A multiplex PCR detection system, kit and application for RH gene typing detection

Abstract

The application discloses a multiplex PCR detection system, a kit and application for RH gene typing detection. The application can efficiently and accurately identify common Rh genotypes in Asian population through a single PCR amplification reaction. The application not only effectively makes up for the limitations of traditional serological methods in detecting complex blood types such as D variant, but also has the advantages of high standardization, low cost, simple operation and low clinical promotion threshold.

Description

Technical Field

[0001] This invention relates to the field of gene detection, specifically to a multiplex PCR detection system, kit, and application for RH genotyping detection, and particularly to a multiplex PCR detection system, kit, and application for RH genotyping based on the melting curve of a fluorescent probe. Background Technology

[0002] The Rh blood group system is one of the most clinically significant blood group systems, renowned for its high complexity and strong immunogenicity. Its antigens are encoded by two highly homologous, tightly linked genes: the RHD gene and the RHCE gene. The RHD gene encodes the D antigen, and its presence or absence is crucial for determining RhD positivity or negativity. Most RhD-positive individuals possess both the RHD and RHCE genes, while the most common RhD-negative type is due to the complete deletion of the RHD gene. In Africa, RhD negativity is often caused by RHDψ. Furthermore, there are cases where RHD gene mutations, partial deletions, or rearrangements lead to weakened D antigen expression or altered antigenic epitopes; these are collectively referred to as D variants, mainly including partial D, weak D, and DEL types. Due to the limitations of traditional serological methods, this technique struggles to accurately distinguish between different types of weak D or partial D.

[0003] Among the Chinese population, RHD*01EL.01 (c.1227G>A) RHD * DⅥ and RHD * weak partial 15 The D variant is the most common allele. In particular, type DVI, as the most prevalent partial D variant in the Chinese population, accounts for a major proportion of partial D variants in Shanghai and Zhejiang. Studies have reported that over 30 D variants can induce anti-D antibodies, with types DIV and DVI having the most reported cases. Type DIV is mainly concentrated in Caucasian populations and some African populations, and is rare in East Asian populations; while type DVI has been recorded in multiple populations worldwide. DVI is further divided into four types based on the different regions where the RHD gene is replaced by the RHCE gene, all of which are RHD-CE-D hybrid genes. Therefore, accurate typing of individuals with type DVI is particularly important. Furthermore, although reports of certain variants (such as weak D15) producing anti-D antibodies in the Chinese population are rare, considering their population frequency and related cases abroad, they still require attention in clinical practice.

[0004] Traditional serological methods and some genotyping methods designed solely to detect the presence or absence of the RhD gene risk insufficient identification capability or misidentification when faced with RhD variants resulting from partial gene deletions, point mutations, etc. Therefore, developing detection technologies capable of comprehensively and accurately identifying RhD blood types and their variants is of great significance.

[0005] Currently, the conventional PCR-SSP method for RhD gene detection typically first determines the presence of the RhD gene, then amplifies only a subset of key exons to identify whether RhD exons have been replaced by RHCE exons. However, this strategy does not cover all 10 exons of the RhD gene. Relying solely on the amplification results of a single RhD gene fragment is highly susceptible to misdiagnosis or missed detection due to exon deletions, substitutions, or sequence variations.

[0006] The information in the background section is merely intended to illustrate the general background of the invention and should not be construed as an admission or implication in any way that such information constitutes prior art known to those skilled in the art. Summary of the Invention

[0007] To address at least some of the technical problems existing in the prior art, this invention provides an Rh molecular blood typing system, kit, and application based on multiplex asymmetric PCR combined with probe melting curve analysis technology. The aim is to efficiently and accurately identify common Rh genotypes in Asian populations through a single PCR amplification reaction. This invention not only effectively overcomes the limitations of traditional serological methods in detecting complex blood types such as the D variant, but also possesses significant advantages such as high standardization, low cost, ease of operation, and low barriers to clinical application. Specifically, this invention includes the following:

[0008] In a first aspect, the present invention provides a multiplex PCR detection system for RH genotyping detection, wherein the detection system includes primers with sequences as shown in SEQ ID No. 1-26 and probes with sequences as shown in SEQ ID No. 27-44.

[0009] In some embodiments, according to the multiplex PCR detection system for RH genotyping according to the present invention, the primers and the probes form independent reaction systems.

[0010] In some embodiments, the multiplex PCR detection system for RH genotyping detection according to the present invention includes a first reaction system and a second reaction system for RHCE genotyping detection, and a third reaction system, a fourth reaction system, a fifth reaction system, and a sixth reaction system for RHD genotyping detection. The first reaction system includes primers shown in SEQ ID No. 1-4 and probes shown in SEQ ID No. 27-29; the second reaction system includes primers shown in SEQ ID No. 1-8 and probes shown in SEQ ID No. 30-33; the third reaction system includes primers shown in SEQ ID No. 9-16 and probes shown in SEQ ID No. 34-37; the fourth reaction system includes primers shown in SEQ ID No. 11, 12, 15-20 and probes shown in SEQ ID No. 38-41; the fifth reaction system includes primers shown in SEQ ID No. 21-24 and probes shown in SEQ ID No. 42, 43; and the sixth reaction system includes primers shown in SEQ ID No. 25, 26 and probe shown in SEQ ID No. 44.

[0011] In some embodiments, the multiplex PCR detection system for RH genotyping according to the present invention includes a probe containing a fluorescent group selected from FAM, VIC, CY5, ROX, BHQ2, BHQ1, MGB, ATTO425, or CY5.5.

[0012] In some embodiments, the multiplex PCR detection system for RH genotyping detection according to the present invention is in liquid form or lyophilized powder form.

[0013] In some embodiments, the multiplex PCR detection system for RH genotyping detection according to the present invention further includes an internal reference primer and a probe, wherein the sequences of the internal reference primer are shown in SEQ ID No. 45 and 46, and the sequence of the internal reference probe is shown in SEQ ID No. 47.

[0014] A second aspect of the present invention provides a kit for RH genotyping detection, comprising the detection system described in the first aspect. Those skilled in the art will understand that the kit of the present invention may further contain any suitable components suitable for PCR reactions, such as, but not limited to, dNTPs, Mg... 2+ Heat-resistant DNA polymerase, etc. The added dNTPs and Mg... 2+ The concentrations of forward and reverse primers, probes, and enzymes are not particularly limited and can be adjusted as needed.

[0015] In addition to the components described above, the kit of the present invention may also include instructions on how to perform the typing method and interpretation rules of the present invention. It is understood that the kit may further include precautions related to the regulation of manufacturing, use, or sale of the diagnostic kit. Furthermore, the kit of the present invention may also provide detailed instructions on storage and troubleshooting. The kit may optionally be housed in a suitable device, preferably for robotic operation at high throughput.

[0016] In some embodiments, the components of the kit of the present invention may be disposed in a container. The container typically includes at least one vial, test tube, flask, bottle, syringe, and / or other container means, wherein the solvent may optionally be placed in equal portions. The kit may also include means for containing a second container of sterile, pharmaceutically acceptable buffers and / or other solvents.

[0017] In some embodiments, the components of the kit of the present invention may be provided in solution form, such as an aqueous solution. When present in aqueous solution form, the concentration or content of these components can be readily determined by those skilled in the art according to different needs. For example, for storage purposes, the component concentration may be higher, and when in operation or in use, the concentration can be reduced to the working concentration, for example, by diluting the higher concentration solution. In some embodiments, the components of the kit of the present invention may be provided in powder form, such as a lyophilized powder.

[0018] A third aspect of the present invention provides a method for RH genotyping based on the melting curve of multiplex PCR probes, comprising the step of performing asymmetric PCR using the detection system described in the first aspect or the kit described in the second aspect.

[0019] In some embodiments, the method according to the present invention further includes a step of joint interpretation based on the fluorescence channel and the melting curve.

[0020] The genotyping method of this invention can be used for commercial experimental testing for non-diagnostic purposes, as well as for diagnostic testing. The genotyping method for diagnostic purposes can be understood as being for diagnostic use. This invention achieves RH genotyping of samples with only six reaction systems through joint interpretation of fluorescence channels and melting curves, offering advantages such as speed, ease of operation, and high throughput.

[0021] The typing method of this invention does not particularly limit the specific process or steps, as long as the detection system or kit described in this invention is used. In an exemplary method, the method of this invention includes a PCR amplification step, particularly an asymmetric PCR amplification step. The instrument used for the above amplification process can be a real-time fluorescence PCR instrument known in the art.

[0022] In the method of the present invention, the type of sample used for detection is not particularly limited. Preferably, it is a blood sample, such as serum or plasma.

[0023] A fourth aspect of the present invention provides the use of a reagent in the preparation of a kit for RH genotyping detection, characterized in that the reagent comprises the detection system described in the first aspect.

[0024] This invention utilizes multiplex asymmetric PCR probe melting curve technology to establish a six-tube multiplex PCR system, including two tubes for the RHCE blood group system and four tubes for the RHD blood group system. The entire detection process takes only 100 minutes, which is not only simple and rapid but also accurate and efficient, possessing high practical value. Regarding result analysis, for SNP-specific probes, only the Tm value of the melting curve of the fluorescent probe corresponding to each SNP locus needs to be determined to obtain the base results for different loci, further allowing for the inference of the RH genotype: when the locus is homozygous, a single melting curve will appear for the corresponding wild-type or mutant Tm value; while when the locus is heterozygous, both wild-type and mutant Tm values ​​will appear simultaneously, with the melting curve exhibiting a double peak or a "bun peak" where two Tm values ​​can be identified using tools. For exon presence discrimination probes, only the presence or absence of an RH exon needs to be determined by observing whether a melting curve exists at the corresponding Tm value. Attached Figure Description

[0025] Figure 1 Specificity verification of primers for exons 1-5 of the RHCE gene (plasmid template). The top image shows the amplification results of the RHCE plasmid template, and the bottom image shows the amplification results of the RHD plasmid template; the marker is a 100 bp DNA ladder; ①-⑧ represent different primer pairs designed for each exon. The red circles indicate the feasible specific primers after screening, and the rest are non-specific amplification or background signals.

[0026] Figure 2 Specificity verification of primers for exons 6-9 of the RHCE gene (plasmid template). The top image shows the amplification results of the RHCE plasmid template, and the bottom image shows the amplification results of the RHD plasmid template; the marker is a 100 bp DNA ladder; ①- This indicates different primer pairs designed for each exon. The red circles represent the specific primers that are feasible after screening, while the rest represent non-specific amplification or background signals.

[0027] Figure 3RHD gene exon 1-5 primer specificity verification (plasmid template). The top image shows the amplification results of the RHCE plasmid template, and the bottom image shows the amplification results of the RHD plasmid template; the marker is a 100 bp DNA ladder; ①-⑩ represent different primer pairs designed for each exon. The red circles indicate the feasible specific primers after screening, and the rest are non-specific amplification or background signals.

[0028] Figure 4 RHD gene exon 6-9 primer specificity verification (plasmid template). The top image shows the amplification results of the RHCE plasmid template, and the bottom image shows the amplification results of the RHD plasmid template; the marker is a 100 bp DNA ladder; ①- This indicates different primer pairs designed for each exon. The red circles represent the specific primers that are feasible after screening, while the rest represent non-specific amplification or background signals.

[0029] Figure 5 Hybrid rhesus box primer specificity verification. Marker: Trans2K Plus II DNA Marker. Primers ④ and ⑥ showed the expected amplification bands in completely deleted gDNA samples. Negative plasmids and RHD-positive gDNA samples (i.e., negative samples) did not amplify the expected bands, indicating that the primers have high specificity.

[0030] Figure 6 Melting curves of primers and probes for the RHCE blood group system.

[0031] Figure 7 Melting curves of RHD blood group system primers and SNP-specific probes.

[0032] Figure 8 Melting curves of RHD blood group system primers and exon presence discrimination probes.

[0033] Figure 9 Melting curves of hybrid rhesus box primers and probes.

[0034] Figure 10 Validation of multiple systems for RHCE and RHD blood group systems.

[0035] Figure 11 Optimized melting curves of the RHCE blood group system.

[0036] Figure 12 Melting curves of the optimized Hybrid rhesus box primers and probes.

[0037] Figure 13 Optimized melting curves of the RHD blood group system. Detailed Implementation

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0040] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention.

[0041] Traditional serological testing methods have several limitations. First, there are risks of missed detections and false positives: Serological reagents typically contain blood group antigens with a prevalence greater than 1% in the local population, posing a risk of missed detections for low-frequency antigens. Furthermore, for specific blood types, such as Rh weak D and Rh partial D subtypes, changes in antigen expression or protein modification can easily lead to missed detections or false positives. In cases of inconsistent forward and reverse typing or uncertain screening results, more complex confirmatory tests are required, increasing testing time and cost. Second, they are susceptible to interference from sample quality and medications: For example, serum from patients with altered albumin / globulin ratios or those who have received high-molecular-weight plasma substitutes can cause nonspecific rouleaux formation of red blood cells, interfering with agglutination results. Third, the reagent kits are derived from human red blood cells: The cells in serological testing kits are derived from human red blood cells, which are more difficult to obtain and standardize compared to chemical raw materials. Fourth, result interpretation is highly subjective: Although fully automated blood typing instruments are widely used, their basic principle is still antigen-antibody reaction, and result interpretation is based on determining the "+" degree according to the agglutination result, which is easily affected by the operator's experience and skill level. Fifth, it does not meet the development trend of precision medicine: serological test results are reactive and cannot provide precise information at the molecular level.

[0042] This invention designs highly specific primers based on the sequence differences between the RHD and RHCE genes in their intron regions. Simultaneously, it develops and systematically optimizes various fluorescently labeled probes targeting key single nucleotide polymorphism (SNP) sites. Based on this, the optimized primers and probes are rationally combined, and specific primers and probes for the internal reference gene GAPDH are introduced to monitor the stability and effectiveness of the reaction system. Through further fine optimization of the multiplex PCR system conditions, it was finally possible to perform RHCE gene genotyping in two reaction tubes and RHD gene genotyping in four reaction tubes. For result interpretation, the genotyping results are obtained by interpreting the melting temperature (Tm) value of the melting curve of the corresponding fluorescence channel in each tube. This method is not only simple and rapid but also accurate and efficient, possessing significant practical value.

[0043] The multiplex asymmetric PCR probe melting curve technique used in this invention is a PCR-based technique that combines asymmetric PCR and multiplex probe melting curve analysis. First, a key characteristic of asymmetric PCR is the different ratio of upstream and downstream primers used to amplify gene fragments. When the forward primer is depleted, excess reverse primer can amplify a large amount of single-stranded positive-strand product. When the target gene is present in the single-stranded amplification product, the fluorescent probe can bind to it without competition, resulting in a more pronounced signal change. Second, the fluorescently labeled probe hybridizes with ssDNA in the amplification product. As the temperature gradually increases, the hydrogen bonds between the probe and DNA break, causing melting and forming a melting curve. Double-labeled self-quenching fluorescent probes designed around the mutation site perfectly match the antisense strand, amplifying products with higher melting temperatures (Tm values), while partially mismatched fluorescent probes produce products with lower Tm values. The Tm values ​​of the products can be used to distinguish single nucleotide mutations at the same site. The introduction of the Tm value significantly increases the number of detectable mutations within a single channel. Combining multiple channels achieves an "N×N" amplification effect, effectively increasing detection throughput. Furthermore, it eliminates the need for electrophoresis, simplifying operation. Currently, there are no studies or registered reagents applying this technology for RH genotyping. This invention, based on multicolor probe melting curve analysis, enables the analysis of RH alleles using only six PCR reaction tubes in a single batch, offering advantages such as speed, simplicity, high throughput, and low cost.

[0044] Example 1. Selection of genotyping sites and construction of Rh blood group plasmids The selection criteria for the detection sites chosen in this embodiment fully combine national industry standards with the epidemiological characteristics of Rh blood type variations in the Chinese population. For the RHCE gene (Table 1), the detection sites follow the core polymorphic sites recommended in the group standard "Technical Guidelines for Red Blood Cell Blood Type Genotyping" (T / CSBT 009-2021) issued by the Chinese Blood Transfusion Association, including: c.307T>C, c.676C>G, c.122A>G, c.106G>A, c.733C>G, and c.1006G>T. In addition, this embodiment also covers the detection site c.48C>T, comprehensively covering the major RHCE antigen phenotypes in the Asian population, and can achieve detection of C, c, E, e, and C. w C x Precise typing of key antigens such as V and VS.

[0045] Table 1. Detected RHCE alleles and SNP sites For the RHD gene (Table 2), the design was also based on the mandatory detection sites specified in the T / CSBT 009-2021 standard, and further expanded by incorporating the distribution characteristics of the D variant in the Chinese population. First, deletion of the rhesus box region is the most important genetic mechanism leading to complete deletion of the RHD gene and the formation of the classic RhD-negative phenotype. Second, RHD*01EL.01 (c.1227G>A) RHD * weak partial 15 (c.845G>A) and DVI (partial D) are the three most common D variants in the Chinese population, accounting for over 90% of all D variants. As the most common partial D subtype in China, DVI individuals can produce anti-D antibodies after receiving immune stimulation from normal D-positive antigens. Therefore, this embodiment also includes the detection capability for DVI, thus constructing a comprehensive RHD / RHCE typing scheme that conforms to industry standards and is highly adapted to the genetic background of the Chinese population.

[0046] Table 2 RHD and RHCE are a pair of highly homologous, tightly linked genes located on the short arm of chromosome 1 (1p36.11) with opposite transcription directions, exhibiting sequence similarity exceeding 95%. This embodiment uses human genomic DNA (gDNA) from healthy individuals as a template. Based on the reference sequences of RHD (NG_007494.1) and RHCE (NG_009208.3), specific primer pairs were designed to amplify each exon (except exon 8) and its upstream and downstream introns via PCR. Considering the high homology between RHD and RHCE sequences, some primers can simultaneously amplify fragments corresponding to the two gene regions. After purification, the PCR products were ligated into a B vector, transformed into *E. coli*, and multiple single clones were selected for initial screening by bacterial PCR and verification using Sanger sequencing. By comparing the sequencing results with the reference sequences, the presence of known polymorphic sites in each plasmid was determined. Based on this, for specific blood type phenotypes, site-directed mutagenesis technology is further used to introduce key single nucleotide variants (such as c.845G>A, c.1227G>A, etc.) into the constructed wild-type plasmid backbone, thereby obtaining a plasmid library covering common Rh variants in the Chinese population.

[0047] Furthermore, to accurately simulate the most common clinical mechanism of complete RHD gene deletion, namely the homologous recombination event mediated by the rhesus box, this embodiment also specifically constructed a plasmid containing a hybrid rhesus box. The precise genomic coordinates of the upstream and downstream rhesus boxes were determined by comparison with the NCBI database; subsequently, specific primers were designed to independently amplify the upstream rhesus box fragment and the downstream rhesus box fragment after removing the equivalent region; finally, homologous recombination cloning technology was used to directionally splice the two fragments into the same vector, forming a hybrid rhesusbox plasmid simulating the chromosomal fusion site after RHD deletion.

[0048] All constructed RHD and RHCE plasmids were confirmed by bidirectional Sanger sequencing to ensure sequence accuracy and consistency with the target genotype.

[0049] 2. Preliminary design and validation of primers This embodiment designs highly specific primer pairs based on the sequence differences between the RHD and RHCE genes in their corresponding intron regions. For the RHCE gene, the designed primers not only amplify the specific coding sequence of its exons but also include the non-coding regions flanking the exons (i.e., flanking intron sequences). The primer binding sites are located within the introns and avoid known exon polymorphisms or antigenic determinant variation regions, thus ensuring stable and efficient amplification of the target fragment even in the presence of CE antigen variations. This makes it suitable for reliable detection of various RHCE variant alleles.

[0050] Similarly, for the RHD gene, primer design also covers the D gene exon and its upstream and downstream flanking intron sequences. The primer binding region is strictly limited to conserved sites within the introns without known functional variations, thereby avoiding amplification failure or biased amplification due to template sequence variations.

[0051] However, due to the high homology between RHD and RHCE gene sequences (exon region similarity >95%), with only 1-2 nucleotide differences in some regions, it is difficult to directly design highly specific primer pairs. To address this challenge, this embodiment, based on primer sequences covering RHD / RHCE differential sites, intentionally introduces one or more controllable base mismatches near the 3' end of the primers. These mismatches are not randomly introduced; their purpose is to enhance amplification specificity. The 3' end is a critical region for DNA polymerase extension, and mismatches here can significantly inhibit the elongation efficiency of non-target genes (such as amplifying RHCE templates using RHD primers), thereby effectively distinguishing the highly homologous RHD and RHCE sequences.

[0052] To evaluate the specificity of the initially designed primers, conventional PCR was first performed according to the reaction system shown in Table 3. Specifically, the following components were added to a PCR tube: 10×SSP Buffer, 100 mM MgCl2, 10 mM dNTPs, forward and reverse primers, EagleTaq enzyme, and H2O (Table 3). The constructed RHD, RHCE plasmids, Hybridrhesus box plasmid, and genomic DNA (gDNA) extracted from human samples were used as templates to amplify the target exon sequences.

[0053] The PCR products were analyzed by 1% agarose gel electrophoresis to verify their specificity. Figure 1 and Figure 2 The screening results of primer pairs designed for the RHCE gene are presented; and Figure 3 and Figure 4 This corresponds to the primer pair screening results for the RHD gene; Figure 5This process validates the amplification of hybrid rhesus box-specific primers. During the screening process, cross-testing was performed using both RHD and RHCE plasmids as templates: RHCE-specific primers were expected to produce the target band only in the RHCE plasmid, with no amplification in the RHD plasmid; conversely, RHD-specific primers should amplify only the RHD plasmid. For hybrid rhesus box primers, the design goal was to specifically recognize the fusion sequence formed by recombination of the upstream and downstream rhesus boxes; therefore, it was expected that specific bands would be amplified only in hybrid rhesus box plasmids and completely deleted human gDNA samples. Based on these validation results, highly specific primer pairs were selected. All primer sequences are shown in Tables 4 and 5 for subsequent experimental steps.

[0054] Table 3 SSP Single Test System (1 test) Experimental reaction conditions: 95°C - 3 min; 95°C - 10 s, 60°C - 1 min, 50 cycles.

[0055] Table 4 RHCE and RHD specific primer sequences Note: Bold underlined bases represent the sequence differences between RHCE and RHD, while italicized bases represent artificially introduced mismatched bases.

[0056] Table 5 Hybrid rhesus box and internal control specific primer sequences Note: Bold underlined bases are artificially introduced mismatched bases, and italicized bases are differential bases between upstream and downstream sequences and highly similar sequences.

[0057] 3. Preliminary design and verification of the probe The probe design in this embodiment is based on the functional differences and sequence characteristics of the RHD and RHCE genes. For the RHCE gene, each probe is precisely anchored to a target SNP site, and the Tm value difference is analyzed by melting curve analysis to determine the wild type and mutant type, achieving targeting of C / c, E / e, and C. w C x Accurate interpretation of antigen phenotypes such as V / VS.

[0058] For the RHD gene, probe design is further subdivided into two categories: (1) SNP-specific probes: used to detect D variant-related mutations that are prevalent in the Chinese population (e.g., c.845G>A corresponds to weak D15, c.1227G>A corresponds to Asian type DEL). The design strategy is consistent with that of the SNP probes for RHCE. The probes cover the target SNP sites to ensure that the Tm values ​​of wild-type and mutant templates can be distinguished; (2) Exon presence discrimination probes: used to determine whether a specific exon of the RHD gene is present in its entirety. These probes do not target SNPs, but specifically bind to sites in the corresponding exon regions where RHD and RHCE have sequence differences. Since the base composition of RHD and RHCE is different at these positions, they show clearly separated Tm peaks in the melting curve. Through this Tm difference, it can be clearly determined whether the target exon comes from RHD or RHCE, and thus confirm whether there are structural variations such as deletion, substitution or partial deletion in the RHD gene.

[0059] In addition, a specific probe was designed for the Hybrid rhesus box, with its target sequence located in the equivalent region. When the RHD gene is completely deleted in the sample, the fusion structure will be amplified, and a characteristic melting peak will be generated after the probe binds. If the peak is not observed, it is determined that the RHD gene is present in its entirety.

[0060] All probes underwent rigorous screening, with lengths controlled between 17 and 23 bases. To enable multiplex detection, probes were assigned to four fluorescence detection channels according to the detection target: FAM, VIC, ROX, and Cy5. To further verify the feasibility of the primers and probes, a singlet asymmetric PCR system was used for detection: a 25 μL reaction system containing 10×SSP buffer, 100 mM MgCl2, 10 mM dNTPs, forward / reverse primers, corresponding probes, EagleTaq enzyme, and ddH2O (Table 6).

[0061] Table 6. Singleton asymmetric PCR detection system (1 test) The experiment employed a two-step reaction method. The reaction conditions were: 95°C - 3 min, 95°C - 10 s, 60°C - 1 min, 50 cycles, with fluorescence signal acquisition at 60°C. Denaturation occurred at 95°C - 1 min, hybridization at 45°C - 5 min, and the temperature was gradually increased from 45°C to 90°C, with a 1°C increase every 5 s (melting curve).

[0062] Probes designed for the RHCE gene SNP site were amplified and validated using the following templates: (1) RHCE exon plasmid containing the wild-type sequence of the SNP; (2) RHCE mutant plasmid with an artificially introduced mutation at the corresponding SNP site; (3) human gDNA sample; and (4) corresponding RHD exon plasmid. The expected results were: the first three templates could all produce specific amplification and corresponding melting curve peaks. Since the primers all targeted the RHCE sequence, the RHD plasmid could not be effectively amplified due to the lack of a perfectly matching sequence, so no melting peak appeared, thus verifying the high specificity of the system for RHCE.

[0063] Probes designed for RHD gene SNP sites are validated using template combinations similar to those described above, including RHD wild-type plasmids, RHD mutant plasmids, human gDNA samples, and corresponding RHCE exon plasmids. For RHD exon presence discrimination probes, the templates used include: (1) RHD exon plasmids; (2) human gDNA samples; and (3) corresponding RHCE exon plasmids. These probes aim to distinguish between RHD and RHCE-derived amplification products based on melting temperature (Tm) differences, thereby determining whether the target exon belongs to the RHD gene. Hybrid rhesus box probes use templates including: (1) Hybrid rhesus box negative plasmids; (2) Hybrid rhesus box positive plasmids; and (3) human gDNA samples.

[0064] Singleton asymmetric PCR was performed on different templates, and the detection results of the RHCE probe are as follows: Figure 6 As shown in the figure. The results showed that the RHCE primer-probe combination had good specificity: the RHD plasmid did not produce a melting curve peak, indicating no cross-amplification; both the RHCE wild-type plasmid and the human gDNA sample showed clear melting peaks; when using the RHCE mutant plasmid, the Tm value of its melting peak was significantly different from that of the wild type, which was as expected. The detection results verified the high specificity of this system for recognizing RHCE SNP sites.

[0065] The results of RHD SNP specificity detection are as follows: Figure 7 As shown, the results indicated that the RHCE plasmid did not produce a melting curve peak, indicating no cross-amplification; both the RHD wild-type plasmid and the human gDNA sample showed clear melting peaks; when using the RHD mutant plasmid, its melting peak Tm value was significantly different from that of the wild type, which was as expected. The RHD blood group system primer and exon presence discrimination probe combination ( Figure 8 It also exhibits good specificity: the RHCE plasmid did not produce a melting curve peak, indicating no cross-amplification; both the RHD wild-type plasmid and the human gDNA sample showed clear melting peaks, and the Tm values ​​were as expected. The detection results validated the ability of this system to distinguish RHD blood group genes.

[0066] Hybrid rhesus box probe detection results are as follows: Figure 9 As shown, negative plasmids did not produce melting curve peaks, while positive plasmids exhibited clear melting peaks, and the Tm values ​​were as expected. Completely deleted samples underwent homologous recombination, resulting in sequence fusion and the formation of a hybrid rhesus box; probe detection revealed the expected melting peak. RHD positive samples that did not produce a hybrid rhesus box, however, did not exhibit melting peaks.

[0067] The complete sequences of RHCE, RHD, and Hybrid rhesus box probes are shown in Table 7 and will be used in subsequent experimental steps.

[0068] Table 7. Singleton asymmetric PCR detection system (1 test) 4. Preliminary combination and verification of multiple systems After completing the design, verification and optimization of primers and probes for the RH blood group system, this embodiment conducted preliminary multiplex system construction based on the optimized components, forming a two-tube RHCE multiplex asymmetric PCR system and a three-tube RHD multiplex asymmetric PCR system. The preliminary primer and probe schemes are shown in Tables 8 and 9.

[0069] The Magnetic Bead Blood Genomic DNA Extraction Kit (catalog number: DP329) from Beijing Tiangen Biotech Co., Ltd. was used to extract gDNA according to the manufacturer's instructions, and gDNA was then used as a template for detection. To evaluate the feasibility of primer-probe combinations in various multiplex systems under co-amplification conditions, preliminary detection was performed using the constructed plasmid and human gDNA as templates, respectively. The results are as follows: Figure 10 As shown in the figure. The results show that most probes produced the expected melting curve peaks in the RHCE system, except for probe P-307, which did not produce a signal. It is speculated that it was inhibited by other components in the multiple reactions. It may be necessary to adjust the primer or probe concentration corresponding to this probe, or transfer it to other reaction tubes to reduce interference.

[0070] In contrast, the RHD multiplex system exhibited more complex characteristics: several probes that amplified well in singlet reactions failed to produce significant melting peaks in multiplex combinations. Analysis suggests this is primarily due to competitive amplification interference between primers in the multiplex system. Primer pairs with shorter amplification products have higher amplification efficiency, preferentially consuming dNTPs, DNA polymerase, and template resources in the reaction system, thus inhibiting the amplification efficiency of primer pairs with longer fragments, leading to the loss or significant weakening of some target signals. Furthermore, the presence of multiple primer pairs in each reaction tube and the resulting variety of amplification products after PCR may also affect the melting curve peaks.

[0071] In summary, further adjustments to primer concentrations and reaction components are needed, or the reaction tubes may need to be replaced, in order to achieve simultaneous and efficient amplification of each target in a multiplex system.

[0072] Table 8. Probe sequences of each tube after preliminary assembly Table 9 Primer sequences after preliminary assembly 5. Application and further optimization of multiplex detection systems in human whole blood samples. For the probes that performed poorly, this study prioritized the optimization strategy of replacing fluorescent labels and redistributing across tubes. Specifically, the fluorescent dyes (such as FAM, VIC, ROX, Cy5) attached to each probe were adjusted, and the probe distribution was recombined between different reaction tubes to reduce inter-channel interference and improve amplification balance.

[0073] In the RHCE reaction system, the P-307 probe and the internal control probe were exchanged to different reaction tubes, and the concentrations of the primers and probes corresponding to this site were appropriately increased. The optimized results showed that P-307 clearly produced a specific melting peak, and the Tm value was consistent with expectations. Figure 11 ).

[0074] In the RHD reaction system, the amplification product of the Hybrid rhesus box (HRB) specific primers is as long as 2286 bp, which is an extremely long fragment in this multiplex asymmetric PCR system, making amplification significantly more difficult than other short-fragment targets. When coexisting with other shorter-fragment primers with higher amplification efficiency in the same reaction tube, HRB amplification is easily inhibited, resulting in no signal output from the probe. Given the important clinical significance of HRB detection in determining whether the RHD gene is completely deleted, it was ultimately decided to set up the HRB detection unit as a separate reaction tube to minimize competitive interference from other components. In addition, the P-HRB probe sequence was redesigned (sequence details are in Table 10), and its fluorescent label was changed from the original dye to the ROX fluorescent group with higher signal intensity and lower background to further improve detection sensitivity. After multiple rounds of screening (results are shown in Table 10), the HRB detection unit was successfully tested. Figure 12 Ultimately, P-HRB3 was selected as the optimal probe.

[0075] Besides HRB, this invention systematically optimized the remaining primer and probe combinations in the RHD system, including adjusting the distribution of each primer-probe pair in the reaction tube, optimizing primer and probe concentrations, and adjusting substrate components such as dNTPs. The final optimization results are as follows: Figure 13As shown in the figure. After the above adjustments, each fluorescence channel produced clear and distinct melting curve peaks, and the melting temperature was completely consistent with the expectation. Among them, probes P-845 and P-1227, designed for key SNP sites, both showed two well-separated melting peaks when detecting wild-type and mutant plasmids, respectively, indicating that they can effectively distinguish different genotypes.

[0076] Furthermore, compared to the initial combination, the amplification curves of each optimized tube all exhibited a typical S-shape, with a clear start-up and stable plateau phase, fully demonstrating that multiplex asymmetric PCR amplification is efficient and specific, and the overall performance of the system is significantly improved. The final adjusted primer and probe sequences for each tube are shown in Tables 11 and 12.

[0077] Table 10 Hybrid rhesus box probe sequences Table 11 Optimized probe sequences for each tube Table 12 Optimized primer sequences for each tube 6. Multiplex detection systems are applied to the detection of human whole blood samples. After comprehensive optimization of the multiplex detection system, this embodiment collected 70 clinical samples (Table 13), whose RHD genotyping results were clearly confirmed by second-generation sequencing or third-generation sequencing. Using the gDNA of these samples as templates, the results showed that all reaction tubes produced clear and stable melting curves, and the genotyping results were consistent with the second-generation / third-generation sequencing results, indicating that the system has good applicability and reliability in real clinical samples.

[0078] Table 13 Genotyping and Detection Sites of Clinical Samples Although the invention has been described with reference to exemplary embodiments, it should be understood that the invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments described in this specification without departing from the scope or spirit of the invention. The scope of the claims should be interpreted in the broadest possible sense to cover all modifications and equivalent structures and functions.

Claims

1. A multiplex PCR detection system for RH genotyping, characterized in that, The detection system includes primers with sequences as shown in SEQ ID No. 1-26 and probes with sequences as shown in SEQ ID No. 27-44.

2. The multiplex PCR detection system for RH genotyping detection according to claim 1, characterized in that, The primers and the probe form an independent reaction system.

3. The multiplex PCR detection system for RH genotyping detection according to claim 2, characterized in that, The reaction system includes a first reaction system and a second reaction system for RHCE genotyping detection, and a third reaction system, a fourth reaction system, a fifth reaction system, and a sixth reaction system for RHD genotyping detection. The first reaction system includes primers shown in SEQ ID No. 1-4 and probes shown in SEQ ID No. 27-29; the second reaction system includes primers shown in SEQ ID No. 1-8 and probes shown in SEQ ID No. 30-33; the third reaction system includes primers shown in SEQ ID No. 9-16 and probes shown in SEQ ID No. 34-37; the fourth reaction system includes primers shown in SEQ ID No. 11, 12, 15-20 and probes shown in SEQ ID No. 38-41; the fifth reaction system includes primers shown in SEQ ID No. 21-24 and probes shown in SEQ ID No. 42, 43; and the sixth reaction system includes primers shown in SEQ ID No. 25, 26 and probe shown in SEQ ID No.

44.

4. The multiplex PCR detection system for RH genotyping detection according to claim 1, characterized in that, The probe contains a fluorescent group.

5. The multiplex PCR detection system for RH genotyping detection according to claim 1, characterized in that, The detection system is in liquid form or lyophilized powder.

6. The multiplex PCR detection system for RH genotyping detection according to claim 1, characterized in that, The detection system further includes an internal reference primer and a probe, the sequences of which are shown in SEQ ID No. 45 and 46, and the sequence of which is shown in SEQ ID No.

47.

7. A kit for RH genotyping detection, characterized in that, The detection system includes any one of claims 1-6.

8. A method for RH genotyping based on multiplex PCR probe melting curves, characterized in that, The step includes performing asymmetric PCR using the detection system according to any one of claims 1-6 or the kit according to claim 7.

9. The method according to claim 8, characterized in that, It further includes a step of joint interpretation based on fluorescence channels and melting curves.

10. The application of the reagent in the preparation of a kit for RH genotyping detection, characterized in that, The reagent comprises the detection system according to any one of claims 1-6.

Images