CN117737222B - Methods, compositions and kits for genotyping Rh blood group system antigen - Google Patents
Methods, compositions and kits for genotyping Rh blood group system antigen Download PDFInfo
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Abstract
The invention discloses a method, a composition and a kit for genotyping Rh blood group system antigen. The method of the present invention includes the steps of constructing a reaction system and performing a PCR amplification reaction in the reaction system. Wherein the reaction system comprises a first oligonucleotide set and a second oligonucleotide set simultaneously, the first oligonucleotide set can amplify fragments aiming at a first mutation site, the second oligonucleotide set can amplify fragments aiming at a second mutation site and fragments aiming at a third mutation site, the first oligonucleotide set contains a first fluorescent probe, the second oligonucleotide set contains a second fluorescent probe, and the Tm values of the first and second oligonucleotide sets and the third mutation site are different. The invention can realize three types of different genotypes by only combining two oligonucleotides, and can realize the genotyping of up to 12 different genotypes at a time by a single tube.
Description
Technical Field
The invention relates to multiple genotyping, in particular to a method, a composition and a kit for genotyping Rh blood group system antigen.
Background
The irregular antibody is the blood group antibody except the ABO blood group system, clinically blood group measurement refers to ABO blood group and RhD blood group measurement, and the irregular antibody can prevent immune reaction caused by combining the irregular antibody with corresponding antigen during blood transfusion or production, heat is caused when the irregular antibody is light, hemolysis is caused when the irregular antibody is heavy, and life is endangered. The Rh system is an important component in irregular antibodies. The antigenicity strength sequence of Rh system is D > E > C > C > E. anti-D, anti-C, anti-E and some complex antibodies can all cause acute and chronic hemolytic transfusion reactions and neonatal hemolysis.
The research on Rh blood group genotyping is mainly based on a typing method based on PCR, and comprises the following 4 methods: (1) PCR-sequence specific primers (PCR-SSP): the sequence specific primer designed according to the Rh gene specific locus amplifies a certain exon or intron, which is one of the most commonly used Rh blood group genotyping technologies at present. (2) PCR-restriction fragment Length polymorphism (PCR-RFLP): restriction enzyme PCR amplified products are subjected to length polymorphism analysis, single nucleotide substitution of restriction sites can be detected, but compared with PCR-SSP, PCR-RFLP is more cumbersome and time-consuming, and is not suitable for multiplex PCR. (3) real-time quantitative PCR: the method is often used for detecting trace fetal DNA in the peripheral blood of pregnant women, and is a main method for fetal Rh blood typing. (4) DNA sequencing: the polymorphism of Rh gene can be detected by directly sequencing the PCR product, and the sites of various base variations can be determined. In addition to PCR-based genotyping methods, high resolution melting curve (HRM) techniques may also genotype the RhCcEe locus. HRM is a new technique for gene analysis that forms melting curves of different forms based on the difference in melting temperature of single nucleotides. The use of HRM in certain human blood types has been reported in the literature, including ABO, rhD, duffy and Kidd blood types.
Taking widely used PCR-SSP as an example, the whole blood genome DNA extraction kit is adopted to extract genome DNA from EDTA anticoagulated venous peripheral blood. And respectively designing 4 PCR reaction systems aiming at specific sequences of RHC, c, E and E, and carrying out PCR amplification. Separating the amplified product by agarose gel electrophoresis, judging the size of the band, and carrying out RhCcEe genotyping. In HRM technology, genomic DNA extraction was also performed on EDTA-anticoagulated venous peripheral blood using whole blood genomic DNA extraction kit. Then, a plurality of pairs of primers for detecting RhCE genotyping are designed based on RhCE gene sequences, and melting curves of different gene loci are analyzed through a fluorescent quantitative PCR instrument, so that RhCcEe genotyping is identified.
However, PCR-based methods have disadvantages of complicated operation, time consumption, easy contamination, multi-tube detection, etc., and DNA sequencing has high cost and long time consumption, which is difficult to be widely used in clinic. HRM technology is more sensitive than traditional PCR and sequencing, but when detecting RHEe alleles, two HRM are needed to detect EE, EE and EE genotypes, which is not beneficial to clinical application.
The information in the background section is only for the purpose of illustrating the general background of the invention and is not to be construed as an admission or any form of suggestion that such information forms the prior art that is well known to those of ordinary skill in the art.
Disclosure of Invention
In order to solve at least part of the technical problems in the prior art, the invention provides a method, a composition and a kit for genotyping Rh blood group system antigen. Specifically, the present invention includes the following.
In a first aspect of the present invention, there is provided a method for genotyping Rh blood group system antigen comprising the steps of constructing a reaction system and performing a PCR amplification reaction in said reaction system;
Wherein the reaction system comprises a first oligonucleotide set comprising a first forward oligonucleotide, a first reverse oligonucleotide and a first fluorescent probe, thereby being capable of amplifying a fragment directed to a first mutation site, the first fluorescent probe being capable of specifically binding to the first mutation site, and a second oligonucleotide set comprising a second forward oligonucleotide, a second reverse oligonucleotide and a second fluorescent probe, thereby being capable of amplifying a fragment directed to a second mutation site and a fragment directed to a third mutation site, the second fluorescent probe being capable of binding to the second mutation site and the third mutation site, the first fluorescent probe and the second fluorescent probe being different in Tm value from the binding of different mutation sites.
In certain embodiments, the method for genotyping Rh blood group system antigens according to the invention, wherein the first mutation site comprises a Cc mutation, the second mutation site comprises an Ee mutation, and the third mutation site is a Dd mutation.
In certain embodiments, the method for Rh blood group system antigen genotyping according to the invention, wherein the Tm value of the first fluorescent probe and CC mutant fragment is at least 2 ℃ greater than the Tm value of the first fluorescent probe and CC mutant fragment; the Tm value of the second fluorescent probe and the EE mutant fragment is at least 2 ℃ greater than the Tm value of the second fluorescent probe and the EE mutant fragment; the Tm value of the second fluorescent probe and the ee mutant fragment is at least 2 ℃ greater than the Tm value of the second fluorescent probe and the DD mutant fragment.
In certain embodiments, the method for genotyping Rh blood group system antigens according to the invention, wherein the ratio of the concentration of the first forward oligonucleotide to the concentration of the first reverse oligonucleotide in the reaction system is 1 (8-15); and/or the ratio of the concentration of the second forward oligonucleotide to the concentration of the second reverse oligonucleotide is 1 (8-15).
In certain embodiments, the method for Rh blood group system antigen genotyping according to the invention, wherein the first fluorescent probe comprises a ROX fluorescent group; the second fluorescent probe comprises an ATTO-647 fluorescent group.
In certain embodiments, the method for genotyping Rh blood group system antigens according to the invention, wherein the reaction conditions comprise performing PCR at 95-3 min, 95-10 s, 60-1 min,50 cycles; collecting fluorescent signals at 60 ℃, carrying out denaturation at 95-1 min, hybridization at 45-5 min, gradually heating at 45-90 ℃ and heating at 5 s to 1 ℃ to obtain a melting curve.
In a second aspect of the invention, there is provided a composition for genotyping Rh blood group system antigens comprising a first oligonucleotide set comprising a first forward oligonucleotide, a first reverse oligonucleotide and a first fluorescent probe capable of amplifying fragments directed to a first mutation site, the first fluorescent probe being capable of binding to the first mutation site, and a second oligonucleotide set comprising a second forward oligonucleotide, a second reverse oligonucleotide and a second fluorescent probe capable of amplifying fragments directed to a second mutation site and fragments directed to a third mutation site, the second fluorescent probe being capable of binding to the second mutation site and the third mutation site, the first fluorescent probe and the second fluorescent probe being different in Tm value to the different mutation sites.
In certain embodiments, the composition for genotyping of Rh blood group system antigens according to the invention, wherein the first forward oligonucleotide, the first reverse oligonucleotide and the first fluorescent probe are each present alone or in mixture and the second forward oligonucleotide, the second reverse oligonucleotide and the second fluorescent probe are each present alone or in mixture.
In a third aspect of the invention, there is provided a reaction system for genotyping an Rh blood group system antigen comprising a first set of oligonucleotides comprising a first forward oligonucleotide, a first reverse oligonucleotide and a first fluorescent probe capable of amplifying a fragment directed to a first mutation site, the first fluorescent probe being capable of specifically binding to the first mutation site, and a second set of oligonucleotides comprising a second forward oligonucleotide, a second reverse oligonucleotide and a second fluorescent probe capable of amplifying a fragment directed to a second mutation site and a fragment directed to a third mutation site, the second fluorescent probe being capable of binding to the second mutation site and the third mutation site, the first fluorescent probe and the second fluorescent probe being different in Tm value from binding to different mutation sites.
In a fourth aspect of the invention, there is provided a kit for genotyping Rh blood group system antigens comprising a first oligonucleotide set comprising a first forward oligonucleotide, a first reverse oligonucleotide and a first fluorescent probe capable of amplifying fragments directed to a first mutation site, the first fluorescent probe being capable of specifically binding to the first mutation site, and a second oligonucleotide set comprising a second forward oligonucleotide, a second reverse oligonucleotide and a second fluorescent probe capable of amplifying fragments directed to a second mutation site and fragments directed to a third mutation site, the second fluorescent probe being capable of binding to the second mutation site and the third mutation site, the first fluorescent probe and the second fluorescent probe being different in Tm value to different mutation sites.
The invention only adopts two different marked fluorescent probes and two pairs of specific primers to realize the detection in a single reaction tube, thereby saving time and labor. And the synthesis of more fluorescent probes is not needed, so that money is saved. The melting curve has high sensitivity through interpretation of different Tm values. Compared with the two repetition of RhEe alleles detected by the HRM method, the method not only achieves the identification of DD/Dd, DD, CC, cc, cc, EE, ee and ee multiple genotypes detected by a single tube, but also improves the experimental efficiency and shortens the reaction time.
In application, more and more hospitals currently take RhCcEe genotyping of patients and blood donors as routine detection experiments. The method has the advantages that retrospective research and analysis are carried out on patients needing repeated blood transfusion, the Rh phenotype cooperation infusion can reduce the probability of antigen exposure, the blood transfusion curative effect is enhanced, and the important link of the blood transfusion safety of the patients is also ensured. The genotyping accuracy is higher and more sensitive than the genotyping accuracy, and is one of the directions of future Rh-matched infusion, so that the invention is widely applied to clinical detection.
Drawings
FIG. 1 shows the results of the detection under different ratio concentrations.
FIG. 2 is a test result after optimization for RhCcEe.
FIG. 3 is a graph showing the result of distinguishing D positive from D negative by the second fluorescent probe.
FIG. 4 signal intensities of different fluorescently labeled RHE/e probes.
FIG. 5 comparison of different pairs of RhE/e primers.
FIG. 6 RhC/c optimization of primers and probes.
FIGS. 7-9 are the typing of RhCcEe for multiplex PCR detection of clinical samples.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in the present invention, it is understood that the upper and lower limits of the ranges and each intermediate value therebetween are specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, 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 belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
The term "Rh blood group system" herein refers to a system that divides different blood groups according to the presence of antigenic material of the Rh blood group on red blood cells in a human body. Rh-positive results when Rh agglutinogens (corresponding to DD or DD genotypes) were present on blood erythrocytes. Otherwise negative (corresponding to dd genotype). The human population with Rh positive or negative is further classified into CC, cc, cc, EE, ee, ee genotypes according to genotypes.
Herein, the term "oligonucleotide" refers to a plurality of linked nucleotides (i.e., molecules comprising a sugar (e.g., ribose or deoxyribose)) linked to an organic base, wherein the organic base comprises a pyrimidine (e.g., cytosine (C), thymine (T) or uracil (U)) or a purine (e.g., adenine (a) or guanine (G)). Oligonucleotides include DNA and RNA and various modifications thereof. Modifications include base modifications, sugar modifications, and backbone modifications.
[ Method for genotyping antigen of Rh blood group System ]
In a first aspect of the present invention, there is provided a method for genotyping Rh blood group system antigens comprising the steps of constructing a reaction system and performing a PCR amplification reaction in said reaction system.
In the present invention, the reaction system means a reaction mixture, particularly a reaction solution, which may contain both the first oligonucleotide set and the second oligonucleotide set. Wherein "first" and "second" are used herein for the sole purpose of distinguishing between different oligonucleotide sets. The first oligonucleotide set and the second oligonucleotide set are combinations of oligonucleotides directed respectively against different mutation sites. For example, the first set of oligonucleotides corresponds to a first mutation site, the second set of oligonucleotides corresponds to a second mutation site or further corresponds to a third mutation site. The first mutation site, the second mutation site and the third mutation site are sites having differences between different genotypes. The present invention has unexpectedly found that there are specific mutation sites or discrimination sites between the numerous genotypes of the Rh blood group system after extensive analysis. Almost all genotypes can be detected simultaneously by designing a specific combination of two oligonucleotides.
For convenience of explanation herein, the first oligonucleotide set and the second oligonucleotide set are sometimes collectively referred to as an oligonucleotide set. The oligonucleotide set of the present invention comprises at least an oligonucleotide as a primer (sometimes referred to herein as a "primer oligonucleotide") and a fluorescent probe (sometimes referred to herein as a "probe oligonucleotide") as a detection indicator.
In the present invention, the primer oligonucleotide is capable of specifically binding to at least a portion of a contiguous sequence of a target sequence. "specifically binds" herein means at least one of the following: (1) The target DNA region hybridized to the oligonucleotide in one template is only one, and each base in the oligonucleotide is paired with a corresponding base of the target DNA, i.e., the oligonucleotide is perfectly matched to the target DNA; (2) Under conditions suitable for PCR reactions, only one detectable fragment between 50 and 350bp in length can be obtained after hybridization of the paired primer oligonucleotides to the same template. The "specific binding" of the present invention is also referred to as "specific hybridization". The primer oligonucleotide is generally a linear structure, and its length is usually 10 to 50 nt, preferably 15 to 40 nt, more preferably 18 to 30 nt. The paired primer oligonucleotides generally include an oligonucleotide as an upstream primer and an oligonucleotide as a downstream primer, and the paired primer oligonucleotides can be amplified by PCR to obtain fragments containing a specific mutation site.
In the present invention, the primer oligonucleotide is preferably a synthetic oligonucleotide. In synthesizing the primer oligonucleotide, it is preferably designed to be substantially complementary, preferably 100% complementary, to the target sequence so that the target sequence hybridizes thereto and the 3' end base pairs and an amplification extension reaction can occur. "substantially complementary to" a target sequence means that an oligonucleotide is complementary to the target sequence under conventional reaction conditions to effect hybridization, as long as such complementarity is insufficient to completely block hybridization. Preferably, means known in the art are used to design the annealing temperature of the primer oligonucleotide to the target sequence and to control the GC content of the primer oligonucleotide to be between 45-60%.
In the present invention, the probe oligonucleotide is capable of binding to the first mutation site or the second mutation site of the present invention and comprises a fluorescent group. Illustratively, the probe oligonucleotide capable of binding to the first mutation site is a first fluorescent probe and the probe oligonucleotide capable of binding to the second mutation site is a second fluorescent probe.
In certain embodiments, the first mutation site is a Cc genotype discrimination site, in which case the first fluorescent probe is preferably capable of binding to a different Cc genotype, but has a different Tm value, and the difference in Tm value when binding to a different genotype is 2 ℃ or more, preferably 3 ℃ or more, more preferably 4 ℃ or more, 5 ℃ or more.
In certain embodiments, the second mutation site is an Ee genotype discrimination site, in which case the second fluorescent probe is preferably capable of binding to a different Ee genotype. The second fluorescent probe is further preferably capable of binding to a different Dd genotype, e.g. to a Dd genotype or Dd genotype, but not to a Dd genotype. The second fluorescent probe should have a different Tm value when bound to a different genotype, and the difference in Tm value when bound to a different genotype should generally be 2℃or more, preferably 3℃or more, more preferably 4℃or more, 5℃or more, whereby the different genotypes of Ee and Dd can be distinguished only by the melting curve obtained by the second fluorescent probe. The present invention unexpectedly found that there was a second mutation site and a second fluorescent probe between a multitude of different genotypes that met such a need. Thus, the probe oligonucleotide of the present invention is not necessarily fully complementary to the DNA to which it hybridizes, and in the case of different genotypes, the probe oligonucleotide may not be fully complementary to the DNA to which it hybridizes.
In the present invention, the fluorescent groups contained in the probe oligonucleotide are not limited, and any fluorescent groups known in the art may be used, and illustrative examples include, but are not limited to, FAM (6-carboxyfluorescein), TET (tetrachlorofluorescein), HEX (hexachlorofluorescein; rhodamine-based fluorescent groups such as ROX (6-carboxy-X-rhodamine) and TAMRA (6-carboxytetramethyl rhodamine), ATTO-647, cy dye families, especially Cy3 and Cy5, for example, and other fluorescent groups such as fluorescent groups having different emission spectra such as NED and JOE may be used, for example, fluorescent groups in the Alexa, atto, dyomics, dyomicsMegastokes and Thilyte dye families.
In the present invention, the probe oligonucleotide preferably further comprises a quenching group, and preferably, a fluorescent group and a quenching group are provided at both ends of the probe oligonucleotide sequence, respectively. Before the start of the PCR reaction, the probe oligonucleotide is designed to have sufficient proximity between the fluorescent groups and the quenching groups to allow fluorescence to be transferred via resonance energy and to attenuate or not fluoresce. For this purpose, the length of the probe oligonucleotide is generally 10 to 50 nt, preferably 15 to 30 nt. When PCR is performed, the primer and probe bind to the template, and during the extension phase, the exonuclease activity of the polymerase 5 'to 3' hydrolyzes the template-bound probe from the template, separating the fluorescent and quenching groups in the probe oligonucleotide, thereby releasing a fluorescent signal, and obtaining a melting curve based on the change in the fluorescent signal.
In the present invention, the first fluorescent probe and the second fluorescent probe preferably have different fluorescent groups, respectively, thereby enabling two different sites to be detected simultaneously in the same reaction system.
In the reaction system of the present invention, the concentration of the forward oligonucleotide is smaller than that of the reverse oligonucleotide, so that the desired signal can be obtained. The ratio of the concentration of forward oligonucleotide to the concentration of reverse oligonucleotide is generally 1 (8-15), such as 1:9, 1:10, 1:12, 1:14, etc.
The step of performing a PCR amplification reaction in a reaction system of the present invention generally includes PCR amplification and melting curve preparation. Wherein the PCR amplification comprises performing PCR at 95-3 min, 95-10 s, 60-1 min,50 cycles. Melting curve preparation includes, for example, denaturation of 95-1 min, hybridization of 45-5 min, gradual heating of 45-90℃and heating of 5 s by 1 ℃.
[ Composition ]
In a second aspect of the invention, there is provided a composition for genotyping Rh blood group system antigens comprising a first oligonucleotide set comprising a first forward oligonucleotide, a first reverse oligonucleotide and a first fluorescent probe capable of amplifying fragments directed to a first mutation site, the first fluorescent probe being capable of specifically binding to the first mutation site, and a second oligonucleotide set comprising a second forward oligonucleotide, a second reverse oligonucleotide and a second fluorescent probe capable of amplifying fragments directed to a second mutation site and fragments directed to a third mutation site, the second fluorescent probe being capable of binding to the second mutation site and the third mutation site, the first fluorescent probe and the second fluorescent probe being different in Tm value to bind to different mutation sites.
The form of the composition of the present invention is not limited, and the composition is illustratively a powder mixture of different oligonucleotides, or a solution form of different oligonucleotides dissolved in a buffer. In the case of mixing the paired primer oligonucleotides, the concentration or amount of the forward oligonucleotide is required to be smaller than the concentration or amount of the reverse oligonucleotide. Preferably, the ratio of the concentration of forward oligonucleotide to the concentration of reverse oligonucleotide is generally 1 (8-15), such as 1:9, 1:10, 1:12, 1:14, etc.
[ Reaction System ]
In a third aspect of the invention, there is provided a reaction system for genotyping an Rh blood group system antigen comprising a first set of oligonucleotides comprising a first forward oligonucleotide, a first reverse oligonucleotide and a first fluorescent probe capable of amplifying a fragment directed to a first mutation site, the first fluorescent probe being capable of specifically binding to the first mutation site, and a second set of oligonucleotides comprising a second forward oligonucleotide, a second reverse oligonucleotide and a second fluorescent probe capable of amplifying a fragment directed to a second mutation site and a fragment directed to a third mutation site, the second fluorescent probe being capable of binding to the second mutation site and the third mutation site, the first fluorescent probe and the second fluorescent probe being different in Tm value from binding to different mutation sites.
The reaction system of the present invention is the reaction system obtained in the method of the first aspect, wherein the first oligonucleotide set and the second oligonucleotide set are described in detail hereinabove, and are not described in detail herein.
In certain embodiments, the reaction system of the present invention further comprises a buffer, exemplary buffers comprising Tris (ph=8.5), KCl, mgCl 2, dntps, tween20, glycerol, a reactive enzyme. Preferably, the reaction system comprises 20 mM Tris (ph=8.5), 100 mM KCl, 12 mM MgCl 2, 0.4 mM dNTP, 0.2% Tween20, 10% glycerol, 1U/T Taq hot start enzyme.
[ Kit ]
In a fourth aspect of the invention there is provided a kit for genotyping Rh blood group system antigens comprising the oligonucleotide set of the first aspect of the invention. The description of the oligonucleotides is as described above and will not be repeated here.
In addition to oligonucleotides, kits of the invention may include precautions related to regulating manufacturing, use, or marketing of diagnostic kits in a form prescribed by a government agency. In addition, the kits of the invention may also be provided with detailed instructions for use, storage and troubleshooting. The kit may also optionally be provided in a suitable device, preferably for robotic operation in a high throughput setting.
In certain embodiments, the components (e.g., oligonucleotides) of the kits of the invention can be provided as a dry powder. When the reagents and/or components are provided as dry powders, the powders may be restored by the addition of a suitable solvent. It is contemplated that the solvent may also be disposed in another container. The container will typically include at least one vial, test tube, flask, bottle, syringe, and/or other container means, with the solvent optionally being placed in aliquots. The kit may further comprise means for a second container comprising a sterile, pharmaceutically acceptable buffer and/or other solvent.
In certain embodiments, the components of the kits of the invention may be provided in solution, e.g., in aqueous solution. Where present in aqueous solution, the concentration or amount of these ingredients can be readily determined by one skilled in the art according to various needs. For example, for storage purposes, the concentration of oligonucleotides may be present in a higher form, and when in operation or in use, the concentration may be reduced to an operating concentration by, for example, diluting a higher concentration solution as described above.
The kits of the invention may further comprise other reagents or components. For example, DNA polymerase, various types of dNTPs, and ions such as Mg 2+, etc., required for performing PCR. These other reagents or components are known to those skilled in the art and are readily known from publications such as the fourth edition of the molecular cloning Experimental guidelines in Cold spring harbor.
Where more than one component is present in a kit, the kit will also typically contain a second, third or other additional container in which additional components may be placed separately. In addition, combinations of components may be contained in the container.
Kits of the invention may also include components that retain or maintain DNA, such as agents that are resistant to degradation by nucleic acids. Any of the compositions or reagents described herein may be a component in a kit.
In a preferred embodiment, the reaction solution of the present invention comprises both the first oligonucleotide set and the second oligonucleotide set, preferably, the forward oligonucleotide concentration or amount is smaller than the reverse oligonucleotide in the reaction system, either the first oligonucleotide set or the second oligonucleotide set.
Examples
1. Experimental method
1. Sample DNA extraction
1.1, Taking 200 mu L of blood sample into a 2mL centrifuge tube, and sequentially adding 300 mu L of pyrolysis liquid and 350 mu L of isopropanol;
1.2 adding 20 mu L of protease K solution;
1.3 adding 300 mu L of cracking liquid GIL, and oscillating and uniformly mixing;
1.4 placing the centrifuge tube at 65 ℃ for incubation for 15 minutes, and reversing and uniformly mixing for 3 times, wherein each time is 3-5 times; standing at room temperature for 5min;
Adding 350 mu L of isopropanol into the mixture, and uniformly mixing the mixture for 10sec by shaking;
1.6 adding 20 mu L of magnetic bead suspension G, shaking and mixing for 1min, standing for 9min, and shaking and mixing for 1min every 3 min;
1.7 placing the centrifuge tube on a magnetic rack for standing for 30sec, and carefully sucking off the liquid after the magnetic beads are completely adsorbed;
1.8, taking the centrifuge tube off the magnetic rack, adding 700 mu L buffer GDA (absolute ethyl alcohol is added in the use process), and shaking and uniformly mixing for 5min;
1.9 placing the centrifuge tube on a magnetic rack for standing for 30sec, and carefully sucking off the liquid after the magnetic beads are completely adsorbed;
1.10, taking the centrifuge tube off the magnetic rack, adding 700 mu L of rinsing liquid PWD (absolute ethyl alcohol is added in use), and shaking and uniformly mixing for 2min;
1.11 placing the centrifuge tube on a magnetic rack for standing for 30sec, and carefully sucking off the liquid after the magnetic beads are completely adsorbed;
1.12 repeating steps 1.10 and 1.11 once;
1.13, placing the centrifuge tube on a magnetic rack, and airing for 10-15min at room temperature;
1.14 taking the centrifuge tube off the magnetic rack, adding 50-100 mu L of elution buffer solution TB, shaking and mixing uniformly, and placing at 56 ℃. Incubating for 10min, and mixing for 3 times during the incubation, wherein each time is 3-5 times;
1.15 placing the centrifuge tube on a magnetic rack for standing for 2min, after the magnetic beads are completely adsorbed, carefully transferring the DNA solution into a new centrifuge tube, detecting the ratio of the concentration of DNA to OD260/OD280, and preserving the sample at-20 ℃ for a long time.
2. Genotyping detection procedure
Taking whole genome DNA as a template, adding the following reaction components according to TAKARA TAQTM Hot Start Version instruction manual, and placing the reaction components in 1 reaction tube:
TABLE 1
PCR amplification was performed on an SLAN-96P real time PCR system instrument under the following reaction conditions: fluorescent signals are collected at 60 ℃ in 50 cycles of 95-3 min, 95-10 s and 60-1 min. Denaturation of 95-1 min, hybridization of 45-5 min, gradual heating of 45-90 ℃, and increasing of 5 s by 1 ℃, thereby obtaining a melting curve.
3. Interpretation of results
The corresponding Tm value was determined based on RhCC, rhcc, rhEE, rhee, rhDD, rhdd homozygotes. For RhEe heterozygotes, the melting curve will appear as both RhE and Rhe peaks, and for RhE or Rhe homozygotes, a single melting curve will appear for the corresponding Tm values. In the case of RhCc heterozygotes, the peak separation treatment is further performed by Origin software because the Tm value of the RhCC heterozygote is similar to that of the melting curve of RhCC, the RhCC homozygote is unimodal after treatment, and the RhCc heterozygote is bimodal after treatment. For D positive (including RhDD homozygote and RhDd heterozygote), a peak corresponding to Tm will appear, and for D negative (i.e. Rhdd homozygote), there is no peak corresponding to Tm.
TABLE 2
2. Experimental results
1. Probes and corresponding primers with ROX and CY5 marks are designed for specific mutation site sequences.
TABLE 3 Table 3
PCR amplification was performed on an SLAN-96P real time PCR system instrument using the TaKaRa reagent kit. The reaction system is shown in Table 1. Amplification conditions: firstly, a single-tube single system is adopted for pre-experiment, and the reaction conditions are as follows: fluorescent signals are collected at 60 ℃ in 50 cycles of 95-3 min, 95-10 s and 60-1 min. Denaturation of 95-1 min, hybridization of 45-5 min, gradual heating of 45-90 ℃, and rise of 5 s by 1 ℃ (melting curve).
Surprisingly, when the forward primer and the reverse primer were 1:1, the resulting melting curve did not exhibit the expected peak corresponding to the Tm value. After intensive studies, it was found that no peak appears when the concentration of the forward primer is greater than or equal to that of the reverse primer, and a peak corresponding to the Tm value appears gradually when the concentration of the reverse primer is gradually greater than that of the forward primer, regardless of whether the primer pair for the first site or the primer for the second site (see FIG. 1). Further analysis gave an optimal ratio of forward primer to reverse primer of 1:10. Therefore, next, in the reaction system shown in Table 1, the amount of the forward primer mixture was 0.2. Mu.L, the amount of the reverse primer mixture was 2. Mu.L (the forward primer concentration and the reverse primer concentration were equal), and the subsequent experiments were performed. The results show that: in the RHE-676-5'-CY5-3' -BHQ2 probe set, genotyping was identical to serotyping. In the RHC-307-5'-ROX-3' -BHQ2 probe set, genotyping was also completely identical to serotyping as judged by Tm value and peak-to-peak fusion (FIGS. 2A-B).
As can be seen from FIG. 3, when D is positive, i.e., DD or DD genotype, there is a peak at 63℃and when D is negative, i.e., DD genotype, there is no corresponding peak around 63 ℃. The primers of the invention can therefore be used to distinguish whether D is positive or negative.
2. Since the fluorescence signal for RhE/e is weak, the inventors further optimized analysis found that excellent signal intensity can be obtained when modifying the fluorescent group for RhE/e probe. Specifically, the substitution of CY5 for FAM, HEX and ATTO-647, with unchanged system and reaction conditions, resulted in the finding that FAM and HEX-labeled fluorescent probes did not exhibit the expected melting curve, while ATTO-647-labeled fluorescent probes corresponded to a melting curve that was much higher than that of CY5 (FIG. 4).
3. To further increase the signal intensity of RhE/e detection, the inventors further designed a pair of RHE primers with Tm of 68℃around their probes (see Table 4).
TABLE 4 Table 4
By comparing the two pairs of RHE primers, it was found that the combination of the second pair of primers with ATTO-647-labeled probe resulted in a higher specific peak height and a smaller Ct value (fig. 5). Thus the final primers and corresponding fluorescent probes for detection of the RhE/e locus were determined.
4. For the RhC/c site, the inventors further carried out an optimization design, specifically, in order to better judge the corresponding site according to Tm and peak separation treatment, a new probe sequence and primer sequence were designed without changing the ROX label (see Table 5).
TABLE 5
As a result, it was found that the two pairs of primers were not able to distinguish the RhC/c gene locus in the second probe ratio, and therefore the first probe was selected. When comparing the two pairs of primers, the results showed better reproducibility of the second pair of primers, and finally the combination of RHC-primer-2 and RHC-307-5'-ROX-3' -BHQ2-1 probe was selected to identify the gene locus of RhC/c (FIG. 6).
5. Single tube multiplex PCR detection of RhCcEe genotyping of sample gDNA
Collecting 40 clinical blood samples, extracting gDNA by a magnetic bead method, mixing primers and probes for distinguishing different genotypes by taking gDNA as a template, and amplifying according to the same reaction conditions. The results showed that the results of RhCcEe typing by this method are consistent with their serological results (FIGS. 7-9).
Although two pairs of primers and two different fluorescently labeled probes are placed in one tube to detect different signals to distinguish different genotypes, the two pairs of primers and the respective fluorescently labeled probes are placed in two tubes respectively to detect the genotypes of RHC/c and RhE/e respectively.
While the invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. Various modifications or changes may be made to the exemplary embodiments of the present disclosure without departing from the scope or spirit of the invention. The scope of the claims is to be accorded the broadest interpretation so as to encompass all modifications and equivalent structures and functions.
Claims (7)
1. The method for genotyping the Rh blood group system antigen is characterized by comprising the steps of constructing a reaction system and performing PCR amplification reaction in the reaction system;
Wherein the reaction system comprises a first oligonucleotide set comprising a first forward oligonucleotide, a first reverse oligonucleotide and a first fluorescent probe capable of amplifying a fragment of a first mutation site for a Cc mutation, the first fluorescent probe being capable of specifically binding to the first mutation site, and a second oligonucleotide set comprising a second forward oligonucleotide, a second reverse oligonucleotide and a second fluorescent probe capable of amplifying a fragment of a second mutation site for an Ee mutation and a fragment of a third mutation site for a Dd mutation, the second fluorescent probe being capable of binding to the second mutation site and the third mutation site, the first fluorescent probe and the second fluorescent probe being different in Tm value from the binding of the different mutation sites, the first fluorescent probe comprising a ROX fluorophore at the 5' end; the second fluorescent probe comprises an ATTO-647 fluorescent group at the 5' end;
the first forward oligonucleotide is a sequence shown as SEQ ID No.9, the first reverse oligonucleotide is a sequence shown as SEQ ID No.10, the first fluorescent probe is a sequence shown as SEQ ID No.13, the second forward oligonucleotide is a sequence shown as SEQ ID No.7, the second reverse oligonucleotide is a sequence shown as SEQ ID No.8, and the second fluorescent probe is a sequence shown as SEQ ID No. 5;
The ratio of the concentration of the first forward oligonucleotide to the concentration of the first reverse oligonucleotide in the reaction system is 1 (8-15); and the ratio of the concentration of the second forward oligonucleotide to the concentration of the second reverse oligonucleotide is 1 (8-15).
2. The method for genotyping Rh blood group system antigens according to claim 1, wherein said Tm value of said first fluorescent probe and CC mutant fragment is at least 2 ℃ greater than said Tm value of said first fluorescent probe and CC mutant fragment; the Tm value of the second fluorescent probe and the EE mutant fragment is at least 2 ℃ greater than the Tm value of the second fluorescent probe and the EE mutant fragment; the Tm value of the second fluorescent probe and the ee mutant fragment is at least 2 ℃ greater than the Tm value of the second fluorescent probe and the DD mutant fragment.
3. The method for genotyping Rh blood group system antigens according to claim 2, wherein said reaction conditions comprise performing PCR at 95-3 min, 95-10 s, 60-1 min,50 cycles; collecting fluorescent signals at 60 ℃, carrying out denaturation at 95-1 min, hybridization at 45-5 min, gradually heating at 45-90 ℃ and heating at 5s to 1 ℃ to obtain a melting curve.
4. A composition for genotyping of Rh blood group system antigens, comprising a first oligonucleotide set comprising a first forward oligonucleotide, a first reverse oligonucleotide and a first fluorescent probe capable of amplifying a fragment of a first mutation site directed against a Cc mutation, said first fluorescent probe being capable of binding to the first mutation site, and a second oligonucleotide set comprising a second forward oligonucleotide, a second reverse oligonucleotide and a second fluorescent probe capable of amplifying a fragment of a second mutation site directed against an Ee mutation and a fragment of a third mutation site directed against a Dd mutation, said second fluorescent probe being capable of binding to the second mutation site and the third mutation site, said first fluorescent probe and said second fluorescent probe being different in Tm value from the binding of the different mutation sites, said first fluorescent probe comprising a ROX fluorophore at the 5' end; the second fluorescent probe comprises an ATTO-647 fluorescent group at the 5' end;
the first forward oligonucleotide is a sequence shown as SEQ ID No.9, the first reverse oligonucleotide is a sequence shown as SEQ ID No.10, the first fluorescent probe is a sequence shown as SEQ ID No.13, the second forward oligonucleotide is a sequence shown as SEQ ID No.7, the second reverse oligonucleotide is a sequence shown as SEQ ID No.8, and the second fluorescent probe is a sequence shown as SEQ ID No. 5;
the ratio of the concentration of the first forward oligonucleotide to the concentration of the first reverse oligonucleotide is 1 (8-15); and the ratio of the concentration of the second forward oligonucleotide to the concentration of the second reverse oligonucleotide is 1 (8-15).
5. The composition for genotyping an Rh blood group system antigen according to claim 4 wherein said first forward oligonucleotide, first reverse oligonucleotide and first fluorescent probe are each present alone or in mixture and said second forward oligonucleotide, second reverse oligonucleotide and second fluorescent probe are each present alone or in mixture.
6. A reaction system for genotyping of Rh blood group system antigens, comprising a first oligonucleotide set comprising a first forward oligonucleotide, a first reverse oligonucleotide and a first fluorescent probe capable of amplifying a fragment of a first mutation site directed against Cc mutation, said first fluorescent probe being capable of specifically binding to the first mutation site, and a second oligonucleotide set comprising a second forward oligonucleotide, a second reverse oligonucleotide and a second fluorescent probe capable of amplifying a fragment of a second mutation site directed against Ee mutation and a fragment of a third mutation site directed against Dd mutation, said second fluorescent probe being capable of binding to the second mutation site and the third mutation site, said first fluorescent probe and said second fluorescent probe being different in Tm value to the different mutation sites, said first fluorescent probe comprising a ROX fluorescent group at the 5' end; the second fluorescent probe comprises an ATTO-647 fluorescent group at the 5' end;
the first forward oligonucleotide is a sequence shown as SEQ ID No.9, the first reverse oligonucleotide is a sequence shown as SEQ ID No.10, the first fluorescent probe is a sequence shown as SEQ ID No.13, the second forward oligonucleotide is a sequence shown as SEQ ID No.7, the second reverse oligonucleotide is a sequence shown as SEQ ID No.8, and the second fluorescent probe is a sequence shown as SEQ ID No. 5;
The ratio of the concentration of the first forward oligonucleotide to the concentration of the first reverse oligonucleotide in the reaction system is 1 (8-15); and the ratio of the concentration of the second forward oligonucleotide to the concentration of the second reverse oligonucleotide is 1 (8-15).
7. Kit for genotyping of Rh blood group system antigens, characterized in that it comprises a first oligonucleotide set comprising a first forward oligonucleotide, a first reverse oligonucleotide and a first fluorescent probe capable of amplifying a fragment of a first mutation site directed against Cc mutation, said first fluorescent probe being capable of specifically binding to the first mutation site, and a second oligonucleotide set comprising a second forward oligonucleotide, a second reverse oligonucleotide and a second fluorescent probe capable of amplifying a fragment of a second mutation site directed against Ee mutation and a fragment of a third mutation site directed against Dd mutation, said second fluorescent probe being capable of binding to the second mutation site and the third mutation site, said first fluorescent probe and said second fluorescent probe being different in Tm value from the binding of the different mutation sites, said first fluorescent probe comprising a ROX fluorescent group at the 5' end; the second fluorescent probe comprises an ATTO-647 fluorescent group at the 5' end;
the first forward oligonucleotide is a sequence shown as SEQ ID No.9, the first reverse oligonucleotide is a sequence shown as SEQ ID No.10, the first fluorescent probe is a sequence shown as SEQ ID No.13, the second forward oligonucleotide is a sequence shown as SEQ ID No.7, the second reverse oligonucleotide is a sequence shown as SEQ ID No.8, and the second fluorescent probe is a sequence shown as SEQ ID No. 5;
the ratio of the concentration of the first forward oligonucleotide to the concentration of the first reverse oligonucleotide is 1 (8-15); and the ratio of the concentration of the second forward oligonucleotide to the concentration of the second reverse oligonucleotide is 1 (8-15).
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