A method for chromosome copy number counting based on relative fluorescence intensity between chromosome monochromatids

Abstract

This invention provides a chromosome copy number counting method based on the relative fluorescence intensity between chromosome monosoms. This method leverages the built-in genetic markers of different chromosomes in multiplex STR typing systems. By introducing a control sample with known ploidy, and considering that the relative fluorescence intensity between two chromosome monosoms is a constant value, the ploidy of chromosomes with unknown ploidy in the test sample is calculated. This method yields accurate results that can be cross-validated with ploidy determinations based on genetic marker typing, eliminating the need for further screening of other polymorphic genetic markers or additional experimental studies to determine the ploidy of the chromosome. Furthermore, it can serve as a basis for developing corresponding software for multiplex STR typing systems. Developing corresponding analytical software based on this calculation scheme can significantly reduce manual intervention in data analysis, improve the accuracy of analytical results, and significantly reduce experimental workload.

Description

Technical Field

[0001] This invention relates to the field of gene detection, specifically to a chromosome copy number counting method based on the relative fluorescence intensity between chromosome monosoms. Background Technology

[0002] Short tandem repeats (STRs) are highly polymorphic genetic markers widely distributed in eukaryotic genomes. They typically consist of a 2–6 bp core repeat unit arranged in tandem repeats, with the repeat count usually between 10 and 60. Different alleles of a STR are usually named using the variable number of repeats in the core repeat unit. Due to their wide distribution, high polymorphism, and ease of detection, STRs have been widely used in many fields, including genetic mapping, gene localization, cell line identification, forensic kinship and individual identification, and genetic disease diagnosis.

[0003] In medical genetics, one of the important applications of STRs is to rapidly determine the ploidy (ploidy number) of a chromosome by typing multiple STR genetic markers on that chromosome. Since 2001, Kathy Mann et al. from St. Thomas' Hospital in London, UK, have been dedicated to the application of STRs in chromosome ploidy research, and in 2012 published a landmark review article in this field (QF-PCR: application, overview and review of the literature. Kathy Mann and Caroline Mackie Ogilvie, Prenatal Diagnosis 2012, 32, 309-314.). In this review article, Kathy Mann et al. summarized the test results of more than 95,000 cases and presented a schematic diagram of STR genotype peaks and chromosome ploidy. Figure 1Based on this, an international guideline for the diagnosis of aneuploidy using QF-PCR was developed: QF-PCR for the diagnosis of aneuploidy best practice guidelines (2012) v3.01; and published by the British Association for Clinical Cytogenetics and Clinical Molecular Genetics (hereinafter referred to as the International Guideline). In 2016, the China Collaborative Group on the Application of Quantitative Fluorescent PCR Technology in Prenatal Diagnosis, based on the International Guideline, developed the Chinese version of "Expert Opinions on the Application of Quantitative Fluorescent PCR Technology in Prenatal Diagnosis" (hereinafter referred to as Expert Opinions), which was published in the May 2016 issue of the Chinese Journal of Obstetrics and Gynecology, Volume 51, No. 5. It can be said that the STR typing system based on multiplex fluorescent PCR combined with capillary electrophoresis (QF-PCR) has become one of the fundamental molecular testing tools for chromosome ploidy testing.

[0004] pass Figure 1 As can be seen from the schematic diagram, when multiple alleles are detected on a STR, the ploidy of the chromosome it belongs to can be intuitively indicated. When a bimodal pattern with a relative fluorescence intensity of approximately 1:1 is detected, it indicates that the chromosome it belongs to is disomy. When a relative fluorescence intensity of approximately 1:1:1 or 2:1 is detected, it indicates that the chromosome it belongs to is trisomy.

[0005] However, as Figure 1 As shown, when a STR detects only one allele (i.e., when the STR exhibits a single-peak pattern), the STR will not provide effective information about the ploidy of its chromosome. This is because when the chromosome containing the STR is monosomic, only a single-peak pattern will be detected; when the chromosome containing the STR is disomic or trisomic, if the STR's genotype is homozygous, its peak pattern will also be single-peaked.

[0006] Therefore, to improve the efficiency of STR testing, multiple STR genetic markers are usually tested simultaneously for the same chromosome. Both international guidelines and Chinese expert opinions require that at least four highly polymorphic STR genetic markers be tested for the same chromosome.

[0007] Even so, taking a heterozygosity of 0.6 for each STR (meaning 60% of normal individuals show an approximately 1:1 bimodal pattern on this STR) as an example, even if four STRs are tested, approximately 2.56% of individuals may still show homozygosity for all four STRs, i.e., a unimodal pattern, making it impossible to determine the ploidy of the chromosomes containing these four STRs. Based on international guidelines and Chinese expert opinions regarding ploidy criteria (e.g., in determining diploidy, at least two STR genetic markers need to show an approximately 1:1 bimodal pattern), approximately 25.6% of individuals will show only one approximately 1:1 bimodal pattern among the four STRs, with the others showing unimodal patterns, making a definitive determination impossible. Even if STRs with higher heterozygosity are selected, such as four STR loci with a heterozygosity of 0.7, the probabilities of the above two scenarios still reach 0.8% and 10.8%, respectively.

[0008] In such cases, one solution is to test the sample for more STR genetic markers in order to provide more information about the chromosome ploidy. However, this solution inevitably increases the workload and requires the development of more STR genetic markers. Summary of the Invention

[0009] To overcome the shortcomings of existing technologies, this invention provides a chromosome copy number counting method based on the relative fluorescence intensity between chromosome monosoms. Using this method, when all STRs on a chromosome exhibit a single-peak pattern during multiple STR genotyping, the ploidy of the chromosome containing that STR can be counted based on the relative fluorescence intensity of the single-peak pattern, thereby avoiding additional testing workload. The principle underlying this method is as follows:

[0010] Even when all tested STR genetic markers exhibit a single-peak pattern, the relative fluorescence intensity (peak height or peak area) of this single-peak pattern still contains ploidy information of the chromosome; that is, the relative fluorescence intensity of the single-peak pattern is related to the ploidy number of the chromosome. It is known that 1 ng of human genomic DNA is approximately equivalent to 150 cells. When a chromosome is haploid, 1 ng of genomic DNA contains approximately 150 copies; when it is diploid, approximately 300 copies; and when it is triploid, approximately 900 copies. Therefore, when a fixed amount of genomic DNA template is added, for single-peak genotyping results, the initial copy number of the PCR template will be significantly different depending on whether the chromosome containing the STR is monosomic, disomymic, or trisomic, resulting in different relative fluorescence intensities of the obtained single-peak pattern.

[0011] To achieve the above objectives, the present invention adopts the following technical solution:

[0012] A first aspect of the present invention provides a chromosome copy number counting method based on the relative fluorescence intensity between chromosome monosoms, comprising the following steps:

[0013] Step 1: Select a sample with known ploidy numbers of chromosome A and chromosome B as a control sample. Use a multiplex STR typing system to detect the sum of relative fluorescence intensities H1 of M1 genetic markers on chromosome A with ploidy number K1 and the sum of relative fluorescence intensities H2 of M2 genetic markers on chromosome B with ploidy number K2 in the control sample. Calculate the relative fluorescence intensity RFUM between chromosome monosoms using the following formula (1):

[0014]

[0015] Step two: Simultaneously, the test sample is examined using the aforementioned multiple STR genotyping system. Based on the genotyping results, chromosome B, whose ploidy can be clearly determined, is used as the internal reference chromosome, and its ploidy number is denoted as K. ref The sum of the relative fluorescence intensities of the above M2 genetic markers is denoted as H. ref In this sample to be tested, the ploidy number of chromosome A, whose ploidy cannot be clearly determined based on the typing results, is denoted as K. un The sum of the relative fluorescence intensities of the above M1 genetic markers is denoted as H. 待测 ;

[0016] Step 3: Calculate the ploidy number K of the chromosome with unknown ploidy using formula (2). un :

[0017]

[0018] Furthermore, both M1 and M2 mentioned above are greater than or equal to 4.

[0019] Furthermore, the ploidy number in the sample to be tested is not determined by the criteria for determining the ploidy number of chromosomes.

[0020] K un When the value is ≤1.45, the ploidy number of the chromosome being tested is 1;

[0021] 1.55≤K un When the value is ≤2.45, the ploidy number of the chromosome being tested is 2;

[0022] 2.55≤K un When the value is ≤3.45, the ploidy number of the chromosome to be tested is 3.

[0023] Furthermore, in step one above, RFUM was measured using multiple control samples, and the average value was substituted into formula (2) to calculate K. un .

[0024] Furthermore, the aforementioned relative fluorescence intensity refers to peak height or peak area.

[0025] Furthermore, the control samples mentioned above were normal samples with a chromosome ploidy number of 2.

[0026] A second aspect of the present invention is to provide a product for chromosome copy number counting based on the above-described method.

[0027] The present invention adopts the above technical solution and has the following technical effects compared with the prior art:

[0028] This invention provides a chromosome ploidy counting scheme based on the relative fluorescence intensity of genetic markers for chromosome ploidy testing. According to this scheme, even when all tested genetic markers are unimodal, the ploidy of the chromosome containing the genetic marker can be accurately determined without the need for screening other polymorphic genetic markers or conducting additional experimental studies to determine the ploidy of the chromosome.

[0029] In this scheme, the inventors creatively proposed and proved an inherent characteristic parameter of a multiplex STR genotyping system—the relative fluorescence intensity (RFUM) between chromosome monosoms. Based on this characteristic parameter, combined with an external reference sample, the ploidy number of the chromosome to be tested in the sample can be obtained accurately and can be cross-validated with the ploidy determination results based on the genetic marker genotyping results themselves. This scheme can also serve as a basis for the development of corresponding software for multiplex genotyping systems. Developing corresponding analytical software based on this calculation scheme will significantly reduce manual intervention in data analysis, improve the accuracy of analytical results, and significantly reduce experimental workload. Attached Figure Description

[0030] Figure 1 It is a schematic diagram of STR genotype peaks and chromosome ploidy in existing technology;

[0031] Figure 2 This is a schematic diagram of the genetic marker typing pattern on the chromosome with undetermined ploidy of the sample to be tested and the internal reference chromosome in one embodiment of the present invention;

[0032] Figure 3 This is an STR typing result diagram of chromosomes 18 and 13 of sample 242 in one embodiment of the present invention;

[0033] Figure 4 This is an STR typing result diagram of chromosomes 18 and 13 of sample 241 in one embodiment of the present invention;

[0034] Figure 5 This is an STR typing result diagram of chromosomes 18 and 13 of sample 243 in one embodiment of the present invention;

[0035] Figure 6 This is an STR typing result diagram of chromosomes 18 and 13 of sample 244 in one embodiment of the present invention;

[0036] Figure 7 This is an STR typing result diagram of chromosomes 18 and 13 of sample 245 in one embodiment of the present invention;

[0037] Figure 8 This is a diagram showing the STR typing results of the X chromosome and chromosome 21 in the FAM and HEX fluorescence channels of the sample 2-1 to be tested in one embodiment of the present invention.

[0038] Figure 9 This is a diagram showing the STR typing results of the X chromosome and chromosome 21 in the FAM and HEX fluorescence channels of control sample 3-1 in one embodiment of the present invention.

[0039] Figure 10 This is a diagram showing the typing results of the X chromosome and chromosome 21 in the FAM and HEX fluorescence channels of control sample 8-1 in one embodiment of the present invention.

[0040] Figure 11 This is a diagram showing the STR typing results of chromosome 11-1X and chromosome 21 in one embodiment of the present invention;

[0041] Figure 12 This is a diagram showing the STR typing results of the X chromosome and chromosome 21 in the FAM and HEX fluorescence channels of sample 9-1 in one embodiment of the present invention.

[0042] Figure 13 This is an STR typing result diagram of chromosome 5-1X and chromosome 21 of the sample to be tested in one embodiment of the present invention;

[0043] Figure 14 This is the STR typing result of chromosomes 18 and 13 in the TAMRA and ROX fluorescence channels of sample 3-1 in one embodiment of the present invention;

[0044] Figure 15 This is the STR typing result of chromosomes 18 and 13 in the TAMRA and ROX fluorescence channels of control sample 8-1 in one embodiment of the present invention;

[0045] Figure 16 This is the STR typing result of chromosomes 18 and 13 in the TAMRA and ROX fluorescence channels of control sample 11-1 in one embodiment of the present invention;

[0046] Figure 17 This is the STR typing result of chromosomes 18 and 13 in the TAMRA and ROX fluorescence channels of control sample 9-1 in one embodiment of the present invention;

[0047] Figure 18This is the STR typing result of chromosomes 18 and 13 in the TAMRA and ROX fluorescence channels of sample 10-1 in one embodiment of the present invention. Detailed Implementation

[0048] This invention provides a method for chromosome copy number counting based on the relative fluorescence intensity between chromosome monosoms. The invention is described in detail below with specific embodiments to facilitate a better understanding; however, these embodiments do not limit the scope of the invention.

[0049] Example 1

[0050] This embodiment provides a chromosome copy number counting method based on the relative fluorescence intensity between chromosome monosoms. This method leverages the characteristic of multiplex STR genotyping systems that include internal reference genetic markers for different chromosomes. When a normal external reference sample (i.e., a control sample) with normal ploidy is introduced, the ploidy number is determined by combining the internal reference genetic marker with the normal external reference sample, based on the relative fluorescence intensity of alleles of genetic markers on chromosomes with undetermined ploidy. During this process, when different samples are tested using the same multiplex STR genotyping system, since the multiplex STR genotyping system is fixed, the ratio of the relative fluorescence intensity (H1) of multiple genetic markers on chromosome A (ploidy number K1) to the relative fluorescence intensity (H2) of multiple genetic markers on chromosome B (ploidy number K2) will be a relatively constant. The inventors creatively introduce the concept of relative fluorescence intensity (RFUM) between chromosome monosoms, which can be defined as the ratio of the relative fluorescence intensity of one chromosome monosomy A to that of another chromosome B. RFUM can be measured using the following formula:

[0051]

[0052] Based on the above, the specific steps of this method are as follows:

[0053] Suppose a multiplex STR genotyping system contains genetic markers from multiple chromosomes. This system is used to perform genotyping on a sample. If the peak patterns of M1 genetic markers on a single chromosome cannot determine its ploidy, its ploidy number is denoted as K. un (Karyotype of undetermined chromosome), the sum of the relative fluorescence intensities of the M1 genetic markers on the chromosome under test is denoted as H. 待测 .

[0054] In the sample to be tested, the ploidy can be determined based on the M2 genetic markers on another chromosome. This chromosome can then be used as an internal reference chromosome, and its ploidy number is denoted as K. ref (Karyotype of reference chromosome), the sum of the relative fluorescence intensities of all genetic marker alleles on the internal reference chromosome is denoted as H. ref .

[0055] refer to Figure 2 If a chromosome in a sample has four genetic markers, and only one marker shows a 1:1 peak morphology while the other three show a single peak morphology (or all three show a single peak morphology), then the ploidy of that chromosome in the sample cannot be determined based solely on the genetic marker peak morphology results. The ploidy number is denoted as K. un Of the four genetic markers on the internal reference chromosome in the test sample, three showed a 1:1 peak pattern, confirming that the ploidy of this chromosome in the test sample is 2-somatic, i.e., K. ref =2. H1 is the sum of the relative fluorescence intensities of the five alleles of the four genetic markers on the chromosome with unknown ploidy; H2 is the sum of the relative fluorescence intensities of the seven alleles of the four genetic markers on the internal reference chromosome.

[0056] According to the above formula (1), K is obtained. un The calculation formula is as follows (2):

[0057]

[0058] RFUM can be obtained by using the multiple typing system to measure the sum of the relative fluorescence intensities of the M1 genetic markers on chromosome A with known ploidy and the M2 genetic markers on chromosome B with known ploidy, and then substituting these values ​​into equation (1) above. Specifically, at least three normal control samples can be tested. The RFUM value of each control sample is calculated using equation (1), and the arithmetic mean of the RFUM values ​​of each sample is recorded as follows: Will Substituting into equation (2) yields the ploidy number K of chromosomes with unknown ploidy in the sample to be tested. un Since the multiples can only take the integers 1, 2, and 3, the theoretical limit values ​​can be given as follows:

[0059] K un When the value is ≤1.45, the ploidy number of the chromosome with unknown ploidy is 1;

[0060] 1.55≤K un When the value is ≤2.45, the ploidy number of the chromosome with unknown ploidy is 2;

[0061] 2.55≤K un When the value is ≤3.45, the ploidy number of the unknown ploidy chromosome is 3.

[0062] Specifically:

[0063] 1. If a multiplex amplification system consisting of four fluorescence channels, FAM, HEX, TAMRA, and ROX, is used, RFUM calculations can be performed between the two fluorescence channels TAMRA and ROX, and between the two fluorescence channels FAM and HEX. However, mixed channel calculations between FAM, HEX, TAMRA, and ROX are not recommended.

[0064] 2. The typing tests of the samples to be tested and the normal control samples should be conducted using reagents from the same batch.

[0065] 3. The internal reference chromosomes in the test sample and the normal control sample must be identical and their ploidy number must be known.

[0066] To better understand the method of Example 1, several examples are now provided to further verify the effectiveness of the method. The kit used to verify the chromosome copy number counting scheme proposed in the following examples is the AneuFiler kit developed by a certain company, which includes 6 STR genetic markers on chromosome 13, 6 STR genetic markers on chromosome 18, 8 STR genetic markers on chromosome 21, and 10 genetic markers for determining the ploidy of sex chromosomes (X and Y chromosomes).

[0067] Verification Example 1

[0068] Five samples (samples 241-245) were tested using this kit. In sample 242, the TAMRA channel showed that among the six STR genetic markers on chromosome 18, only one STR exhibited an approximately 1:1 bimodal pattern, while the other five were unimodal. Based on international guidelines, no conclusion could be drawn regarding the ploidy of chromosome 18 in this sample. In the ROX channel of this sample, all six STR genetic markers on chromosome 13 showed an approximately 1:1 bimodal pattern. Based on international guidelines, this confirmed that chromosome 13 in this sample was diploid, i.e., ploidy number K. ref =2.

[0069] Samples 243, 244, and 245 were used as control samples, and sample 241 was used as a known ploidy sample for verification. According to international guidelines, the ploidy of chromosomes 18 and 13 in these four samples can be clearly identified as disomy, i.e., the ploidy number is 2.

[0070] Figures 3 to 7 The genotyping results for these 5 samples are shown in Table 1. Using the calculation scheme provided in Example 1, the ploidy number of chromosome 18 in sample 242 is calculated.

[0071] Table 1. Calculation results of ploidy of chromosome 18 in sample 242.

[0072]

[0073] The calculated ploidy number of chromosome 18 in sample 242 was 2; the calculated ploidy number of chromosome 18 in verification sample 241 was also 2, which is consistent with the judgment result based on the STR genotyping result.

[0074] Verification Example 2

[0075] Normal males have one X chromosome; normal females have two X chromosomes. That is, the ploidy number of the X chromosome in normal females is 2, while in males it is 1. Humans are diploid; in normal individuals, chromosome 21 is disomyized, meaning the ploidy number of chromosome 21 is 2.

[0076] This kit examines the distribution of genetic markers on the X and 21 chromosomes in the FAM and HEX fluorescence channels. Figures 8-12 To determine the test results of a set of samples using this multityping system, it is proposed to... Figure 8 The ploidy number of the X chromosome in the sample to be tested is shown. Figures 9-11 Three male control samples were used, with an X chromosome ploidy of 1; the normal female X chromosome ploidy used to validate the results was 2. Figure 12 In all 5 samples, the internal reference chromosome 21 was identified as disomy (K) based on the 8 STR typing results. ref =2.

[0077] Based on the genotyping results, the ZFXY locus in sample 2-1 showed no Y signal, indicating it was a female sample. The ploidy count of the X chromosome in sample 2-1, calculated using the method provided in Example 1, is shown in Table 2.

[0078] Table 2.2-1 Calculation results of X chromosome ploidy in the sample

[0079]

[0080] In Table 2: genetic markers located in the FAM fluorescence channel are shown in italics, and genetic markers located in the HEX fluorescence channel are shown in bold. Among the X chromosome genetic markers, according to the kit instructions, the alleles corresponding to the gray background do not belong to the X chromosome segment, but are Y chromosome segments (ZFXY site) or other chromosomes (c3 or c11 alleles), and therefore are not included in the calculation of H1.

[0081] Based on the above calculation results, the ploidy number of chromosome X in sample 2-1 is 1; the ploidy number of chromosome X in verification sample 9-1 is 2, which is consistent with the results of STR genotyping and phenotype.

[0082] Verification Example 3: Calculation of Chromosome 21 ploidy number

[0083] The kit was used to perform genotyping on a sample 5-1. Figure 13 The results show that the typing of the eight genetic markers on chromosome 21 is unimodal, and the chromosome ploidy cannot be confirmed according to international guidelines.

[0084] This sample and the sample in Example 2 are used as synchronous test samples, and the control sample in Example 2 is also used for comparison. Figure 13 The ploidy of chromosome 21 in sample 5-1 was tested, and the results are shown in Table 3. When testing the ploidy of chromosome 21, the X chromosome was used as an internal reference chromosome. In this case, the ploidy of the X chromosome in males was 1, and the ploidy of the X chromosome in females was 2.

[0085] Table 3. Calculation results of ploidy of chromosome 21 in sample 5-1.

[0086]

[0087] In Table 3: genetic markers located in the FAM fluorescence channel are shown in italics, and genetic markers located in the HEX fluorescence channel are shown in bold. For X chromosome genetic markers, according to the kit instructions, the alleles corresponding to the gray background do not belong to the X chromosome segment, but are Y chromosome segments (ZFXY site) or other chromosomes (c3 or c11 alleles), and therefore are not included in the calculation of H2.

[0088] Sample 5-1, ZFXY, shows Y chromosome signal and X chromosome ploidy number K. ref Take 1; verify that sample 9-1 is a normal female sample, X chromosome ploidy number K ref Take 2.

[0089] Using the scheme of this invention, the ploidy number of chromosome 21 in sample 5-1 was calculated to be 1; the ploidy number of chromosome 21 in normal female sample 9-1 was calculated to be 2, which is consistent with the STR typing result of chromosome 21 in this sample and the phenotype is also consistent.

[0090] Verification Example 4: Calculation of Trisomy Plurals

[0091] Figures 14 to 17 The kit was used to determine the STR typing results of chromosomes 18 and 13 in the TAMRA and ROX fluorescence channels of the control and validation samples in Example 2. Figure 18 To verify the STR typing results of sample 10-1.

[0092] Based on STR typing and international guidelines, chromosome 13 of the sample to be tested (3-1) can be identified as trisomy 13, i.e., its ploidy number is 3. This embodiment uses this sample to verify the calculation effect of the present invention when the ploidy number is 3. The calculation results are shown in Table 4.

[0093] Table 4. Calculation results of ploidy of chromosome 21 in sample 3-1.

[0094]

[0095] Based on the STR typing of chromosome 18 and international guidelines, the ploidy number of the internal reference chromosome 18 (test sample, control sample, and validation sample) can all be determined to be 2, i.e., K. ref The value is 2.

[0096] Based on STR typing and international guidelines, the ploidy number of chromosome 13 in the control sample was confirmed to be 2.

[0097] The sample tested showed an approximately 1:1:1 trimodal chromosome 13 pattern. According to international guidelines, the ploidy number of chromosome 13 can be confirmed as 3.

[0098] Based on STR typing and international guidelines, the ploidy number of chromosome 13 in the verification sample was confirmed to be 2.

[0099] Using the scheme provided in Example 1, the ploidy number of chromosome 13 in sample 3-1 was calculated to be 3; the ploidy number of chromosome 13 in validation sample 10-1 was calculated to be 2, both of which are consistent with the judgment results based on STR typing and international guidelines.

[0100] The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. A method for counting chromosome copy numbers based on the relative fluorescence intensity between chromosome monosoms, characterized in that, Includes the following steps: Step 1: Select samples with known ploidy numbers of chromosome A and chromosome B as control samples. Use a multiplex STR typing system to detect the sum of relative fluorescence intensities (H1) of M1 genetic markers on chromosome A with ploidy number K1 and the sum of relative fluorescence intensities (H2) of M2 genetic markers on chromosome B with ploidy number K2 in the control samples. Calculate the relative fluorescence intensity (RFUM) between chromosome monosoms using the following formula: Formula 1; Step two: Simultaneously, the multiple STR genotyping system is used to examine the test sample. Based on the genotyping results, chromosome B, whose ploidy can be clearly determined, is used as the internal reference chromosome, and its ploidy number is denoted as K. ref The sum of the relative fluorescence intensities of the M2 genetic markers mentioned above is denoted as H. ref In the sample to be tested, the ploidy number of chromosome A, for which the ploidy cannot be clearly determined based on the typing results, is denoted as K. un， The sum of the relative fluorescence intensities of the M1 genetic markers mentioned above is denoted as H. 待测 ; Step 3: Calculate the ploidy number K of the chromosome with unknown ploidy using Equation 2. un : Formula 2; Among them, M1 and M2 are both greater than or equal to 4; Among them, the ploidy number in the sample to be tested is not determined by the criteria for determining the ploidy number of chromosomes: At that time, the ploidy number of the chromosome to be tested was 1; At that time, the ploidy number of the chromosome to be tested was 2; At that time, the ploidy number of the chromosome to be tested was 3.

2. The method according to claim 1, characterized in that, In step one, RFUM is measured using multiple control samples, and the average value is substituted into Equation 2 to calculate K. un .

3. The method according to claim 1, characterized in that, The relative fluorescence intensity is the peak height or peak area.

4. The method according to claim 1, characterized in that, The control sample was a normal sample with a chromosome ploidy of 2.

5. A product for chromosome copy number counting based on the method described in any one of claims 1-4.

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