Primer probe group and kit for micro-chimeric detection and application of primer probe group and kit

Abstract

The invention discloses a primer probe group for micro-chimeric detection, a kit and application of the primer probe group and the kit, and belongs to the technical field of gene detection. 29 InDel loci are screened for the first time, the loci are firstly subjected to fragment analysis and then subjected to typing, then specific primers and probes are designed for site amplification and detection, the typing loci are detected based on qPCR, and the micro-chimeric rate in a biological sample of a recipient is obtained; the detection method disclosed by the invention has the advantages of strong specificity, high sensitivity and the like, so that the detection method has a relatively good application prospect in micro-chimeric detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene detection, and in particular relates to a primer probe set and a kit for microchimerism detection and applications thereof. Background Art

[0002] Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is increasingly used to treat hematopoietic diseases, malignant solid tumors, and immune system diseases, but relapse after transplantation or graft rejection has become the main cause of transplant failure. After hematopoietic stem cell transplantation, whether the donor cells are successfully implanted and proliferate and differentiate stably for a long time is of great significance for clinical prognosis and treatment guidance. Post-transplant chimerism refers to the presence of donor cells in the recipient's body after receiving an allogeneic or xenogeneic transplant, and the presence of recipient cells in the transplant. This phenomenon of the mutual existence of donor and recipient cells is called chimerism; among them, microchimerism refers to donor cells <1%, which is mainly seen in solid organ transplantation. Therefore, monitoring the chimerism after transplantation is an important method to evaluate the effect of donor cell implantation.

[0003] At present, the conventional method for chimera detection is SNP combined with RT-PCR technology. SNP refers to changes such as conversion, inversion, insertion or deletion at a specific nucleotide position in genomic DNA, and the frequency of any allele in the population is not less than 1%. It is generally believed that there are only two types of SNPs, namely biallelic. Therefore, when detecting known mutation sites, it is only necessary to confirm and analyze different types of bases of SNPs without the need to perform full sequence determination on DNA fragments. SNPs have the characteristics of large number, wide distribution and genetic stability. SNP polymorphism can fully reflect the genetic differences between individuals, so the genotyping of donors and recipients at SNP sites can be used to evaluate the chimera state.

[0004] However, existing methods for chimera detection still have problems such as poor accuracy and low sensitivity. Summary of the invention

[0005] The purpose of the present invention is to provide a primer probe set, a kit and application thereof for microchimerism detection, so as to solve the problems of poor accuracy and low sensitivity in existing methods for chimerism detection.

[0006] In a first aspect, the present invention provides a primer-probe set for microchimerism detection, comprising a primer set and a probe set; wherein the primer set comprises a first primer set having a nucleotide sequence as shown in SEQ ID NO.1-3 and amplifying a sequence of rs59841142 site, a second primer set having a nucleotide sequence as shown in SEQ ID NO.5-7 and amplifying a sequence of rs34076006 site, a third primer set having a nucleotide sequence as shown in SEQ ID NO.9-11 and amplifying a sequence of rs3049448 site, a fourth primer set having a nucleotide sequence as shown in SEQ ID NO.13-15 and amplifying a sequence of rs3029189 site, a fifth primer set having a nucleotide sequence as shown in SEQ ID NO.17-19 and amplifying a sequence of rs35309403 site, a sixth primer set having a nucleotide sequence as shown in SEQ ID NO.21-23 and amplifying a sequence of rs2307696 site, and a sixth primer set having a nucleotide sequence as shown in SEQ ID NO. The seventh primer set as shown in SEQ ID NOs.25-27 and amplifies the rs1610937 site sequence, the eighth primer set as shown in SEQ ID NOs.29-31 and amplifies the rs2308010 site sequence, the ninth primer set as shown in SEQ ID NOs.33-35 and amplifies the rs16663 site sequence, the tenth primer set as shown in SEQ ID NOs.37-39 and amplifies the rs34209360 site sequence, the eleventh primer set as shown in SEQ ID NOs.41-43 and amplifies the rs1610932 site sequence, the twelfth primer set as shown in SEQ ID NOs.45-47 and amplifies the rs35453727 site sequence, the thirteenth primer set as shown in SEQ ID NOs.49-51 and amplifies the rs5787309 site sequence, the nucleotide sequence as shown in SEQ ID NOs. ID NO.53-55 and amplify the rs60564093 site sequence, the 14th primer set with nucleotide sequences as shown in SEQ ID NO.57-59 and amplify the rs3038530 site sequence, the 16th primer set with nucleotide sequences as shown in SEQ ID NO.61-63 and amplify the rs60867863 site sequence, the 17th primer set with nucleotide sequences as shown in SEQ ID NO.65-67 and amplify the rs56024302 site sequence, the 18th primer set with nucleotide sequences as shown in SEQ ID NO.69-71 and amplify the rs140790 site sequence, the 19th primer set with nucleotide sequences as shown in SEQ ID NO.73-75 and amplify the rs769299 site sequence, the 20th primer set with nucleotide sequences as shown in SEQ ID NO.77-79 and amplify the rs67405073 site sequence, the 21st primer set with nucleotide sequences as shown in SEQ ID NO.81-83 and amplify the rs16458 site sequence, the 21st primer set with nucleotide sequences as shown in SEQ ID NOs.85-87 and amplify the rs4646006 site sequence, the 23rd primer set with nucleotide sequences as shown in SEQ ID NOs.89-91 and amplify the rs771783753 site sequence, the 24th primer set with nucleotide sequences as shown in SEQ ID NOs.93-95 and amplify the rs34419736 site sequence, the 25th primer set with nucleotide sequences as shown in SEQ ID NOs.97-99 and amplify the rs2308112 site sequence, the 26th primer set with nucleotide sequences as shown in SEQ ID NOs.101-103 and amplify the rs2067204 site sequence, the 27th primer set with nucleotide sequences as shown in SEQ ID NOs.105-107 and amplify the rs34855933 site sequence, the 28th primer set with nucleotide sequences as shown in SEQ ID NOs. The 28th primer set shown in SEQ ID NO.109-111 and amplifying the sequence of rs34076006 site, and the 29th primer set with nucleotide sequences shown in SEQ ID NO.113-115 and amplifying the sequence of rs72031009 site.

[0007] In some embodiments, the probe group includes a first probe having a nucleotide sequence as shown in SEQ ID NO.4 and detecting a sequence of rs59841142, a second probe having a nucleotide sequence as shown in SEQ ID NO.8 and detecting a sequence of rs34076006, a third probe having a nucleotide sequence as shown in SEQ ID NO.12 and detecting a sequence of rs3049448, a fourth probe having a nucleotide sequence as shown in SEQ ID NO.16 and detecting a sequence of rs3029189, a fifth probe having a nucleotide sequence as shown in SEQ ID NO.20 and detecting a sequence of rs35309403, a sixth probe having a nucleotide sequence as shown in SEQ ID NO.24 and detecting a sequence of rs2307696, a seventh probe having a nucleotide sequence as shown in SEQ ID NO.28 and detecting a sequence of rs1610937, an eighth probe having a nucleotide sequence as shown in SEQ ID NO.32 and detecting a sequence of rs2308010, a ninth probe having a nucleotide sequence as shown in SEQ ID NO.36 and detecting a sequence of rs16663, and a nucleotide sequence as shown in SEQ ID NO. NO.40 and detecting the rs34209360 site sequence, the eleventh probe whose nucleotide sequence is shown in SEQ ID NO.44 and detecting the rs1610932 site sequence, the twelfth probe whose nucleotide sequence is shown in SEQ ID NO.48 and detecting the rs35453727 site sequence, the thirteenth probe whose nucleotide sequence is shown in SEQ ID NO.52 and detecting the rs5787309 site sequence, the fourteenth probe whose nucleotide sequence is shown in SEQ ID NO.56 and detecting the rs60564093 site sequence, the fifteenth probe whose nucleotide sequence is shown in SEQ ID NO.60 and detecting the rs3038530 site sequence, the sixteenth probe whose nucleotide sequence is shown in SEQ ID NO.64 and detecting the rs60867863 site sequence, the seventeenth probe whose nucleotide sequence is shown in SEQ ID NO.68 and detecting the rs56024302 site sequence, the eighteenth probe whose nucleotide sequence is shown in SEQ ID NO.72 and detecting the rs140790 site sequence, the The nucleotide sequence of the nineteenth probe shown in SEQ ID NO.76 and detecting the sequence of the rs769299 site, the nucleotide sequence of the twentieth probe shown in SEQ ID NO.80 and detecting the sequence of the rs67405073 site, the nucleotide sequence of the twenty-first probe shown in SEQ ID NO.84 and detecting the sequence of the rs16458 site, the nucleotide sequence of the twenty-second probe shown in SEQ ID NO.88 and detecting the sequence of the rs4646006 site, the nucleotide sequence of the twenty-second probe shown in SEQ ID NO.92 and detecting the 23rd probe of the rs771783753 site sequence, the 24th probe of the nucleotide sequence as shown in SEQ ID NO.96 and detecting the rs34419736 site sequence, the 25th probe of the nucleotide sequence as shown in SEQ ID NO.100 and detecting the rs2308112 site sequence, the 26th probe of the nucleotide sequence as shown in SEQ ID NO.104 and detecting the rs2067204 site sequence, the 27th probe of the nucleotide sequence as shown in SEQ ID NO.108 and detecting the rs34855933 site sequence, the 28th probe of the nucleotide sequence as shown in SEQ ID NO.112 and detecting the rs34076006 site sequence, and the 29th probe of the nucleotide sequence as shown in SEQ ID NO.116 and detecting the rs72031009 site sequence.

[0008] In the present invention, the inventors screened out 29 InDel sites (with wide coverage, located on 16 autosomes and the Y chromosome). For the above sites, the sites were first typed after fragment analysis, and then specific primers and probes were designed to amplify and detect the sites. The typed sites were detected based on qPCR to obtain the microchimerism rate in the recipient's biological sample.

[0009] In some embodiments, the 5' end of the first probe, the second probe, the third probe, the fourth probe, the fifth probe, the sixth probe, the seventh probe, the eighth probe, the ninth probe, the tenth probe, the eleventh probe, the twelfth probe, the thirteenth probe, the fourteenth probe, the fifteenth probe, the sixteenth probe, the seventeenth probe, the eighteenth probe, the nineteenth probe, the twentieth probe, the twenty-first probe, the twenty-second probe, the twenty-third probe, the twenty-fourth probe, the twenty-fifth probe, the twenty-sixth probe, the twenty-seventh probe, the twenty-eighth probe, and the twenty-ninth probe contain a fluorescent group and a quencher group at the 3' end.

[0010] In some embodiments, the fluorescent group includes at least one of FAM, 6-FAM, HEX, VIC, Cy5, and ROX; and the quenching group includes at least one of BHQ1, BHQ2, and MGB.

[0011] In some embodiments, internal reference gene primers and probes are also included. The nucleotide sequences of the internal reference gene primers are shown in SEQ ID NOs.117-118, and the nucleotide sequence of the probe is shown in SEQ ID NO.119.

[0012] In some embodiments, the 5' end of the probe of the internal reference gene contains a fluorescent group, and the 3' end contains a quenching group; wherein the fluorescent group includes at least one of FAM, 6-FAM, HEX, VIC, Cy5, and ROX; and the quenching group includes at least one of BHQ1, BHQ2, and MGB.

[0013] In a second aspect, the present invention provides a qPCR quantitative kit for microchimerism detection, comprising any one of the above-mentioned primer probe sets.

[0014] In some embodiments, the concentration of the primer set is 300-500 μM, for example, 300 μM, 350 μM, 400 μM, 450 μM, 500 μM or other values ​​within this range; the concentration of the probe set is 150-400 μM, for example, 150 μM, 200 μM, 250 μM, 300 μM, 350 μM, 400 μM or other values ​​within this range.

[0015] It should be noted that the qPCR quantitative kit may also include other conventional components, such as whole blood genomic DNA extraction reagents and reagents required for PCR reactions.

[0016] In a third aspect, the present invention provides use of any of the above primer probe sets or any of the above qPCR quantitative kits in microchimerism detection.

[0017] In a fourth aspect, the present invention provides a method for detecting microchimerism, comprising the following steps: S1, using a primer sequence such as a nucleotide sequence shown in SEQ ID NO.120-179 to detect the InDel site combination of the donor and the recipient of stem cell transplantation to obtain the differentiated sites of the donor and the recipient; S2, using any of the above-mentioned qPCR quantitative kits to detect the differentiated sites to obtain the microchimerism rate in the recipient's biological sample.

[0018] In some embodiments, the present invention provides a method for continuously monitoring the microchimerism rate in a post-transplantation sample, comprising the following steps: 1) when there is no donor in the post-transplantation sample, performing NGS sequencing on the post-transplantation sample to obtain 901 SNP sites and 29 InDel site information, fitting a probability density map based on the 901 SNP sites, analyzing the InDel sites according to the probability density map, and obtaining the microchimerism rate in the post-transplantation sample without a donor (which is 2 -ΔΔCt×100%, wherein ΔΔCT=ΔCT1-ΔCT2), and simultaneously analyzing the deviation of the InDel sites; 2) selecting 1-2 InDel sites with small deviation in the above step 1), and continuing to use the qPCR quantitative kit for detection to obtain the ΔCT1 value, thereby calculating the ΔCT2 value; continuing to use the qPCR quantitative kit for detection to obtain the re-test result of the microchimerism rate in the transplanted sample.

[0019] The beneficial effects of the present invention are as follows: different from the prior art, the present invention screens out 29 InDel sites for the first time (with wide coverage, located on 16 autosomes and the Y chromosome respectively), and for the above sites, firstly, after fragment analysis, the sites are typed, and then specific primers and probes are designed to amplify and detect the sites, and the typed sites are detected based on qPCR to obtain the microchimerism rate in the biological sample of the recipient; the detection method of the present invention has the advantages of strong specificity and high sensitivity, and therefore, has a good application prospect in microchimerism detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a graph of the typing results of random samples in Example 1 of the present invention, wherein "+" represents an insertion type, "-" represents a deletion type, del represents "-", and in represents "+";

[0021] Figure 2 This is a diagram showing the typing results of sample C of the present invention, wherein "+" represents an insertion type, "-" represents a deletion type, del represents "-", and in represents "+";

[0022] Figure 3 This is the typing result diagram of sample D of the present invention, where "+" represents insertion type, "-" represents deletion type, del represents "-", and in represents "+";

[0023] Figure 4 This is a graph of the qPCR test results in Example 2 of the present invention, wherein a specific primer probe set and a primer probe set of an internal reference gene are added to each well (taking the first well on the upper left as an example, this well is the result obtained by amplifying the "+ / +" specimen with the B1(+) primer probe and the internal reference genome, the green value is the CT value of the specific site, and the purple value is the CT value of the internal reference gene;

[0024] Figure 5 This is a graph of the amplification curve of qPCR detection in Example 3 of the present invention;

[0025] Figure 6 This is a graph showing the CT value results of qPCR detection in Example 3 of the present invention, wherein the green value is the CT value of the site after the recipient transplantation, and the purple value is the CT value of the reference gene after the recipient transplantation;

[0026] Figure 7 This is a probability density diagram of NGS sequencing in Example 4 of the present invention. DETAILED DESCRIPTION

[0027] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] The experimental methods without specific conditions in the examples are usually carried out according to conventional experimental methods in the field of molecular biology, including but not limited to the experimental methods described in Molecular Cloning: A Laboratory Manual by MR Green, Molecular Biology by Robert F. Weaver, etc., or according to the experimental methods recommended by the kit and instrument manufacturers. The reagents and biological materials used in the examples can be obtained from commercial channels unless otherwise specified.

[0029] Example 1 Site design and fragment analysis

[0030] Screening suitable InDel sites from population frequency databases such as gnomad, HapMap, Exac, 1000G 1000 Genomes, and UCSC literature requires the following screening conditions to be met:

[0031] 1. Minimum allele frequency (MAF) is between 0.30-0.50;

[0032] 2. The insertion / deletion bases are between 3-10bp;

[0033] 3. The selected loci should be located on different chromosomes as much as possible;

[0034] 4. The selected loci are located on the same chromosome, ensuring that the distance is greater than 10Mb and does not form linkage, and conform to Hardy-Weinberg equilibrium in the population;

[0035] 5. The number of selected sites combined with the gene frequency ensures that the feasibility of donor-free sample detection is greater than 99.99%.

[0036] Based on the above principles, 29 InDel sites were finally selected, located on 16 different autosomes and the Y chromosome, and divided into four groups: A, B, C, and D, as shown in Table 1 below:

[0037] Table 1 InDel site information table

[0038]

[0039]

[0040] According to the above grouping, A, B, C, and D were assigned to 4 fluorescent channels (spectral calibrants were independently developed, and the 4 channels were fluorescently labeled with FAM, HEX, TAMRA, and ROX, respectively), and amelogenin gender determination sites were added, a total of 30 sites, and fragment analysis amplification primers were designed (as shown in Table 5 below, "*" represents fluorescently labeled primers). The product size was between 100-300 bp, and the size spacing of adjacent site fragments was >10 bp. The site layout and the final concentration of the primers for single-well reactions are shown in Tables 3 and 4 below, respectively:

[0041] Table 3 Site layout and final concentration of site primers

[0042] FAM A1 A2 A3 A4 A5 A6 A7 A8 HEX B1 B2 B3 B4 B5 B6 B7 B8 TAMRA C0 C1 CY C2 C3 C4 C5 C6 ROX D1 D2 D3 D4 D5 D6

[0043] Table 4 Final concentration of primers in single well (μM)

[0044] Location A1 A2 A3 A4 A5 A6 A7 A8 Primers 45 50 50 75 60 40 100 275 Location B1 B2 B3 B4 B5 B6 B7 B8 Primers 80 45 300 50 60 40 50 120 Location C0 C1 CY C2 C3 C4 C5 C6 Primers 125 60 225 200 50 150 200 200 Location D1 D2 D3 D4 D5 D6 Primers 50 50 50 50 75 300

[0045] Table 5 Fragment analysis amplification primers

[0046]

[0047]

[0048] First, a DNA extraction kit was used to obtain random sample peripheral blood template DNA, and then a PCR amplification reaction was performed, as shown below:

[0049] 10μl amplification system: 5μl 2×PCR buffer; 0.3μl ARMS PCR Taq; 1.0μl Primers Mix; 2ng DNA; RNase-free Water to complete the reaction system.

[0050] PCR program: 95°C pre-denaturation for 30 s, 95°C denaturation for 15 s, 60°C annealing for 20 s, 72°C extension for 40 s, for a total of 32 cycles, and finally 72°C extension for 14 min.

[0051] The PCR products obtained above were directly loaded into the ABI 3500 capillary electrophoresis instrument for analysis, and the typing results of all loci of the random sample could be obtained at one time. Figure 1 shown.

[0052] from Figure 1It can be seen that 12 of the InDel sites are homozygous and the other sites are heterozygous. The results show that the fragment analysis method of the present invention can quickly type the sites.

[0053] Further, illustratively, the above-mentioned fragment analysis method is used to detect sample C (recipient) and sample D (donor), and the typing results are as follows: Figure 2 and 3 shown.

[0054] from Figure 2 and 3 It can be seen that there are more InDel sites with different genotypes in sample C and sample D. Therefore, the fragment analysis method in the present invention can quickly and efficiently type the samples (recipients and donors), thereby quickly screening out the differentiated InDel sites in the recipient and the donor, and then detecting the differentiated InDel sites based on qPCR to obtain the microchimerism rate in the recipient sample.

[0055] Among them, according to the above-mentioned InDel site, and using REF as the internal reference gene, the primer probe set for microchimerism detection is designed (wherein, F(+) and R or F and R(+) are used to specifically amplify the inserted fragment; F(-) and R or F and R(-) are used to specifically amplify the deleted fragment; P is the probe sequence; the underlined part in F(+) is the inserted specific base), as shown in Table 6 below:

[0056] Table 6 Primer and probe sequence list

[0057]

[0058]

[0059]

[0060] Example 2 Primer and probe specificity detection

[0061] qPCR was used to detect the specificity of primers and probes. Specifically, sample C was typed in Example 1 (type results are shown in Figure 2 ) and sample D (typing results see Figure 3) were used as recipients and donors, respectively, and any InDel site in the above samples was selected, such as B5, which had two sets of primer probes, B5(+) and B5(-) (specific sequences are shown in Table 6), and qPCR was used to detect the "+ / +" type specimens (sample C) and "- / -" type specimens (sample D) of the B5 site. If B5(+) amplifies "+ / +" with a signal and "- / -" with no signal, it can be considered that the B5(+) primer probe set has good specificity; if B5(-) amplifies "- / -" with a signal and "+ / +" with no signal, it can be considered that the B5(-) primer probe set has good specificity.

[0062] The concentration of the primer probe set in each InDel site is shown in Table 7 below:

[0063] Table 7 Concentration of primer probe sets in InDel sites

[0064]

[0065]

[0066]

[0067] The qPCR amplification reaction system and procedure are as follows:

[0068] 20μl amplification system: 10μl 2×Advanced Master Mix; 1-2μl Primers Mix; 0.2-1μl Probe; Genomic DNA 20-100ng; RNase-free Water to supplement the reaction system.

[0069] qPCR program: 95°C pre-denaturation for 20 s, 95°C denaturation for 3 s, 60°C annealing for 30 s, and 60°C extension for 30 s, for a total of 40 cycles.

[0070] Taking B1-B8 loci as an example, qPCR detection was performed, and the internal reference genes were used as controls. The results are as follows Figure 4 shown.

[0071] from Figure 4 It can be seen that the primer and probe set of the present invention has good specificity.

[0072] Example 3 Primer and probe accuracy and sensitivity testing

[0073] Sample C was typed in Example 1 (type results are shown in Figure 2 ) and sample D (typing results see Figure 3) were used as recipients and donors, respectively, and mixed in different proportions (donor D / recipient C: 100%, 40%, 10%, 5%, 0.8%, 0.6%, 0.4%, 0.25%, 0.05%, 0.025%, 0.01%) to obtain standards with different concentrations of 20 ng / μL. The standard prepared above was used as a template for quantitative analysis, and RNase-Free ddH2O was used as a template as a blank control. The qPCR reaction system and conditions were the same as in Example 2.

[0074] The CT values ​​of the InDel sites (eg, B5) and the reference gene obtained by different ratios of standard samples were obtained by qPCR, and the difference (ΔCT) between the Ct values ​​obtained by the two was calculated.

[0075] With C as the sample before surgical transplantation and CD as the sample after transplantation, the CD chimerism rate (%) can be calculated based on the difference between the ΔCT before and after transplantation (ΔΔCT) = 2 -ΔΔCt ×100%, where ΔΔCT=ΔCT1-ΔCT2=(CT of recipient site after transplantation-CT of recipient internal reference after transplantation)-(CT of donor site-CT of donor internal reference). By comparing the theoretical value and the test value obtained by qPCR calculation, it can be used to judge the sensitivity and accuracy of each site. Taking B5(-) as an example for detection, the amplification curves and CT value results of each sample mixed in different proportions are shown as follows: Figure 5 and 6 shown.

[0076] from Figure 5 and 6 It can be seen that the amplification curve of each sample mixed in different proportions has a good shape, and the CT value results are relatively accurate.

[0077] The ΔΔCt calculation formula was used to calculate the sample mixed in different proportions. The results are shown in Table 8 below:

[0078] Table 8 Chimerism results of samples mixed in different proportions

[0079]

[0080]

[0081] As can be seen from Table 8, for the B5(-) site, the test values ​​are 36.10% (theoretical 40%), 11.58% (theoretical 10%), 3.02% (theoretical 5%), 0.62% (theoretical 0.8%), 0.56% (theoretical 0.6%), 0.38% (theoretical 0.4%), 0.24% (theoretical 0.2%), 0.043% (theoretical 0.05%), 0.018% (theoretical 0.02%), and 0.0066% (theoretical 0.01%), respectively. The results show that the primer and probe tests have good accuracy and the sensitivity can reach about 0.01%.

[0082] Example 4 Method for Continuously Monitoring Microchimerism Rate in Post-Transplantation Samples

[0083] This embodiment provides a method for continuously monitoring the microchimerism rate in a sample after transplantation, comprising the following steps:

[0084] 1) When there is no donor for the post-transplantation sample, NGS sequencing is performed on the post-transplantation sample to obtain 901 SNP sites and 29 InDel site information. The probability density map is obtained based on the fitting of the 901 SNP sites (such as Figure 7 As shown in the figure, the x-axis is the minor allele frequency, the y-axis is the density, Peak1 is the first peak, Peak2 is the second peak, the first peak (Peak1) corresponds to the P2 event (i.e., R = "+ / +", D = "+ / -" or R = "- / -", D = "+ / -"), and the second peak (Peak2) corresponds to the P1 event (R = "+ / +", D = "- / -" or R = "- / -", D = "+ / +")). According to the probability density diagram, the InDel site is analyzed to obtain the microchimerism rate in the transplanted sample without a donor (which is 2 -ΔΔCt ×100%, where ΔΔCT=ΔCT1-ΔCT2), and the deviation of InDel sites was analyzed at the same time; the results are shown in Table 9 below;

[0085] Table 9 InDel site quantitative information and deviation

[0086]

[0087]

[0088] 2) Select 1-2 InDel sites with small deviation in the above step 1), continue to use the qPCR quantitative kit for detection, obtain the ΔCT1 value, and then calculate the ΔCT2 value; continue to use the qPCR quantitative kit for detection to obtain the re-test result of the microchimerism rate in the transplanted sample.

[0089] In this embodiment, the microchimerism rate in the post-transplantation sample can be continuously monitored by using the SNP sequencing method for initial detection and then using the qPCR quantitative kit for re-detection.

[0090] It should be noted that the above embodiments all belong to the same inventive concept, and the description of each embodiment has its own focus. For matters that are not described in detail in some embodiments, reference may be made to the description in other embodiments.

[0091] The above-mentioned embodiments only express the implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A primer probe set for microchimerism detection, characterized in that: Includes primer sets and probe sets; Wherein, the primer set includes a first primer set having a nucleotide sequence as shown in SEQ ID NO.1-3 and amplifying a sequence of rs59841142 site, a second primer set having a nucleotide sequence as shown in SEQ ID NO.5-7 and amplifying a sequence of rs34076006 site, a third primer set having a nucleotide sequence as shown in SEQ ID NO.9-11 and amplifying a sequence of rs3049448 site, a fourth primer set having a nucleotide sequence as shown in SEQ ID NO.13-15 and amplifying a sequence of rs3029189 site, a fifth primer set having a nucleotide sequence as shown in SEQ ID NO.17-19 and amplifying a sequence of rs35309403 site, a sixth primer set having a nucleotide sequence as shown in SEQ ID NO.21-23 and amplifying a sequence of rs2307696 site, a seventh primer set having a nucleotide sequence as shown in SEQ ID NO.25-27 and amplifying a sequence of rs1610937 site, and a seventh primer set having a nucleotide sequence as shown in SEQ ID NO. The eighth primer set as shown in SEQ ID NOs.29-31 and amplifies the rs2308010 site sequence, the ninth primer set as shown in SEQ ID NOs.33-35 and amplifies the rs16663 site sequence, the tenth primer set as shown in SEQ ID NOs.37-39 and amplifies the rs34209360 site sequence, the eleventh primer set as shown in SEQ ID NOs.41-43 and amplifies the rs1610932 site sequence, the twelfth primer set as shown in SEQ ID NOs.45-47 and amplifies the rs35453727 site sequence, the thirteenth primer set as shown in SEQ ID NOs.49-51 and amplifies the rs5787309 site sequence, the fourteenth primer set as shown in SEQ ID NOs.53-55 and amplifies the rs60564093 site sequence, the nucleotide sequence as shown in SEQ ID NOs. No. 57-59 and amplifies the rs3038530 site sequence, the 15th primer set with nucleotide sequences as shown in SEQ ID NOs. 61-63 and amplifies the rs60867863 site sequence, the 17th primer set with nucleotide sequences as shown in SEQ ID NOs. 65-67 and amplifies the rs56024302 site sequence, the 18th primer set with nucleotide sequences as shown in SEQ ID NOs. 69-71 and amplifies the rs140790 site sequence, the 19th primer set with nucleotide sequences as shown in SEQ ID NOs. 73-75 and amplifies the rs769299 site sequence, the 20th primer set with nucleotide sequences as shown in SEQ ID NOs. 77-79 and amplifies the rs67405073 site sequence, the 21st primer set with nucleotide sequences as shown in SEQ ID NOs. 81-83 and amplifies the rs16458 site sequence, the 21st primer set with nucleotide sequences as shown in SEQ ID NOs.85-87 and amplify the rs4646006 site sequence, the 22nd primer set with a nucleotide sequence as shown in SEQ ID NO.89-91 and amplify the rs771783753 site sequence, the 24th primer set with a nucleotide sequence as shown in SEQ ID NO.93-95 and amplify the rs34419736 site sequence, the 25th primer set with a nucleotide sequence as shown in SEQ ID NO.97-99 and amplify the rs2308112 site sequence, the 26th primer set with a nucleotide sequence as shown in SEQ ID NO.101-103 and amplify the rs2067204 site sequence, the 27th primer set with a nucleotide sequence as shown in SEQ ID NO.105-107 and amplify the rs34855933 site sequence, the 28th primer set with a nucleotide sequence as shown in SEQ ID NO.109-111 and amplify the rs34076006 site sequence, the 29th primer set with a nucleotide sequence as shown in SEQ ID NO. The 29th primer set shown in IDNO.113-115 and amplifying the rs72031009 site sequence.

2. The primer probe set according to claim 1, characterized in that: The probe group includes a first probe having a nucleotide sequence as shown in SEQ ID NO.4 and detecting a sequence of rs59841142, a second probe having a nucleotide sequence as shown in SEQ ID NO.8 and detecting a sequence of rs34076006, a third probe having a nucleotide sequence as shown in SEQ ID NO.12 and detecting a sequence of rs3049448, a fourth probe having a nucleotide sequence as shown in SEQ ID NO.16 and detecting a sequence of rs3029189, a fifth probe having a nucleotide sequence as shown in SEQ ID NO.20 and detecting a sequence of rs35309403, a sixth probe having a nucleotide sequence as shown in SEQ ID NO.24 and detecting a sequence of rs2307696, a seventh probe having a nucleotide sequence as shown in SEQ ID NO.28 and detecting a sequence of rs1610937, an eighth probe having a nucleotide sequence as shown in SEQ ID NO.32 and detecting a sequence of rs2308010, a ninth probe having a nucleotide sequence as shown in SEQ ID NO.36 and detecting a sequence of rs16663, and a NO.40 and detecting the rs34209360 site sequence, the eleventh probe whose nucleotide sequence is shown in SEQ ID NO.44 and detecting the rs1610932 site sequence, the twelfth probe whose nucleotide sequence is shown in SEQ ID NO.48 and detecting the rs35453727 site sequence, the thirteenth probe whose nucleotide sequence is shown in SEQ ID NO.52 and detecting the rs5787309 site sequence, the fourteenth probe whose nucleotide sequence is shown in SEQ ID NO.56 and detecting the rs60564093 site sequence, the fifteenth probe whose nucleotide sequence is shown in SEQ ID NO.60 and detecting the rs3038530 site sequence, the sixteenth probe whose nucleotide sequence is shown in SEQ ID NO.64 and detecting the rs60867863 site sequence, the seventeenth probe whose nucleotide sequence is shown in SEQ ID NO.68 and detecting the rs56024302 site sequence, the eighteenth probe whose nucleotide sequence is shown in SEQ ID NO.72 and detecting the rs140790 site sequence, the The nucleotide sequence of the nineteenth probe shown in SEQ ID NO.76 and detecting the sequence of the rs769299 site, the nucleotide sequence of the twentieth probe shown in SEQ ID NO.80 and detecting the sequence of the rs67405073 site, the nucleotide sequence of the twenty-first probe shown in SEQ ID NO.84 and detecting the sequence of the rs16458 site, the nucleotide sequence of the twenty-second probe shown in SEQ ID NO.88 and detecting the sequence of the rs4646006 site, the nucleotide sequence of the twenty-second probe shown in SEQ ID NO.92 and detecting the rs771783753 site sequence, the 23rd probe whose nucleotide sequence is shown in SEQ ID NO.96 and detecting the rs34419736 site sequence, the 25th probe whose nucleotide sequence is shown in SEQ ID NO.100 and detecting the rs2308112 site sequence, the 26th probe whose nucleotide sequence is shown in SEQ ID NO.104 and detecting the rs2067204 site sequence, the 27th probe whose nucleotide sequence is shown in SEQ ID NO.108 and detecting the rs34855933 site sequence, the 28th probe whose nucleotide sequence is shown in SEQ ID NO.112 and detecting the rs34076006 site sequence, and the 29th probe whose nucleotide sequence is shown in SEQ ID NO.116 and detecting the rs72031009 site sequence.

3. The primer probe set according to claim 2, characterized in that: The 5' end of the first probe, the second probe, the third probe, the fourth probe, the fifth probe, the sixth probe, the seventh probe, the eighth probe, the ninth probe, the tenth probe, the eleventh probe, the twelfth probe, the thirteenth probe, the fourteenth probe, the fifteenth probe, the sixteenth probe, the seventeenth probe, the eighteenth probe, the nineteenth probe, the twentieth probe, the twenty-first probe, the twenty-second probe, the twenty-third probe, the twenty-fourth probe, the twenty-fifth probe, the twenty-sixth probe, the twenty-seventh probe, the twenty-eighth probe, and the twenty-ninth probe contain a fluorescent group, and the 3' end contains a quenching group.

4. The primer probe set according to claim 3, characterized in that: The fluorescent group includes at least one of FAM, 6-FAM, HEX, VIC, Cy5, and ROX; The quenching group includes at least one of BHQ1, BHQ2, and MGB.

5. The primer probe set according to claim 1, characterized in that: It also includes internal reference gene primers and probes. The nucleotide sequences of the primers of the internal reference genes are shown in SEQ ID NOs.117-118, and the nucleotide sequence of the probes is shown in SEQ ID NO.

119.

6. The primer probe set according to claim 5, characterized in that: The 5' end of the probe of the internal reference gene contains a fluorescent group, and the 3' end contains a quenching group; The fluorescent group includes at least one of FAM, 6-FAM, HEX, VIC, Cy5 and ROX; the quenching group includes at least one of BHQ1, BHQ2 and MGB.

7. A qPCR quantitative kit for microchimerism detection, characterized in that: A primer probe set comprising any one of claims 1-6.

8. The qPCR quantitative kit according to claim 7, characterized in that The concentration of the primer set is 300-500 μM, and the concentration of the probe set is 150-400 μM.

9. Use of the primer-probe set according to any one of claims 1 to 6 or the qPCR quantitative kit according to any one of claims 7 to 8 in microchimerism detection.

10. A method for detecting microchimerism, characterized in that: The steps include: S1. Using the primer sequences shown in nucleotide sequences such as SEQ ID NO.120-179 to detect the InDel site combination of the donor and the recipient of stem cell transplantation, to obtain the differential sites of the donor and the recipient; S2. Use the qPCR quantitative kit described in any one of claims 7-8 to detect the differential sites to obtain the microchimerism rate in the recipient's biological sample.

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