for 10 minutes; finally hold at 4 °C; (4) Perform model interpretation to determine the Diego blood group.

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