WO2017185758A1 - Primer, probe, kit, and method for microchimerism assay and individual recognition - Google Patents

Abstract

A primer and probe combination used for a microchimerism assay and individual recognition, a kit comprising the primer and probe combination, and a method for implementing, by using the primer and probe combination or the kit, a microchimerism assay and individual recognition. The invention screens, by means of the latest InDel site database, and using an Asian population as the standard, 15 InDel sites with high powers of discrimination for the Asian population. The invention further includes, for each of the InDel sites, a design for a specific primer and probe combination. When compared to the prior art, the method provides the advantages of high polymorphism, good specificity, high sensitivity, ease of use, and rapidity, thereby offering an important method for implementing a microchimerism assay and individual recognition for an Asian population.

Description

Primers, probes, kits and methods for microchimeric detection and individual recognition Technical field

The present invention relates to the field of genetic engineering technology, and in particular to primers, probes, kits and methods for microchimeric detection and individual recognition.

Background technique

Modern geneticists believe that a gene is a generic term for a specific nucleotide sequence with a genetic effect on a DNA (deoxyribonucleic acid) molecule, and is a fragment of a DNA molecule with a genetic effect. Genes can not only transmit genetic information to the next generation through replication, but also enable the expression of genetic information. This genetic information is contained in all human tissues or organs such as human bones, hair, and blood. Genetic testing to identify different individuals through differences in human genomic polymorphism sites has been widely used in forensic and clinical trials.

A variety of genetic markers have recently been developed for individual identification. Among them, Short Tandem Repeats (STR) is considered to be the most standard and authoritative way and method in forensic DNA identification because of its simplicity, rapidity, high accuracy and moderate size. Most practical problems. However, due to high mutation rate STR (typically ^10-2 to ^10-4), a fragment longer detected (100 ~ 400bp), for DNA typing not easy to achieve degraded sample, while the detection sensitivity of the chimeric 5 When the micro-transplant or micro-treatment is clinically performed, the general chimeric rate needs to be maintained at 1 to 2% or less, which cannot meet the requirements.

A chimera refers to a cell mosaic phenomenon in which different genetic traits exist in the same individual, that is, a state in which two or more sets of chromosomes are contained. Among them, microchimerism refers to a phenomenon in which the cellular components of different individual sources are less than 1%. Micro-chimeric detection requires a more sensitive detection method. Therefore, the STR cannot perform micro-chimeric analysis.

With the progress of genomic research, polymorphic sites have received extensive attention. Among them, Single Nucleotide Polymorphism (SNP) has a relatively low spontaneous mutation rate (10 ^-8 ); while a single SNP site amplification product can be controlled below 200 bp, which is beneficial to degradation of samples. Type of. However, the difference between SNP alleles is only one base, so it is difficult to design a highly sensitive detection scheme with a detection sensitivity of only 1%, which is still not suitable for micro-chimeric detection. Moreover, the SNP quantitative method is complicated, the experimental instrument is expensive, the use cost is high, the operation is cumbersome, the flux is small, and it is not suitable for promotion in a conventional forensic laboratory.

In recent years, a new generation of polymorphic loci, Insertion or Deletion Polymorphisms (InDel or DIP), is formed by inserting or deleting DNA fragments of different types and sizes (4-22 bp) in the genome. Polymorphic genetic markers are widely used in forensic evidence identification, paternity testing and other medical tests. From the perspective of forensic evidence testing, the InDel marker has the following characteristics: (1) abundant in the human genome, second only to the SNP; (2) natural mutation rate similar to the SNP (~2.3×10 ^-9 ) (3) The InDel amplified fragment is shorter (less than 160bp), which is more suitable for detecting highly degraded and low-copy DNA templates, increasing the success rate of DNA degradation sample typing; (4) There are significant populations in the InDel locus. And ethnic differences; (5) InDel detection by fluorescence PCR amplification technology, the technical platform is generally applicable; (6) Based on the InDel site, using two-color fluorescence quantitative PCR method to determine the specificity of donor and recipient After the site, quantitative PCR was performed, and the minimum chimerism rate detection was 0.01 to 0.05%. Therefore, in recent years, it has attracted the attention of many forensic scientists at home and abroad. However, to date, commercially available kits for micro-chimeric detection and individual recognition based on InDel genetic markers are rare, and only the Dutch Investigator DIP kit is a relatively mature commercial product. At the same time, the Investigator DIP kit is not particularly applicable to Asian populations due to differences in population and ethnicity of genetic markers. Therefore, it is necessary to develop a set of InDel genetic marker kits for micro-chimeric detection and individual identification for the Asian population.

Summary of the invention

The present invention is directed to the above-mentioned drawbacks existing in the prior art, based on the latest InDel site database, and based on the Asian population, redesigned primers and probes for microchimerism detection and individual recognition, and detection methods. The detection method of the invention has strong polymorphism, good specificity, high sensitivity and is simple and quick.

To this end, one aspect of the invention provides a primer and probe combination for microchimeric detection and individual recognition consisting of the primers and probes set forth in SEQ ID NO. 1-75.

In a preferred embodiment of the invention, the primer and probe combination further consists of a set 1-15 set of primers and probes, wherein the first set consists of the primers set forth in SEQ ID NO. 1-4 and SEQ ID NO. The probe composition shown, the second group consists of the primers shown in SEQ ID NO. 6-9 and SEQ ID NO. The probe composition is shown, the third group consists of the primers shown in SEQ ID NO. 11-14 and the probes shown in SEQ ID NO. 15, and the fourth group consists of the primers shown in SEQ ID NO. 16-19. And probes of SEQ ID NO. 20, group 5 consists of the primers shown in SEQ ID NO. 21-24 and the probes shown in SEQ ID NO. 25, and the sixth group consists of SEQ ID NO. The primer shown in -29 and the probe shown in SEQ ID NO. 30, the seventh group consists of the primer shown in SEQ ID NO. 31-34 and the probe shown in SEQ ID NO. 35, Group 8 It consists of the primers shown in SEQ ID NO. 36-39 and the probes shown in SEQ ID NO. 40, and the ninth group consists of the primers shown in SEQ ID NO. 41-44 and the probe shown in SEQ ID NO. Needle composition, group 10 consists of the primers shown in SEQ ID NO. 46-49 and the probes shown in SEQ ID NO. 50, and the 11th group consists of the primers shown in SEQ ID NO. 51-54 and SEQ ID NO The probe composition shown in .55, the 12th group consists of the primers shown in SEQ ID NO. 56-59 and the probes shown in SEQ ID NO. 60, and the 13th group is represented by SEQ ID NO. 61-64. The primer is composed of the probe shown in SEQ ID NO. 65, and the 14th group is the primer shown in SEQ ID NO. 66-69 and SEQ ID NO. The probe composition shown by 0, the 15th group consists of the primer shown in SEQ ID NO. 71-74 and the probe shown in SEQ ID NO.

The invention is based on the latest InDel locus database, and based on the Asian population, 15 InDel loci for high individual recognition rate of Asian population are selected, which are: N1-1 (rs2308010), N1-2 (rs34855933). , N1-3 (rs4646006), N2-1 (rs3042783), N5-1 (rs1610937), N5-4 (rs1610932), N7-1 (rs16458), N9-1 (rs2308112), N11-1 (rs2307696), N11-2 (rs140790), N13-1 (rs3038530), N13-2 (rs3049448), N14-1 (rs56024302), N16-2 (rs2067204), and N21-1 (rs16663), the rs number in parentheses indicates the bit Point the number in the dbSNP database.

The present invention further designs, for each of the above InDel sites, a common upstream primer (F), a downstream primer (R), an insertion specific primer F (+), and a deletion specific primer F (-), wherein the amplified fragment Amplified Fragment Length (AFL) is the length of the amplified fragment obtained by pairing the primer with the corresponding upstream primer or downstream primer. For example, F(+) and R are used to specifically amplify the insert, R(-) and F. Used to specifically amplify deleted fragments, F and R are used to amplify non-specific fragments (where:

Represents a specific base for insertion/deletion).

The specific primers and probes designed by the present invention are shown in Table 1 for the 15 InDel sites of the high individual recognition rate of the above Asian population.

Table 1. Table of primers and probes of the present invention

Another aspect of the invention provides a kit for microchimeric detection and individual identification, the kit of which A primer and probe combination according to the invention is included. The kit is stored at -20 ° C and consists of the following reagents.

1. Whole blood genomic DNA extraction reagent, purchased from QIAamp DNA Blood Mini kit;

2. RNase-Free ddH ₂ O;

3, 2 × Super Real PreMix reaction solution, purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., article number: FP206;

4, 50 × ROX Reference Dye, purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., article number: FP206;

5. The primer and probe combinations are separately dispensed into the corresponding numbered centrifuge tubes.

In a preferred embodiment of the invention, the concentration of each primer in the kit is 6 [mu]M and the concentration of each probe is 4 [mu]M.

Another aspect of the invention provides the use of the primer and probe combinations of the invention in the preparation of micro-chimeric detection and individual recognition kits.

A further aspect of the invention provides the use of the primers and probe combinations of the invention, or the kits of the invention for micro-chimeric detection and individual recognition.

A final aspect of the invention provides a method of microchimeric detection and individual identification comprising the steps of:

1. Obtain the DNA of the sample to be tested.

DNA extraction can be performed using the QIAamp DNA Blood Mini kit.

2. Using the primer and probe combination of the present invention, or using the kit of the present invention, using the DNA obtained in step 1 as a template, performing fluorescent PCR amplification, and collecting the fluorescent signal.

The system for fluorescence PCR amplification is 20uL: 10uL 2× Super Real PreMix reaction solution (Tiangen Biochemical Technology (Beijing) Co., Ltd., Item No.: FP206); 0.2uL 50×ROX Reference Dye (Tiangen Biochemical Technology (Beijing) Co., Ltd.) , Item No.: FP206); 1uL 6uM primer F(+)/F(-); 1uL 6uM primer R; 1uL 4uM probe; 1uL 20ng/uL DNA; complement the reaction system with RNase-Free ddH ₂ O.

The reaction conditions of the fluorescent PCR were: pre-denaturation at 95 ° C for 15 min, followed by denaturation at 95 ° C for 3 s - 60 ° C for 32 s, for a total of 40 cycles, and the fluorescence signal was collected at 60 ° C.

3. According to the Ct value, the polymorphism difference of the sample to be tested at 15 InDel sites is determined, so that the sample to be tested is subjected to micro-chimeric detection and individual recognition.

The Ct value of each genetic marker is used to judge the range of Ct values of positive and negative samples of each genetic marker. The Ct value of positive samples falls between 24-29, the Ct value of negative samples is above 38, and the Ct values of positive and negative samples differ by more than 12 , indicating that the primers and probes have good specificity.

In a preferred embodiment of the invention, the primers and probes employed are designed for 15 InDel sites with high individual recognition rates for Asian populations.

In a further preferred embodiment of the invention, each primer has a concentration of 6 [mu]M and a concentration of 4 [mu]M per probe.

As apparent from the above description, the present invention has the following advantages as compared with the prior art.

1. Strong polymorphism: Through the 15 InDel loci screened by the present invention, the random frequency selection of 94 samples was tested for gene frequency, and the results showed that the 15 InDel loci alleles were evenly distributed and the polymorphism was strong. Based on the polymorphism information of these 15 InDel sites, microchimeric detection and individual recognition can be performed.

2. Good specificity: The positive and negative samples were detected by the 15 InDel sites screened by the present invention, and the results showed that the specificity was good, the stability was strong, the repeatability was good, and the typing results were accurate (based on the sequencing results). Compare).

3. High sensitivity: When performing chimeric state analysis, 15 InDel sites screened by the present invention are used for fluorescence PCR amplification. When the template amount is only 140 ng, accurate quantitative analysis can be performed, and the minimum chimeric rate detection can be performed. Up to 0.025%.

4. The detection method of the invention adopts double fluorescent label amplification technology, which is simple and fast, and only needs one Applied Biosystems 7500 Real Time PCR System, and the result is automatically analyzed, which can ensure the accuracy of different laboratory data.

DRAWINGS

Figure 1: Schematic diagram of PCR amplification results for the detection of primer and probe specificity of the present invention.

Figure 2: A diagram of PCR amplification results for the individual recognition assay of the kit of the invention.

Fig. 3 is a graph showing the results of detection of the genetic marker N13-1(+) of the present invention.

Figure 4: Amplification plot of the standard (100%, 90%, 50%, 10%, 1%, 0.1%, 0.05%, 0.025%) of the genetic marker N13-1(+) of the present invention.

detailed description

The invention is further illustrated by the following examples, which are intended to illustrate the invention. It is not intended to limit the invention. It is to be noted that a number of modifications and variations of the present invention may be made by those skilled in the art without departing from the scope of the invention.

Example 1: Design and Synthesis of Primers

(1) Screening of InDel sites

The InDel locus is screened using the following principles:

1. The difference in length of the InDel allele fragment (number of inserted or deleted bases) is greater than 3 bp and less than 30 bp;

2, located on 22 human autosomes;

3. The spacing of InDel sites on the same chromosome is greater than 5Mb;

4. The minimum allele frequency (MAF) is between 0.30 and 0.50 (based on the East Asian population);

5, the total number of sites is not less than 30, the cumulative individual recognition rate reaches 0.9999 or more;

6. There is no linkage on the same chromosome, and the distribution in the population is consistent with the Hardy-Weinberg genetic balance;

7. Avoid InDel sites associated with certain disease phenotypes, and polymorphic sites are preferably located in intron regions.

Using the above principles, the polymorphisms are stronger through the URLs shown at http://www.ensembl.org/index.html and http://www.ncbi.nlm.nih.gov/projects/SNP/ respectively. The InDel locus was initially screened, and finally 15 InDel loci were selected: N1-1 (rs2308010), N1-2 (rs34855933), N1-3 (rs4646006), N2-1 (rs3042783), N5- 1 (rs1610937), N5-4 (rs1610932), N7-1 (rs16458), N9-1 (rs2308112), N11-1 (rs2307696), N11-2 (rs140790), N13-1 (rs3038530), N13-2 (rs3049448), N14-1 (rs56024302), N16-2 (rs2067204), N21-1 (rs16663), the rs number in parentheses indicates the number of the site in the dbSNP database.

(2) Sample selection, gene frequency test and primer sequence modification

A total of 94 Chinese population DNA samples were randomly selected for amplification and gene sequencing. 94 healthy human genomic DNA samples were tested using the initially selected primers to assess primer amplification efficiency and gene frequency at each locus.

1, DNA extraction

DNA extraction was performed according to the QIAamp DNA Blood Mini kit, followed by dilution with RNase-Free ddH ₂ O to 20 ng/uL for use.

2. PCR amplification system

25 uL PCR amplification system: 2.5 uL 10×buffer reaction solution; 0.75 uL 10 mM dNTP; 1 uL 5 uM primer F; 1.8 uL 5 uM primer R; 1.4 uL 5 uM primer F(+)/F(-); 2 uL DNA; uL Hot start Taq; 1 uL Mg ²⁺ ; 0.1 uL DMSO, supplemented with RNase-Free ddH ₂ O.

3. PCR reaction conditions

The following PCR reaction conditions were adopted: pre-denaturation at 95 ° C for 30 s, followed by denaturation at 95 ° C for 30 s - 58 ° C for 30 s - 68 ° C for 30 s, for a total of 30 cycles, and finally at 68 ° C for 5 min.

4, electrophoresis detection and primer sequence modification

The PCR product was electrophoresed on 2% agarose for 30 min to verify whether the specific primers were suitable. Based on the sequencing results of the first generation, the primer sequences were modified and retested for the case of electrophoresis false positives and false negatives.

After detection and modification, the primers and probe sequences of the finally determined 15 InDel sites are shown in Table 1. The above primers and probes were synthesized by Shanghai Yingjun Biotechnology Co., Ltd.

94 samples were detected by primers and probes from 15 InDel sites, respectively. The results showed that the allele frequency distribution was relatively balanced. The specific polymorphism information is shown in Table 2. Wherein: "+" stands for insertion polymorphism; "-" stands for deletion polymorphism; "+/+" stands for insertion homozygous polymorphism; "+/-" stands for insertion/deletion heterozygosity polymorphism; "- /-" represents a deletion homozygous polymorphism.

Table 2 Polymorphism information of 15 InDel loci (sequencing results)

Example 2: Primer and probe specificity testing

Under the sequencing results of Example 1, five insertion homozygotes, deletion homozygotes and heterozygotes were selected for fluorescence PCR to detect the specificity of the primers and probes.

1. Fluorescent PCR amplification system

Adopt 20uL fluorescent PCR amplification system: 10uL 2×Super Real PreMix reaction solution (Tiangen Biochemical Technology (Beijing) Co., Ltd., Item No.: FP206); 0.2uL 50×ROX Reference Dye (Tiangen Biochemical Technology (Beijing) Co., Ltd. , Item No.: FP206); 1uL 6uM primer F(+)/F(-); 1uL 6uM primer R; 1uL 4uM probe; 1uL 20ng/uL DNA; complement the reaction system with RNase-Free ddH ₂ O.

2, fluorescent PCR reaction conditions

The following fluorescent PCR reaction conditions were employed: pre-denaturation at 95 ° C for 15 min, followed by denaturation at 95 ° C for 3 s - 60 ° C for 32 s, for a total of 40 cycles, and the fluorescence signal was collected at 60 ° C.

3. Primer and probe specific judgment

The Ct value of each genetic marker is used to judge the range of Ct values of positive and negative samples of each genetic marker. The Ct value of positive samples falls between 24-29, the Ct value of negative samples is above 38, and the Ct values of positive and negative samples differ by more than 12 , indicating that the primers and probes have good specificity.

Taking the N1-2 (+) insertion of the genetic marker as an example, the Ct values of the positive samples (inserted homozygotes and heterozygotes) fall within the range 26-28 (see Figure 1a of the specification), and the negative samples (deleted homozygous) Ct The values were not detectable (i.e., no fluorescence signal was detected at 40 PCR cycles, and the Ct value was greater than 40) (see Figure 1b of the specification), indicating that the primers and probes were of good specificity.

Example 3: Composition of the kit of the present invention

The InDel genetic labeling kit for microchimerism detection and individual recognition of the present invention is stored at -20 ° C and consists of the following reagents.

1. Whole blood genomic DNA extraction reagent: purchased from QIAamp DNA Blood Mini kit.

2. RNase-Free ddH ₂ O.

3, 2 × Super Real PreMix reaction solution, purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., article number: FP206.

4, 50 × ROX Reference Dye, purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., goods No.: FP206.

5. The primer and probe combinations were separately dispensed in the corresponding numbered centrifuge tubes. The concentration of each primer was 6 uM, and the concentration of each probe was 4 uM.

Example 4: The kit of the invention is used for the detection of individual identification

Two different sources of DNA samples (designated A and B, respectively) were randomly selected and polymorphism was detected using the InDel genetic marker in the kit of the present invention.

The fluorescent PCR amplification system and reaction conditions were the same as in Example 2. After the PCR amplification reaction, for each genetic marker, four results may appear after amplification, as shown in Figure 2 of the specification. In Figure 2a, both samples A and B are positive; in Figure 2b, samples A and B are negative; in Figure 2c, sample A is positive and sample B is negative; in Figure 2d, sample A is negative and sample B is negative. Positive. Figures 2a and 2b show no polymorphism differences, and Figures 2c and 2d have polymorphic differences, and it is possible to determine whether the samples are derived from different individuals based on the polymorphism differences.

According to the Ct value, among the 15 InDel sites, sample A and sample B have 14 genetic markers (N1-1(+), N1-2(-), N1-3(-), N2-1( +), N2-1(-), N5-4(+), N7-1(+), N9-1(+), N11-1(-), N11-2(-), N13-1(+ ), N13-1 (-), N14-1 (+), and N16-2 (-) have polymorphic differences. Therefore, it can be judged that the sample A and the sample B are derived from different individuals.

Example 5: Primer and probe accuracy and sensitivity tests

40 pairs of DNA samples were selected for qualitative detection of InDel genetic markers to obtain InDel locus polymorphism information for each pair of samples.

For each pair of samples, select the available genetic markers (the available genetic markers should meet the following characteristics: Ct<28 positive samples are designated as recipients, Ct>38 negative samples are designated as donors), mixed in different proportions, and different ratios are obtained. The standard is tested for sensitivity and accuracy by fluorescence PCR. Meanwhile, in order to achieve a detection sensitivity of 10 ^-5 , the amount of the initial DNA template is at least 140 ng.

Take a pair of samples as an example (the samples are named C and D), and the specific implementation method is as follows.

1. Perform InDel locus polymorphism test on sample C and sample D.

The fluorescent PCR reaction system and conditions were the same as in Example 2. With sample C as the recipient and sample D as the donor, the available genetic markers were selected according to the size of the Ct value (C: Ct < 28 positive samples; D: Ct > 38 negative samples).

2, standard product construction

For sample C and sample D, mix in different proportions (recipient C/donor D: 90%, 50%, 10%, 1%, 0.1%, 0.05%, 0.025%), a standard of 20 ng/uL in different ratios was obtained.

3. Detect the accuracy and sensitivity of primers and probes by quantitative analysis

Standards prepared with chimeric ratios of 100%, 90%, 50%, 10%, 1%, 0.1%, 0.05%, and 0.025% were used as templates for quantitative analysis, and RNase-Free ddH ₂ O was used as a template. As a blank control. In order to reach the 140 ng DNA initial template (detection sensitivity of 10 ^-5 ), 7 uL of 20 ng/uL standard was added, and other fluorescent PCR reaction systems and conditions were the same as in Example 2.

4. Quantitative analysis

By PCR, the Ct values of the genetic markers and the internal reference genes detected by different ratios of the standards were obtained, and the difference (ΔCt) of the Ct values obtained by the two was calculated.

Figure 3 of the specification shows the detection results of the genetic marker N13-1(+). As can be seen from Figure 3, the genetic marker N13-1(+) and the internal reference gene amplification curve were in good shape, the PCR product was highly specific, and there was no non-specific amplification, while the control blank group had no amplification.

Figure 4 is a standard amplification curve of the genetic marker N13-1(+). The figure shows that the standard amplification curve is in good shape. Using C as a pre-transplant sample, CD as a post-transplant sample, according to the difference (ΔΔCt) between post-transplant and pre-transplant ΔCt, the CD mosaic rate %=2 ^-ΔΔCt ×100% can be calculated, where ΔΔCt=ΔCt _{Post-transplantation-} ΔCt pre- _{transplantation} = ( _after Ct- _{tag gene transplantation-} Ct _{internal reference gene transplantation} )-( _pre- Ct _{marker gene transplantation-} Ct _{internal reference gene transplantation} ). The test values obtained by comparing theoretical values with fluorescent PCR can be used to determine the sensitivity and accuracy of each genetic marker.

For the N13-1(+) genetic marker, the test values were 93.34% (theoretical 90%), 65.19% (theoretical 50%), 13.25% (theoretical 10%), 0.9452% (theoretical 1%), and 0.1053% (theoretical). 0.1%), 0.0598% (theoretical 0.05%) and 0.0282% (theoretical 0.025%), indicating that the primers and probes have good test accuracy, and the sensitivity can reach about 0.025%.

Claims (10)

1. A primer and probe combination for microchimeric detection and individual recognition consisting of the primers and probes set forth in SEQ ID NO. 1-75.
2. The primer and probe combination according to claim 1, further comprising a group 1-15 primer and a probe, wherein the first group is the primer set forth in SEQ ID NO. 1-4 and the SEQ ID NO. The probe composition is shown, the second group consists of the primers shown in SEQ ID NO. 6-9 and the probes shown in SEQ ID NO. 10, and the third group consists of the primers shown in SEQ ID NO. 11-14 and The probe composition shown in SEQ ID NO. 15 is composed of the primer set forth in SEQ ID NO. 16-19 and the probe represented by SEQ ID NO. 20, and the fifth group consists of SEQ ID NO. 21- The primer shown in Figure 24 and the probe shown in SEQ ID NO. 25, the sixth group consists of the primer shown in SEQ ID NO. 26-29 and the probe shown in SEQ ID NO. 30, and the seventh group consists of The primer shown in SEQ ID NO. 31-34 and the probe shown in SEQ ID NO. 35, the eighth group consists of the primer shown in SEQ ID NO. 36-39 and the probe shown in SEQ ID NO. Composition, Group 9 consists of the primers shown in SEQ ID NO. 41-44 and the probes shown in SEQ ID NO. 45, and Group 10 consists of the primers shown in SEQ ID NO. 46-49 and SEQ ID NO. The probe composition shown in Figure 50, the 11th group is represented by the primers shown in SEQ ID NO. 51-54 and SEQ ID NO. Probe composition, group 12 consists of the primers shown in SEQ ID NO. 56-59 and the probes shown in SEQ ID NO. 60, and the 13th group consists of the primers and SEQ ID shown in SEQ ID NO. 61-64. The probe composition shown in NO. 65, the 14th group consists of the primers shown in SEQ ID NO. 66-69 and the probe shown in SEQ ID NO. 70, and the 15th group is represented by SEQ ID NO. 71-74. The primer shown is composed of the probe shown in SEQ ID NO.
3. The primer and probe combination according to claim 1 or 2, which is designed for 15 InDel sites with high individual recognition rate in Asian populations.
4. A kit for microchimeric detection and individual recognition comprising the primer and probe combination of any of claims 1-3.
5. The kit according to claim 4, wherein each primer has a concentration of 6 μM and each probe has a concentration of 4 μM.
6. Use of the primer and probe combination of any of claims 1-3 in the preparation of a microchimeric detection and individual recognition kit.
7. Use of the primer and probe combination according to any one of claims 1 to 3, or the kit of claim 4 or 5 in microchimeric detection and individual recognition.
8. A microchimeric detection and individual identification method comprising the following steps:

   (1) obtaining DNA of the sample to be tested;

   (2) using the primer and probe combination according to any one of claims 1 to 3, or using the kit according to claim 4 or 5, using the DNA obtained in the step (1) as a template for performing fluorescent PCR Amplify and collect fluorescent signals;

   (3) According to the Ct value, the polymorphism difference of the sample to be tested at 15 InDel sites is determined, so that the sample to be tested is subjected to micro-chimeric detection and individual recognition.
9. The method of claim 8 wherein the primers and probes employed are designed for 15 InDel sites for high individual recognition rates in Asian populations.
10. The method according to claim 8 or 9, wherein the concentration of each primer is 6 μM, and the concentration of each probe is 4 μM.

Images