Patent Application
20230235390
The present application claims the priority of the Chinese application with the application number of 2022100980235 applied on 2022 Jan. 26, and all the recorded contents serve as a part of the present invention.
The contents of the electronic sequence listing (2023-01-19-SequenceListing.xml; Size: 161,707 bytes; and Date of Creation: Jan. 9, 2023) is herein incorporated by reference in its entirety.
The present invention relates to the field of biomedicine, in particular to a mass spectrometry-based method and a kit for genotyping of platelet and neutrophil antigens and glycoproteins.
Surface antigens of platelets and neutrophils as well as some membrane glycoproteins are closely related to blood transfusion and clinic. Inconsistency of platelet and neutrophil antigen phenotypes between fetuses and mothers or between donors and recipients of blood transfusion, etc. can lead to iso-immunization, and then lead to a series of clinical problems such as platelet transfusion refractoriness, which are important in blood transfusion, transplantation, maternal health care, etc. By conducting accurate genotyping on clinically significant platelet and neutrophil antigens or membrane glycoproteins, corresponding phenotypes can be predicted so that effective clinical measures can be taken.
There are complex and diverse antigen types on surfaces of platelets, mainly including two main types of platelet-associated antigens (antigens that also exist on surfaces of other cells and tissues, such as HLA, ABO antigens, etc.) and platelet-specific antigens (HPA). At present, 35 HPA systems have been found, namely HPA1-35w. HPA iso-antibodies produced by immunization can lead to neonatal iso-immune thrombocytopenia, platelet transfusion refractoriness, post-transfusion purpura, etc.
CD36 is a widely expressed glycoprotein in the human body, and it is expressed in all human platelets, macrophages, endothelial cells, etc. People with CD36 expression deletion may produce anti-CD36 antibodies by immunization in ways such as blood transfusion and pregnancy, which may then lead to neonatal iso-immune thrombocytopenia and even early fetal death, platelet transfusion refractoriness, post-transfusion purpura, transfusion-related acute lung injury and other clinical symptoms. Transfusion of blood components containing anti-CD36 antibodies in patients with a normal CD36 phenotype may also lead to adverse transfusion reactions, resulting in thrombocytopenia and even threatening to life. Since the proportion of people with CD36 expression deletion in Asian populations including China is significantly higher than that in Caucasian populations, the production of anti-CD36 iso-antibodies is an important risk factor for immune-mediated thrombocytopenia in Chinese populations. In addition to the normal phenotype, there are different types of CD36 antigen abnormalities such as type I deletion, type II deletion and weak expression according to different degrees of CD36 deletion and different intensities of expression. Among them, type I deletion refers to the absence of CD36 expression on both platelets and monocytes. Type II deletion refers to that there is no CD36 expression on the surfaces of the platelets, but there is normal expression of CD36 on the surfaces of the monocytes and the macrophages. Weak expression of CD36 refers to that there is expression of CD36 on the surfaces of the platelets, but an expression quantity thereof is significantly lower than a normal level.
Neutrophil antigens (HNA) are a group of glycoproteins expressed on the surfaces of human neutrophils and play an important role in iso-immunization and auto-immunization Certain HNAs also exist on other cells and tissues. At present, 5 HNA systems have been found, namely HNA 1-5. Due to the polymorphism of the HNA, corresponding antibodies can lead to neonatal iso-immune neutropenia, auto-immune neutropenia, febrile transfusion reaction, transfusion-related acute lung injury, etc.
In view of the important clinical significance of platelet and neutrophil antigens and CD36 proteins, accurate typing and identification of these antigens and proteins are necessary. Existing detection methods mainly distinguish different antigen expressions at a protein level or a gene level. Due to the insufficiency or lack of commercial specific antibodies, it is impossible to comprehensively type these antigens or proteins. Therefore, under the premise that the genetic background of the above antigens or proteins is becoming more and more clear at present, genotyping has become a better detection means. To meet clinical needs, it is necessary to establish a high-throughput, rapid and accurate genotyping method for clinically significant platelet and neutrophil antigens and proteins. A nucleic acid mass spectrometry technology has the characteristics of simultaneous detection of SNP and In/Del, short detection time, high detection throughput of a single amplification tube, accuracy and specificity, etc., which can meet the detection needs of the above antigens and proteins.
CN111455027A and CN110172502 provide mass spectrometric detection methods and kits for platelet antigen genotyping, but CN110172502 can only be used for detecting up to 21 HPA sites (HPA1-21w), and CN111455027A can only be used for detecting up to 29 HPA sites (HPA1-29w), so it is difficult to perform one-time typing and detection of all current known HPA sites of platelets (HPA1-35w). Meanwhile, CN111455027A and CN110172502 do not show the corresponding relationship between detection results and phenotypes.
Due to the existence of homologous sequences, rich GC and other problems, up to now, genotyping methods and products for simultaneous detection of 35 platelet antigen genetic sites and simultaneous detection of CD36 glycoproteins by a mass spectrometry technology cannot be achieved, and there are also no genotyping methods and products that can simultaneously detect multiple polymorphic sites such as CD36 glycoproteins, neutrophil antigens and platelet antigens.
Due to the problems that genes of platelet-specific antigens, CD36 glycoproteins and neutrophil antigens have genes with very high homology to the surrounding, moreover, sequences where some SNP sites are located are rich in GC, etc., and genes where some SNP sites are located have highly homologous sequences, resulting in that when the SNP sites of these genes are detected simultaneously based on mass spectrometry, the situations are prone to occurring that some sites do not have peaks and are not detected, or it is easy to amplify to homologous sequences to generate erroneous results, etc., and there is a problem that it is difficult to detect all sites one time.
In view of the problems in the prior art, the present invention provides a mass spectrometry-based method and a kit for genotyping of platelet and neutrophil antigens and glycoproteins, which are used for genotyping of platelet-specific antigens, CD36 glycoproteins and neutrophil antigens. By designing a primer combination and improving amplification reaction conditions, 35 platelet-specific antigen polymorphic sites, 10 CD36 polymorphic sites and 8 neutrophil antigen polymorphic sites can be simultaneously detected one time in one reaction (an amplification reaction and an extension reaction), which have the characteristics of high specificity and sensitivity, and fast and high throughput. The present invention can be used in clinic, scientific research, platelet donor routine screening, etc.
In the present invention, by screening a large number of primer combinations and adjusting an annealing temperature and a primer concentration, finally, 35 platelet-specific antigen polymorphic sites, 10 CD36 polymorphic sites and 8 neutrophil antigen polymorphic sites can be simultaneously detected one time. Moreover, high specificity and sensitivity and fast and high throughput are achieved.
On the one hand, the present invention provides a kit that comprises a mulit-amplificatiion tube that includes primer combination for CD36 genotyping. The primer combination includes amplification primers and extension primers. The amplification primers include forward primers and reverse primers. Sequences and the extension primers of the primer combination are shown in Table 1.
The primer combination for CD36 genotyping can simultaneously detect 10 polymorphic sites of CD36 at one time.
On the other hand, the present invention provides a mulit-amplificatiion tube that includes a primer combination for platelet antigens. The primer combination includes amplification primers and extension primers. The amplification primers include forward primers and reverse primers. Sequences of the primer combination are shown in Table 2.
The primer combination for platelet antigens can simultaneously detect 35 SNP sites of platelet antigens at one time.
On the other hand, the present invention provides a primer combination for platelet antigen and CD36 genotyping. The primer combination includes amplification primers and extension primers. The amplification primers include forward primers and reverse primers. Sequences of the primer combination are shown in Table 3. The primer combination for platelet antigens (35 SNP sites) and CD36(10) can be simultaneously detected at one time.
The primer combination for platelet antigen and CD36 genotyping can simultaneously detect 35 polymorphic sites of platelet antigens and 10 polymorphic sites of CD36.
On yet another hand, the present invention provides a primer combination for neutrophil antigen and CD36 genotyping. The primer combination includes amplification primers and extension primers. The amplification primers include forward primers and reverse primers. Sequences of the primer combination are shown in Table 4.
The primer combination for neutrophil antigen and CD36 genotyping can simultaneously detect 8 polymorphic sites of neutrophil antigens and 10 polymorphic sites of CD36.
On yet another hand, the present invention provides a primer combination for genotyping of platelet antigens and neutrophil antigens. The primer combination includes amplification primers and extension primers. The amplification primers include forward primers and reverse primers. Sequences of the primer combination are shown in Table 5.
The primer combination for genotyping of the platelet antigens and the neutrophil antigens can simultaneously detect 35 polymorphic sites of the platelet antigens and 8 polymorphic sites of the neutrophil antigens.
On yet another hand, the present invention provides a primer combination for genotyping of platelet antigens, neutrophil antigens and CD36. The primer combination includes amplification primers and extension primers. The amplification primers include forward primers and reverse primers. Sequences of the primer combination are shown in Table 6.
The primer combination for genotyping of the platelet antigens, the neutrophil antigens and the CD36 can simultaneously detect 35 polymorphic sites of the platelet antigens, 8 polymorphic sites of the neutrophil antigens and 10 polymorphic sites of the CD36 one time.
A sample of the present invention may be blood, or nucleic acids, such as DNA, extracted from the blood.
Information of the 10 polymorphic sites of the CD36, the 35 polymorphic sites of the platelet antigens and the 8 polymorphic sites of the neutrophil antigens of the present invention is shown in Table 7 respectively, in which, among sequences, sequences in parentheses are polymorphic sites.
Genotype and phenotype information of the 10 polymorphic sites of the CD36, the 35 polymorphic sites of the platelet antigens and 7 polymorphic sites of the neutrophil antigens of the present invention is shown in Table 7.
On yet another hand, the present invention provides a kit for genotyping, and a primer combination includes the primer combination of one of Tables 1 to 6 above.
On yet another hand, the present invention provides a method for genotyping by mass spectrometry detection, mainly including the following steps:
(1) by using an amplification primer mix in the above primer combination (the primer combination in any of Tables 1 to 6, these primers are all added to an amplification tube one time, and multiplex amplification is conducted simultaneously), amplifying genes to be detected by multiplex PCR;
(2) purifying an amplification product obtained in Step (1) by an alkaline phosphatase;
(3) by using an extension primer mix in the above primer combination (the extension primer combination in any of Tables 1 to 6, these primers are all added to an amplification tube one time, and extension of products are conducted simultaneously), extending and amplifying a purified product in Step (2) by a single base; and
(4) conducting sample application on a single-base extended product obtained in Step (3) onto a chip for mass spectrometry detection.
Further, during multiplex PCR reaction in Step (1), a final concentration of each primer in the amplification primer mix used is 0.1 to 1 μM.
Further, a multiplex PCR reaction system in Step (1) is as follows.
The DNA to be detected may be DNA extracted from a blood sample as a template for amplification, such as platelet DNA, glycoprotein DNA or neutrophil DNA, or a mix of these DNAs is used as a template for amplification.
Further, an annealing temperature of the multiplex PCR reaction in Step (1) is 65° C. to 53° C.
Further, cycle conditions of the multiplex PCR reaction in Step (1) are as follows: (97° C., 5 minutes, 15 cycles, decreasing by 0.8° C. each cycle): (97° C., 30 seconds, 65° C. (decreasing by 0.8° C. each cycle from a second cycle), 45 seconds, 15 cycles); 72° C., 2 minutes; (97° C., 30 seconds, 53° C., 45 seconds, 72° C., 2 minutes, 32 cycles); 72° C., 7 minutes; keeping a temperature of 4° C.
Further, the alkaline phosphatase in Step (2) is a shrimp alkaline phosphatase, and a premixed solution system for purification treatment with the alkaline phosphatase in Step (2) is shown in Table 9.
Further, a single-base extension premixed solution system in Step (3) is shown in Table 10.
On yet another hand, the present invention provides use of the above primer combination for preparing a mass spectrometry chip for genotyping of any one or more of platelet antigens, neutrophil antigens and CD36.
On yet another hand, the present invention provides use of the above primer combination or the above kit for genotyping mass spectrometry detection of any one or more of platelet antigens, neutrophil antigens and CD36.
A mass spectrometry-based method and a kit for genotyping of platelet and neutrophil antigens and glycoproteins provided by the present invention have the following beneficial effects.
1. All 35 platelet-specific antigen polymorphic sites, 10 CD36 polymorphic sites and 8 neutrophil antigen polymorphic sites can be simultaneously detected in 2 reactions, and the most comprehensive typing detection for all platelet antigens, CD36 and neutrophil antigens one time is achieved; 2. high specificity and sensitivity, and fast and high throughput are achieved; 3. the present invention can be used in clinic, scientific research, platelet donor routine screening, etc.
The present invention will be further described in detail below in combination with embodiments. It should be pointed out that the following embodiments are intended to facilitate the understanding of the present invention, but do not have any limiting effect on it. Reagents used in the embodiments are all known products, and are obtained by purchasing commercially available products.
In this embodiment, 35 platelet-specific antigen polymorphic sites, 10 CD36 polymorphic sites and 8 neutrophil antigen polymorphic sites are simultaneously detected on 400 cases of blood gene DNAs, so as to conduct genotyping. Although this embodiment only provides that the 35 platelet-specific antigen polymorphic sites, the 10 CD36 polymorphic sites and the 8 neutrophil antigen polymorphic sites are simultaneously detected, it can be understood that primer groups for the 10 CD36 polymorphic sites can also be used for simultaneous detection of the 10 polymorphic sites of CD36; primer groups for any one or more of the 35 platelet-specific antigen polymorphic sites, the 10 CD36 polymorphic sites and the 8 neutrophil antigen polymorphic sites can also be used for genotyping detection of any one or more thereof.
The genotyping detection of this embodiment includes the following steps.
1. Sample Preparation:
Genes (DNAs) of 400 cases of blood samples are extracted, and concentrations thereof are normalized to 5 to 20 ng/μL for subsequent detection experiments.
2. Primer Design
Amplification primers and extension primers are designed to detect 35 polymorphic sites of HPA1-35, 8 polymorphic sites of HNA1-5 and 10 polymorphic sites related to a CD36 deletion phenotype in CD36 protein coding genes. Amplification is divided into 2 test tubes or PCR tubes (repeated 2 times). In each test tube, forward and reverse primers in Table 11 are added one time to detect each site one time, and primer sequences are shown in Table 12.
3. Detection Steps
1) PCR Amplification
All amplification primer combinations shown in Table 12 are used to be added to each amplification test tube (including forward primers and reverse primers), and genes to be detected obtained in Step 1 are amplified by multiplex PCR to obtain target sequence amplification products of samples to be detected.
A PCR amplification reaction system is shown in Table 12.
Cycle conditions of PCR amplification reaction are as follows: (97° C., 5 minutes, 15 cycles, decreasing by 0.8° C. each cycle): (97° C., 30 seconds, 65° C. (decreasing by 0.8° C. each cycle from a second cycle), 45 seconds, 15 cycles); 72° C., 2 minutes; (97° C., 30 seconds, 53° C., 45 seconds, 72° C., 2 minutes, 32 cycles); 72° C., 7 minutes; keeping a temperature of 4° C.
2) Treatment with a Shrimp Alkaline Phosphatase (SAP)
Remaining dNTPs are treated by the shrimp alkaline phosphatase (SAP) to prevent interference with subsequent base extension. An SAP premixed solution system is shown in Table 13.
In Step 1), 2 μl of an SAP premixed solution is added to each reaction well of the 2 test tubes after PCR amplification, a total volume after the mixed solution is added is 7 and then SAP reaction is conducted in an amplification instrument. Reaction programs are as follows: 37° C., 40 minutes; 85° C., 5 minutes; keeping a temperature of 4° C.
3) Base Extension
All extension primer combinations shown in Table 12 are used to be added to test tubes respectively, and purified products in Step 2) are amplified by single-base extension. Through this amplification, a sequence-specific single base is extended at a 3′ end of an extension probe as a molecular weight marker. A single-base extension premixed solution system is shown in Table 14.
In Step 2), 2 μl of an extension premixed solution is added to each test tube after treatment with the shrimp alkaline phosphatase (SAP), a total volume after the mixed solution is added is 9 and then extension reaction is conducted in an amplification instrument.
Single-base extension reaction programs are as follows: 95° C., 30 seconds; (95° C., 5 seconds; (52° C., 5 seconds, 80° C., 5 seconds; 5 cycles) 40 cycles); 72° C., 3 minutes; keeping a temperature of 4° C.
4) Desalination with Resin
41 μl of HPLC water is added to each tube, resin is used for sample desalination, and extension reaction products are purified.
5) Mass Spectrometry Detection
After 2 test tubes of final desalination and purification, samples are subjected to sample application onto a chip (Manufacturer: Agena Bioscience, Model: SpectroCHIP CPM96). Molecular weight detection is performed by a mass spectrometer to determine the species of specific bases and the type of samples to be detected.
6) Result Analysis
Mass spectrometry detection is performed on the 400 cases of samples, and all sites have good results in all the samples (mass spectrometry software is rated A (Conservative) or B (Mordarate)). An obtained representative detection mass spectrogram is shown in
Sensitivity=true positive results/(true positive results+false negative results)*100%=100%.
Specificity=the number of true negatives/(the number of true negatives+the number of false positives)*100%=100%.
HPA-12w is located on a GP1Bbeta gene (NM_000407), and is not on the same gene as other HPA systems. Its SNP site is located at rs375285857, and on a fragment of about 1,000 bp before and after 500 bp and is rich in GC (>75%). In a process of designing a mass spectrometer chip for HPA-1-35, existing primer sequences do not work well for first-step amplification of HPA-12w. This embodiment is optimized by the following steps: (1) available primers for monoplex PCR are screened; (2) PCR conditions are changed (a Touchdown annealing temperature is used); (3) primer concentrations in multiplex PCR reaction are explored and optimized, HPA-12w is successfully introduced into a platelet group panel (gene combination), and conversion efficiency thereof is greater than 70%.
(1) Screening of Available Primers
In this embodiment, three pairs of primers shown in Table 16 are used, of which one pair cannot be amplified in monoplex PCR reaction (the primers are subjected to PCR reaction singly), and the other two pairs have products in monoplex PCR reaction of some samples, but they cannot be amplified in multiplex PCR reaction.
It can be seen from Table 16 that the use of different amplification primers has a significant impact on the amplification effect of HPA12w during multiplex PCR reaction, so it is preferred to use the third group of amplification primers, so that HPA12w can be successfully introduced into a platelet group amplification system. Primer design is particularly important when multiplex amplification is used.
(2) Change of PCR Conditions (Using a Touchdown Annealing Temperature)
In this embodiment, three annealing temperatures shown in Table 17 are respectively used for multiplex PCR (35-plex amplification), to examine the influence of different annealing temperatures on detection results of HPA12w.
It can be seen from Table 17 that different annealing temperatures have a great impact on the conversion efficiency during multiplex PCR (35-plex amplification) reaction by using the above primer groups, and when the annealing temperature is too high, the conversion efficiency will be significantly reduced, therefore, a preferred annealing temperature is 68° C. to 60° C. in a process of designing a mass spectrometer chip for HPA1-35.
(3) Exploring and Optimizing of Amplification Primer Concentrations of an rs375285857 Site in Multiplex PCR Reaction
When the multiplex PCR reaction (35-plex amplification) is performed according to the method shown in Embodiment 1, primer concentrations shown in Table 19 are respectively used for multiplex PCR amplification results. The primer concentrations mentioned here refer to final combinations of all primer combinations, in which, a final concentration of each primer is also 0.1 to 1 μM, as shown in Table 18.
It can be seen from Table 18 that different primer concentrations also have a significant impact on the multiplex PCR amplification reaction, when the primer concentration increases, although the conversion efficiency will also be improved, it will cause mutual influence between detection of different sites, resulting in that some sites cannot be detected, so a preferred primer concentration is 0.1 to 0.3 μM.
In a mass spectrometry kit, HNA-1 genotyping is determined by two SNP sites, namely rs5030738 and rs448740. Among them, rs448740 is located on an FCGR3B gene, and has a homologous gene FCGR3A, and a sequence thereof has a very high homology (˜98%) with a sequence around rs448740. Therefore, rs448740 site amplification primers (V1) in existing mass spectrometry panel are non-specific (homologous sequences can be amplified), resulting in an error in a final typing result of mass spectrometry. In order to specifically amplify a fragment containing the rs448740 site and enable the fragment to be used for subsequent mass spectrometry steps, appropriate PCR primers need to be selected.
This embodiment adopts the following ways: (1) multiple random samples are selected, longer fragments containing rs448740 are amplified and sequencing is conducted to obtain accurate bases of the rs448740 site; (2) multiple PCR primers are designed, the above samples are used for test, and primers that can specifically amplify bands are selected; (3) the primers obtained in (2) are tested, primers with higher conversion efficiency after subsequent mass spectrometry steps are selected, and their concentrations in multiplex mass spectrometry are optimized. The selected primers can specifically amplify the fragment where rs448740 is located. Mass spectrometry typing results are completely consistent with sequencing typing results, and the conversion efficiency is greater than 70%.
(1) Screening of Available Primers
Primers selected in this embodiment are shown in Table 19. Different primers are respectively used for multiple PCR reaction (8 plexes), to examine an amplification effect of the rs448740 site.
It can be seen from Table 19 that the use of different amplification primers has a significant impact on the amplification effect of the rs448740 site during multiplex PCR reaction (8 plexes). When a first group of primers is used, the rs448740 site (8 plexes) cannot be amplified during genotyping detection of 8 sites of neutrophil antigens. When a second group of primers is used, sometimes they cannot be amplified, and sometimes they are non-specifically amplified, that is, a homologous gene FCGR3A is amplified simultaneously. When a third group of primers is used, there is also a problem of non-specific amplification. Only when a fourth group of primers is used, specific amplification can be successfully achieved. Therefore, it is preferable to use the fourth group of amplification primers, so that the rs448740 site can be successfully introduced into the neutrophil panel.
(2) Exploring and Optimizing of Amplification Primer Concentrations of the rs448740 Site in Multiplex PCR Reaction
When the multiplex PCR reaction is performed according to the method shown in Embodiment 1, primer concentrations shown in Table 19 are respectively used for multiplex PCR amplification results, as shown in Table 20.
It can be seen from Table 20 that different primer concentrations also have a significant impact on the multiplex PCR amplification reaction, when the primer concentration increases, although the conversion efficiency will also be improved, it will cause mutual influence between detection of different sites, resulting in that HNA-2 (rs777225032), HPA-5 and HPA-12 cannot be detected in some samples, so a preferred primer concentration is 0.2 to 0.4 μM.
In this embodiment, the primers and primer concentrations obtained by exploration in Embodiment 3 are used for experiments, and it is found that there are 3 sites, namely HPA-5 (rs1801106), HPA-15 (rs10455097), and CD36 (1) (rs550565800) with reduced efficiency or even no peaks. In order to stabilize peak appearance at all sites of the entire panel, no call is eliminated. This embodiment adopts the following ways: (1) two new PCR primer pairs (new1 and new2) are designed for the three sites respectively, and tested to select appropriate primer combinations; (2) PCR conditions are changed according to the characteristics of annealing temperatures of primers to select optimal amplification conditions; (3) a large number of samples are verified.
(1) Screening of available primers for monoplex PCR
In this embodiment, 9 pairs of primers shown in Table 15 are used for three combinations (shown in Table 22), to examine an amplification effect on an overall site.
It can be seen from Tables 21 and 22 that the use of different primer pair combinations has a significant impact on detection results of each site. Only when the primer pair combination of new1 is used, three sites of HPA-5 (rs1801106), (rs10455097), and CD36 (1) (rs550565800) can be successfully detected when 53 sites of the entire panel are detected simultaneously, and simultaneous detection of the overall 53 sites can be successfully carried out.
(2) Change of PCR conditions (using a Touchdown annealing temperature)
In this embodiment, three annealing temperatures shown in Table 18 are respectively used for PCR, to examine effects of different annealing temperatures on the amplification and detection results of the overall site.
It can be seen from Table 23 that when the overall 53 sites are detected, the influence of annealing temperatures is examined again, and it is found that different annealing temperatures still have a great impact on the conversion efficiency of the entire panel during multiple PCR reaction, and the previously determined annealing temperature of 68° C. to 60° C. is still too high, resulting in low conversion efficiency. Therefore, a preferred annealing temperature is 65 to 53° C. in a process of designing a mass spectrometer chip for the entire panel.
(3) Through overall screening and optimization, and by verifying the 53 sites of the entire panel in Embodiment 1 in 400 samples, stable results are obtained, and both detection sensitivity and specificity are very good.
Although the present invention is disclosed above, the present invention is not limited thereto. Any person skilled in the art can make various changes and amendments without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be based on the scope defined by the claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2022100980235 | Jan 2022 | CN | national |
