MNSs blood group rapid detection method based on multiple digital PCR and special probe primer group

Abstract

This invention discloses a rapid detection method for MNSs blood typing based on multiplex digital PCR and a dedicated probe and primer set, belonging to the field of clinical laboratory medicine and molecular diagnostics technology. The method involves mixing a PCR reaction mix, the sample to be tested, enzyme-free purified water, M / N upstream primer, M / N downstream primer, S / s upstream primer, S / s downstream primer, upstream internal control primer, downstream internal control primer, M probe, N probe, S probe, s probe, and internal control probe to obtain a dPCR reaction system. This dPCR reaction system is then added to a microfluidic chip to generate ≥30,000 homogeneous water-in-oil droplets, followed by PCR amplification. The fluorescence signal is then detected using a droplet analyzer, achieving accurate and efficient detection of MNSs blood typing. This method addresses the shortcomings of existing molecular biological detection methods for MNSs blood typing, such as insufficient specificity, sensitivity, and throughput, as well as complex operation and high cost, providing reliable technical support for clinical transfusion safety.

Description

Technical Field

[0001] This invention belongs to the field of clinical laboratory medicine and molecular diagnostics technology, specifically relating to a rapid detection method for MNSs blood types based on multiplex digital PCR and a dedicated probe and primer set. Background Technology

[0002] The human MNSs blood group system is one of the important blood group systems recognized by the International Society of Blood Transfusion (ISBT). Its antigens are encoded by the glycoprotein A (GPA) and glycoprotein B (GPB) genes located on the long arm of chromosome 4 (4q28.2-q31.1). This system exhibits complex antigenic polymorphism, with core antigens including M, N, S, and s, in addition to dozens of rare antigen variants. The difference between M and N antigens depends on the difference in the first and fifth amino acids at the N-terminus of the GPA peptide chain (serine and glycine for M; leucine and glutamic acid for N), determined by single nucleotide polymorphisms (SNPs) on the GYPA gene (encoding GPA). The difference between S and s antigens depends on the difference in the 29th amino acid of the GPB peptide chain (methionine for S; threonine for s), determined by a single base mutation on the GYPB gene (encoding GPB). The GYPA and GYPB genes share approximately 95% high homology, posing a significant challenge to establishing specific detection methods. This blood group system has extremely important clinical and academic value. In the field of clinical blood transfusion, if the recipient has antibodies against MNSs antigens on the donor's red blood cells (such as anti-M, anti-N, anti-S, and anti-s), the transfusion of incompatible blood can trigger acute or delayed hemolytic transfusion reactions, which can lead to disseminated intravascular coagulation, acute renal failure, and even death in severe cases. During pregnancy, if the mother and fetus have incompatible MNSs blood types, the IgG antibodies produced by the mother (especially anti-S and anti-s) can cross the placental barrier and attack the fetal red blood cells, causing hemolytic disease of the fetus and newborn (HDFN). This can lead to anemia, edema, and hepatosplenomegaly in the fetus, and severe jaundice and bilirubin encephalopathy after birth, posing a significant threat to the health and survival of perinatal infants. In addition, the MNSs blood type gene exhibits a characteristic distribution frequency in different ethnic and regional populations, making it an important genetic marker for human genetics, forensic individual identification, and the study of population migration history.

[0003] Currently, methods for detecting MNSs blood types are mainly divided into two categories: serological testing and molecular biological testing. Serological testing is a traditional method that uses indirect antiglobulin tests or direct agglutination tests to determine phenotype by utilizing the binding of anti-M, anti-N, anti-S, and anti-S monoclonal antibodies to erythrocyte surface antigens. However, this method has significant technical bottlenecks: First, the supply of specific antibodies is limited, especially anti-S and anti-S antibodies, which are difficult to prepare and have poor stability, leading to antibody shortages in some laboratories; second, the detection of rare phenotypes (such as MN-) depends on specific antibody combinations, and conventional serological methods are prone to false negatives or misdiagnoses; third, the detection sensitivity is low, making it difficult to obtain reliable results for trace samples (such as trace amounts of peripheral blood) or samples with poor preservation conditions; fourth, it cannot distinguish the correspondence between genotype and phenotype, and the accuracy in determining weakly expressed antigens or mixed blood type samples is insufficient. To overcome the shortcomings of serological testing, molecular biological detection methods have emerged, mainly including sequence-specific primer polymerase chain reaction (PCR-SSP), restriction fragment length polymorphism (PCR-RFLP), and real-time quantitative PCR (qPCR). Among these, PCR-SSP achieves genotyping by designing specific primers targeting different antigen-encoding genes; however, this method requires multiple independent reactions, resulting in low throughput, cumbersome operation, and a high risk of false positives due to primer cross-reactions. This is particularly challenging for genes like GPA and GPB, which exhibit highly homologous sequences, making primer design extremely difficult and prone to non-specific amplification. PCR-RFLP relies on restriction endonuclease digestion, which is cumbersome, time-consuming, and its digestion efficiency is easily affected by experimental conditions, leading to poor reproducibility. While real-time quantitative PCR can achieve multiplex detection in a single tube, it relies on standard curves for relative quantification, and its accuracy is significantly affected by template quality and amplification efficiency. Furthermore, it lacks sufficient sensitivity for low-abundance targets, failing to meet the needs of detecting trace samples. In summary, existing MNSs blood typing technologies have significant shortcomings in terms of specificity, sensitivity, throughput, ease of operation, and cost-effectiveness, making it difficult to meet the growing demands for rapid, accurate, high-throughput, and automated testing in current clinical applications such as precision blood transfusions, high-risk pregnancy monitoring, and large-scale population blood typing. Therefore, there is an urgent need for a highly specific, highly sensitive, rapid, and convenient MNSs blood typing method. Summary of the Invention

[0004] In order to overcome the shortcomings of the prior art, the present invention aims to provide a rapid detection method for MNSs blood type based on multiplex digital PCR and a dedicated probe and primer set, so as to solve the technical problems that the existing MNSs blood type molecular biology detection methods are insufficient in specificity, sensitivity and throughput, and are complicated and costly.

[0005] To achieve the above objectives, the present invention employs the following technical solution: The first aspect of this invention discloses a dedicated probe primer set for rapid detection of MNSs blood types, comprising a dedicated primer pair and a probe composition. The dedicated primer pair comprises an M / N upstream primer, an M / N downstream primer, an S / s upstream primer, an S / s downstream primer, an upstream internal control primer, and a downstream internal control primer. The probe composition comprises an M probe, an N probe, an S probe, an s probe, and an internal control probe. The nucleotide sequences of the M / N upstream primer are shown in SEQ ID NO.1, the M / N downstream primer in SEQ ID NO.2, the S / s upstream primer in SEQ ID NO.3, the S / s downstream primer in SEQ ID NO.4, the upstream internal control primer in SEQ ID NO.5, and the downstream internal control primer in SEQ ID NO.6; the nucleotide sequence of the M probe in SEQ ID NO.7, the N probe in SEQ ID NO.8, the S probe in SEQ ID NO.9, the s probe in SEQ ID NO.10, and the internal control probe in SEQ ID NO.11 are shown.

[0006] Preferably, the M probe, N probe, S probe, s probe and internal control probe are labeled with modifying groups that exhibit different fluorescent colors.

[0007] More preferably, the modifying group includes a fluorescent group and a quenching group; the fluorescent group includes at least one of Cy5 fluorescent group, VIC fluorescent group, FAM fluorescent group, ROX fluorescent group and Q705 fluorescent group; the quenching group includes at least one of BHQ2 quenching group, BHQ3 quenching group and MGB quenching group.

[0008] A second aspect of the present invention discloses a kit for rapid detection of MNSs blood types, comprising the aforementioned dedicated probe and primer set for rapid detection of MNSs blood types.

[0009] Preferably, it also includes a PCR reaction mix and enzyme-free pure water.

[0010] Preferably, the concentrations of the M / N upstream primer, M / N downstream primer, S / s upstream primer, S / s downstream primer, upstream internal control primer, downstream internal control primer, M probe, N probe, S probe, s probe, and internal control probe are all 10 μM.

[0011] A third aspect of the present invention discloses the application of the above-mentioned dedicated probe and primer set for rapid MNSs blood typing or the kit for rapid MNSs blood typing in MNSs blood typing.

[0012] A fourth aspect of the present invention discloses a rapid blood typing method for MNSs based on multiplex digital PCR, comprising the following steps: 1) Using the DNA sample to be tested as a DNA template, the PCR reaction mix, DNA template, enzyme-free pure water, M / N upstream primer, M / N downstream primer, S / s upstream primer, S / s downstream primer, upstream internal control primer, downstream internal control primer, M probe, N probe, S probe, s probe and internal control probe are mixed in a volume ratio of 15:4:15:3:3:3:3:3:3:3:3:3:3:3:3 to obtain the dPCR reaction system; 2) The dPCR reaction system obtained in step 1) was added to a microfluidic chip to generate ≥30,000 homogeneous water-in-oil droplets. After PCR amplification, the signals of different fluorescence channels were detected using a droplet analyzer. 3) By statistically analyzing the positive droplets, negative droplets, and total droplets corresponding to each target gene in the droplet reaction system, combined with the Poisson distribution model, rapid detection of MNSs blood types can be achieved.

[0013] Preferably, in the dPCR reaction system, the final concentrations of the M / N upstream primer, M / N downstream primer, S / s upstream primer, S / s downstream primer, upstream internal control primer, and downstream internal control primer are all 300 nM; the final concentrations of the M probe, N probe, S probe, s probe, and internal control probe are all 266 nM.

[0014] Preferably, the PCR amplification program is as follows: pre-denaturation at 95°C for 30 seconds, followed by 40 cycles, each cycle including denaturation at 94°C for 10 seconds and optimized annealing extension for 30 seconds.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The invention provides a dedicated probe and primer set for rapid MNSs blood typing. 1) Targeting the key sequence differences between the M / N antigen (specific polymorphic site of the GYPA gene) and the S / s antigen (specific polymorphic site of the GYPB gene) in the MNSs blood typing system, highly specific sequence recognition primers (M / N site primer pair, located in the GYPA gene region; S / s site primer pair, located in the GYPB gene region) and fluorescently labeled probes (M probe, N probe, S probe, s probe) are designed to ensure specific binding only to the target genotype, while avoiding cross-reaction with other human genes and GYPA / GYPB homologous sequences, thus guaranteeing specificity; 2) The designed internal control primer pair is located in the GAPDH gene region, which can monitor the effectiveness of the reaction system. Therefore, this dedicated probe primer set has extremely high specificity, capable of specifically identifying and distinguishing the sites on the GYPA gene that determine the M / N antigen and the sites on the GYPB gene that determine the S / s antigen. It enables simultaneous and accurate typing of four targets (M, N, S, and s) within the same reaction system, avoiding cross-reactions. It is used for rapid detection of MNSs blood types, with high sensitivity, strong specificity, high throughput, simple operation, and accurate results, providing a reliable means for clinical transfusion compatibility testing and diagnosis of blood type-related diseases.

[0016] The rapid MNSs blood group detection kit provided by this invention is a detection system that enables single-tube, rapid, accurate, and absolute quantitative detection of the MNSs blood group system core antigens M, N, S, and s corresponding genotypes.

[0017] This invention provides a rapid MNSs blood typing method based on multiplex digital PCR. Targeting the specific coding sequences of the GYPA gene (determining M / N antigens) and GYPB gene (determining S / s antigens) in the MNSs blood typing system, this method integrates the principles of PCR-SSP (Sequence Specific Primer) with the advantages of single-molecule detection in digital PCR (dPCR) on the basis of a dedicated probe and primer set for rapid MNSs blood typing. This method achieves the following: 1) By detecting the endpoint fluorescence signals of positive, negative, and total droplets corresponding to the target genes, the absolute copy number of the target molecule in the original sample can be directly calculated using the Poisson distribution statistical principle, without relying on a standard curve. This method offers advantages such as absolute quantification, extremely high sensitivity, strong anti-interference ability, and good repeatability, enabling automated and standardized genotype reporting; 2) It can simultaneously and evenly detect a stable system of the four core antigen-determining genes (M, N, S, and s) and necessary internal reference genes using different fluorescence channels in a single-tube reaction, greatly improving detection efficiency and cost-effectiveness. By using specially designed probe primer sets combined with dPCR technology for rapid detection of MNSs blood types, interference such as primer dimers and non-specific binding can be effectively avoided. This achieves the goal of accurately detecting human MNSs blood type genotypes and avoiding false positives and false negatives. It enables precise and efficient detection of human MNSs blood type genotypes, solving the problems of insufficient specificity, sensitivity, and throughput, as well as the complexity and high cost of existing molecular biology detection methods for MNSs blood types. This provides reliable technical support for clinical transfusion safety.

[0018] Furthermore, the optimal volume and concentration ratio of the primer-probe mixture system were determined through gradient experiments to reduce primer dimers and mutual interference; the optimized droplet generation conditions of the digital PCR reaction parameters ensured uniformity and stability, effectively inhibiting non-specific amplification. Attached Figure Description

[0019] Figure 1 This is a graph showing the 1D amplitude dimension distribution characteristics of droplets in MNSs blood type samples (M+N+S+s+) detected by the rapid MNSs blood type detection method based on multiplex digital PCR of the present invention; where the vertical axis is the fluorescence intensity of the sample to be tested, and the horizontal axis is the number of droplets. Figure 2 The image shows the 1D amplitude dimension distribution characteristics of droplets in MNSs blood type samples (M+NS-s+) detected by the rapid MNSs blood type detection method based on multiplex digital PCR of the present invention; where the vertical axis represents the fluorescence intensity of the sample to be tested, and the horizontal axis represents the number of droplets. Figure 3The image shows the 1D amplitude dimension distribution characteristics of droplets in MNSs blood type samples (M+N-S+s+) detected by the rapid MNSs blood type detection method based on multiplex digital PCR of the present invention; where the vertical axis represents the fluorescence intensity of the sample to be tested, and the horizontal axis represents the number of droplets. Figure 4 The image shows the 1D amplitude dimension distribution characteristics of droplets in MNSs blood type samples (M-N+S-s+) detected by the rapid MNSs blood type detection method based on multiplex digital PCR of the present invention; where the vertical axis represents the fluorescence intensity of the sample to be tested, and the horizontal axis represents the number of droplets. Figure 5 This is a graph showing the 1D amplitude dimension distribution characteristics of droplets in MNSs blood type samples (M+N+S-s+) detected by the rapid MNSs blood type detection method based on multiplex digital PCR of the present invention; where the vertical axis is the fluorescence intensity of the sample to be tested and the horizontal axis is the number of droplets. Detailed Implementation

[0020] To enable those skilled in the art to understand the features and effects of the present invention, the following description and definitions are only general descriptions of the terms and expressions mentioned in the specification. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in case of conflict, the definitions in this specification shall prevail.

[0021] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0022] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0023] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0024] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0025] This invention provides a dedicated probe and primer set for rapid detection of MNSs blood types. The design references the GYPA and GYPB gene sequences in the National Center for Biotechnology Information (NCBI) database and targets key polymorphic sites of the M / N and S / s antigens. The detection system includes three pairs of site-specific primers (M / N, S / s, and internal control upstream and downstream primers) and five fluorescently labeled probes (corresponding to M, N, S, and s antigens and the internal control, respectively, distinguished by different fluorescent colors). The probes can be modified with locked nucleic acids to enhance binding specificity as needed. The internal control primers and probes are designed according to international standards, as detailed below: 1. M / N site primer pair: The nucleotide sequence of the upstream M / N primer is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream M / N primer is shown in SEQ ID NO.2. The upstream and downstream M / N primers are located in the GYPA gene region. 2. S / s site primer pair: The nucleotide sequence of the upstream S / s primer is shown in SEQ ID NO.3, and the nucleotide sequence of the downstream S / s primer is shown in SEQ ID NO.4. The upstream and downstream S / s primers are located in the GYPB gene region. 3. Internal control primer pair: The nucleotide sequence of the upstream internal control primer is shown in SEQ ID NO.5, and the nucleotide sequence of the downstream internal control primer is shown in SEQ ID NO.6. The upstream and downstream primers of the internal control are located in the GAPDH gene region. The GAPDH gene is stably expressed in various tissues and can be used to monitor the effectiveness of the reaction system.

[0026] Table 1 Nucleotide Sequence List

[0027] 4. Probe composition: including specific probes targeting M, N, S, and s antigen sites and internal control probes; The M probe is a ROX-labeled probe, with the nucleotide sequence shown in SEQ ID NO.7, wherein the 5' end of the nucleotide sequence is attached to a ROX fluorescent group and the 3' end is attached to a BHQ2 quencher group; the N probe is a Cy5-labeled probe, with the nucleotide sequence shown in SEQ ID NO.8, wherein the 5' end of the nucleotide sequence is attached to a Cy5 fluorescent group and the 3' end is attached to a BHQ3 quencher group; the S probe is a FAM-labeled probe, with the nucleotide sequence shown in SEQ ID NO.9, wherein the 5' end of the nucleotide sequence is attached to a FAM fluorescent group and the 3' end is attached to an MGB quencher group; the s probe is a VIC-labeled probe, with the nucleotide sequence shown in SEQ ID NO.10, wherein the 5' end of the nucleotide sequence is attached to a VIC fluorescent group and the 3' end is attached to an MGB quencher group; and the internal control probe is a Q705-labeled probe, with the nucleotide sequence shown in SEQ ID NO.11, wherein the 5' end of the nucleotide sequence is attached to a Q705 fluorescent group and the 3' end is attached to a BHQ3 quencher group.

[0028] The primers and compositions described above have been validated using standard BLAST tools. Each primer and probe binds only to its corresponding target gene and does not cross-react with other human genes or GYPA / GYPB homologous sequences, thus ensuring specificity at the design level.

[0029] The present invention also provides a rapid test kit for MNSs blood typing, comprising 7.5 μL PCR reaction mix, 7.5 μL enzyme-free purified water, 9 μL dedicated primer pair and 7.5 μL probe composition; The dedicated primer pairs include M / N upstream primers, M / N downstream primers, S / s upstream primers, S / s downstream primers, upstream internal control primers, and downstream internal control primers, with each primer added in a volume of 1.5 μL; the probe composition includes M probes, N probes, S probes, s probes, and internal control probes, with each probe added in a volume of 0.8 μL.

[0030] This invention also provides a rapid blood typing method for MNSs based on multiplex digital PCR, comprising the following steps: 1. Mix equal volumes of M / N upstream primer, M / N downstream primer, S / s upstream primer, S / s downstream primer, upstream internal control primer, downstream internal control primer, M probe, N probe, S probe, s probe and internal control probe, and disperse them into tens of thousands of uniform independent microdroplets using droplet generation technology. After PCR amplification, detect the signals of different fluorescence channels using a droplet analyzer. 2. By statistically analyzing the positive droplets, negative droplets, and total droplets corresponding to each target gene in the droplet reaction system, and combining this with the Poisson distribution model, simultaneous quantitative and genotyping screening of multiple target genes can be achieved.

[0031] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading this description, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined in this application.

[0032] In the following examples, the samples used for testing were whole blood from 45 healthy blood donors at the Shaanxi Provincial Blood Center. All samples were serologically typed, and sample collection was approved by the ethics committee. Informed consent was obtained from the subjects, and the samples were anonymized. The serological results were interpreted in a blinded manner by the testing personnel. The main reagents and instruments used in the following examples include: 4 ProbedPCR Prolix (containing UNG enzyme) (purchased from Beijing Xinyi Biotechnology Co., Ltd., batch number: 20250601); sealing oil (purchased from Beijing Xinyi Biotechnology Co., Ltd., batch number: 20250701); high-purity nucleic acid purification kit (purchased from Roche Diagnostics, batch number: 11796828001); microfluidic chip (purchased from Beijing Xinyi Biotechnology Co., Ltd., batch number: 20250201); fully automated digital PCR platform (purchased from Beijing Xinyi Biotechnology Co., Ltd.); all primers and probes used (purity ≥99%) were synthesized by Jiangsu Genscript Biotechnology Co., Ltd.; enzyme-free purified water, proteinase K, etc. were all of analytical grade; the human GAPDH gene was used as the internal control gene to monitor the DNA extraction quality and the effectiveness of the PCR reaction. Other raw materials or instruments used, unless otherwise specified, were all commercially available products or instruments with specifications in accordance with the art. Experimental methods in the following examples that do not specify specific conditions were generally performed under conventional conditions or according to the manufacturer's recommendations.

[0033] I. Rapid Blood Typing Method for MNSs Based on Multiplex Digital PCR 1. Sample processing Forty-five whole blood samples were collected clinically. DNA was extracted using a high-purity nucleic acid purification kit and detected by a Nanodrop spectrophotometer. Samples with an A260 / A280 ratio of 1.8 to 2.0 were stored at -20°C for later use.

[0034] 2. Preparation of dPCR reaction system Referring to Table 2, prepare 30 μL of the corresponding dPCR reaction system according to the experimental groups described below.

[0035] Experimental group: The DNA sample obtained in step 1 was used as the DNA template; Positive control group: using MNSs standard DNA samples as DNA templates; Negative control group: using antigen-negative standard DNA samples as DNA templates; Blank control group: DEPC water was used instead of DNA template.

[0036] Table 2 dPCR reaction system

[0037] 3. Rapid MNSs blood typing based on multiplex digital PCR 1) Add each dPCR reaction system (30 μL) obtained in step 2 to the corresponding reaction well of the microfluidic chip, avoiding the generation of air bubbles; 2) PCR amplification: The microfluidic chip with the sample added is placed into a fully automated digital PCR instrument to generate ≥30,000 homogeneous water-in-oil droplets. The droplet volume is controlled at 0.5 nL, and the coefficient of variation of the droplet homogeneity is ≤5%, ensuring that each droplet is an independent PCR reaction unit. The PCR amplification program is set as follows: 95℃ pre-denaturation for 30 seconds; followed by 40 cycles, each cycle including 94℃ denaturation for 10 seconds and optimized annealing extension for 30 seconds. 3) Fluorescence detection: After the amplification reaction, the fluorescence signal of each droplet was detected by a photomultiplier tube (PMT). The detection channels were set as follows: the fluorescence channels corresponding to the detection probes (FAM channel, excitation wavelength 492 nm, emission wavelength 517 nm; VIC channel, excitation wavelength 538 nm, emission wavelength 554 nm; ROX channel, excitation wavelength 575 nm, emission wavelength 602 nm; cy5 channel, excitation wavelength 646 nm, emission wavelength 664 nm) and the fluorescence channels corresponding to the internal control probes (Q705 channel, excitation wavelength 673 nm, emission wavelength 692 nm). 4) Data Analysis: After fluorescence signal detection, data analysis was performed using dedicated digital PCR analysis software. The highest point of fluorescence amplitude in the negative droplet cluster (containing no sample template, with all other reaction components and procedures identical to the sample) was used as the threshold. Droplets above this threshold were considered positive droplets, and droplets below this threshold were considered negative droplets. Simultaneously, the number of effective droplets in each reaction well was counted, requiring a minimum of 30,000 effective droplets. If the number of effective droplets was less than 30,000, the sample test result was considered invalid, and the reaction system needed to be prepared again and the above steps repeated. 5) For valid detection wells, the target gene copy number was calculated using the Poisson distribution formula. The Poisson distribution calculation parameters were set as follows: reaction system volume 20 μL, droplet volume 0.5 nL (consistent with the actual generated droplet volume). The copy number calculation result was retained to two decimal places, with units of copies / μL. Simultaneously, the sample validity was determined in conjunction with the amplification status of the internal reference gene: if the number of positive droplets of the internal reference gene was ≥ 1% of the total valid droplets, and the copy number of the internal reference gene was within the preset normal range (preset range is 100~300000 copies / μL, determined according to the type of internal reference gene and sample source), then the sample was considered valid, and the target gene copy number calculation result was reliable; if the internal reference gene was not amplified or the copy number exceeded the preset normal range, then the sample was considered invalid, and the sample template needed to be extracted again and the entire detection process repeated. After the experiment, the droplet generation was confirmed to be qualified through quality control documents.

[0038] Table 3. Precision results of digital PCR for different MNSs blood type samples

[0039] Test results as follows Figures 1-5 As shown, all MNSs blood type samples showed signals in the MN detection channels, with no instances of completely no signal in any channel. The Q705 channel was consistently detected in all samples and can be used as an internal control to correct for total droplet count and amplification efficiency. MN typing is interpreted based on the distribution of specific fluorescence signals and the characteristic copy number ratio (rather than simply the presence or absence of specific fluorescence signals). The specific detection characteristics of each blood type sample are as follows: M+N+S+s+ samples: ROX, cy5, FAM, VIC, and Q705 channels all showed positive fluorescence signals, and the signal intensity and copy number were consistent with positive characteristics. The M / N channel copy number ratio was approximately 1:1. Figure 1 (Table 3); M+N S s+ samples: ROX, VIC, and Q705 channels showed positive fluorescence signals with copy numbers consistent with positive characteristics; FAM channel showed negative fluorescence signals; cy5 channel showed signal, but copy number consistent with negative characteristics; M / N channel copy number ratio was approximately 2:1. Figure 2 (Table 3); M+N S+s+ samples: ROX, FAM, VIC, and Q705 channels showed positive fluorescence signals and copy numbers consistent with positive characteristics; although the cy5 channel showed signal, the copy number was consistent with negative characteristics, and the M / N channel copy number ratio was approximately 2:1. Figure 3 (Table 3); M N+S s+ samples: cy5, VIC, and Q705 channels showed positive fluorescence signals with copy numbers consistent with positive characteristics; the FAM channel showed negative fluorescence signals; the ROX channel showed signal, but the copy number was consistent with negative characteristics; the M / N channel copy number ratio was approximately 1:2. Figure 4 (Table 3); M+N+S s+ samples: ROX, cy5, VIC, and Q705 channels showed positive fluorescence signals with copy numbers consistent with positive characteristics; the FAM channel showed negative fluorescence signals; the M / N channel copy number ratio was approximately 1:1. Figure 5 (Table 3). In the S / s genotyping, the copy number of the S site in S-negative samples is 0, which clearly distinguishes S+ / S+ samples. Phenotypic; In the M / N genotyping, the M / N channel copy number ratio of different phenotypic samples shows a characteristic distribution, among which M+N The sample channel copy number ratio is approximately 2:1, M The copy number ratio of the N+ sample channel is approximately 1:2, and the copy number ratio of the M+N+ sample channel is approximately 1:1. Combining the 1D fluorescence amplification pattern and the copy number ratio, MNSs blood types can be accurately distinguished based on the copy number ratio characteristics of each channel, indicating that this digital PCR system has good specificity, stability, and typing accuracy, and the typing results are stable and reliable.

[0040] II. Method Evaluation 1. Repeatability evaluation Three repeated tests were performed on different MNSs blood type samples (sample 4 M-N+S-s+, sample 12 M+NS-s+, and sample 40 M+N-S+s+) (using the same testing method as in step one). The copy number of each fluorescence channel (FAM, VIC, ROX, cy5, Q705) was recorded, and the mean, standard deviation (SD), and coefficient of variation (CV) of each indicator were calculated. The ratio of ROX copy number to cy5 copy number and the corresponding mean and standard deviation were also calculated. Since the FAM copy number was 0, the mean, standard deviation, and CV were not calculated.

[0041] Table 4. Results of digital PCR precision repeatability experiments for different MNSs blood type samples

[0042] The results of the precision and repeatability experiment of digital PCR detection are shown in Table 4. The Q705 channel, as the internal reference channel, showed a low CV value in all three samples. The CV values ​​of each detection index were generally at a low level, indicating that the precision and repeatability of the digital PCR system were good and the detection results were stable and reliable.

[0043] 2. Accuracy Evaluation Using serological results as the gold standard, the results of each antigen test were matched with the results of multiplex digital PCR, further confirming the accuracy of the test.

[0044] This study included 45 samples for testing. Blood typing was performed using a rapid MNSs blood typing method based on multiplex digital PCR (the dPCR reaction system construction method is the same as in step one) and a serological method (following the instructions for anti-M monoclonal antibody and anti-N monoclonal antibody reagents produced by Shanghai Blood Biopharmaceutical Co., Ltd., and anti-S monoclonal antibody and anti-S monoclonal antibody reagents produced by CE-Immundiagnostika GmbH).

[0045] Table 5. Predicted phenotypes from serological and digital PCR detection in 45 selected samples.

[0046] The test results are shown in Table 5. The test results of 45 different MNSs blood type samples are completely consistent with the test results of the traditional gold standard method (serological blood typing), which further proves the accuracy and reliability of this method.

[0047] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of this invention.

Claims

1. A special probe primer set for rapid detection of MNSs blood group, characterized in that, It includes dedicated primer pairs and probe compositions. The dedicated primer pairs include M / N upstream primers, M / N downstream primers, S / s upstream primers, S / s downstream primers, upstream internal control primers, and downstream internal control primers. The probe compositions include M probes, N probes, S probes, s probes, and internal control probes. The nucleotide sequences of the M / N upstream primer are shown in SEQ ID NO.1, the M / N downstream primer in SEQ ID NO.2, the S / s upstream primer in SEQ ID NO.3, the S / s downstream primer in SEQ ID NO.4, the upstream internal control primer in SEQ ID NO.5, and the downstream internal control primer in SEQ ID NO.6; the nucleotide sequence of the M probe in SEQ ID NO.7, the N probe in SEQ ID NO.8, the S probe in SEQ ID NO.9, the s probe in SEQ ID NO.10, and the internal control probe in SEQ ID NO.11 are shown.

2. The dedicated probe and primer set for rapid MNSs blood typing according to claim 1, characterized in that, The M probe, N probe, S probe, s probe, and internal control probe were labeled with different fluorescent groups.

3. The dedicated probe and primer set for rapid MNSs blood typing according to claim 2, characterized in that, The modifying groups include fluorescent groups and quenching groups; the fluorescent groups include at least one of Cy5 fluorescent groups, VIC fluorescent groups, FAM fluorescent groups, ROX fluorescent groups and Q705 fluorescent groups; the quenching groups include at least one of BHQ2 quenching groups, BHQ3 quenching groups and MGB quenching groups.

4. A rapid test kit for MNSs blood typing, characterized in that, Includes the dedicated probe and primer set for rapid MNSs blood typing as described in any one of claims 1 to 3.

5. The rapid MNSs blood typing kit according to claim 4, characterized in that, It also includes PCR reaction mix and enzyme-free pure water.

6. The rapid MNSs blood typing kit according to claim 4, characterized in that, The concentrations of the M / N upstream primer, M / N downstream primer, S / s upstream primer, S / s downstream primer, upstream internal control primer, downstream internal control primer, M probe, N probe, S probe, s probe, and internal control probe were all 10 μM.

7. The application of the dedicated probe and primer set for rapid MNSs blood typing as described in claim 1 or the rapid MNSs blood typing kit as described in any one of claims 4 to 6 in MNSs blood typing.

8. A rapid blood typing method for MNSs based on multiplex digital PCR, characterized in that, Includes the following steps: 1) Using the DNA sample to be tested as a DNA template, the PCR reaction mix, DNA template, enzyme-free pure water, M / N upstream primer, M / N downstream primer, S / s upstream primer, S / s downstream primer, upstream internal control primer, downstream internal control primer, M probe, N probe, S probe, s probe and internal control probe are mixed in a volume ratio of 15:4:15:3:3:3:3:3:3:3:3:3:3:3:3 to obtain the dPCR reaction system; 2) Add the dPCR reaction system obtained in step 1) into the microfluidic chip, perform PCR amplification, and detect the fluorescence signal by microdroplets to realize MNSs blood type detection.

9. The rapid blood typing method for MNSs based on multiplex digital PCR according to claim 8, characterized in that, In the dPCR reaction system, the final concentrations of the M / N upstream primer, M / N downstream primer, S / s upstream primer, S / s downstream primer, upstream internal control primer, and downstream internal control primer were all 300 nM; the final concentrations of the M probe, N probe, S probe, s probe, and internal control probe were all 266 nM.

10. The rapid blood typing method for MNSs based on multiplex digital PCR according to claim 8, characterized in that, The PCR amplification program was as follows: pre-denaturation at 95°C for 30 seconds, followed by 40 cycles, each cycle consisting of denaturation at 94°C for 10 seconds and optimized annealing extension for 30 seconds.

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