A primer and probe combination for detecting extremely unbalanced mixed samples using non-recombinant locus on X and Y chromosomes and its application

Abstract

The present invention discloses a primer and probe combination for detecting extremely imbalanced mixed samples by using non-recombinant region loci of X and Y chromosomes, and an application thereof, belonging to the technical field of forensic detection. The primer and probe combination of the present invention includes a primer and probe for amplifying the X chromosome, and a primer and probe for amplifying the Y chromosome. The present invention has developed a primer and probe combination for the non-recombinant regions of X and Y chromosomes, and established a simple, efficient and sensitive digital PCR detection method, providing a new technical means for the detection of extremely imbalanced mixed samples in forensic medicine.

Description

Technical Field

[0001] The present invention relates to the technical field of forensic detection, and particularly to a primer and probe combination for detecting extremely unbalanced mixed samples by using X and Y chromosome non-recombinant region loci and an application thereof. Background Art

[0002] Detecting DNA genetic markers in biological samples for individual identification and kinship determination is an important part of forensic research. In the process of forensic practice, a small amount of DNA traces of a criminal suspect collected during a crime scene investigation may be masked by a large amount of DNA of the victim, and these small samples can provide key information to prove the guilt of the criminal. Especially in sexual crime cases and violent cases, mixed stains composed of male semen and female victim's vaginal fluid and mixed bloodstains of multiple individuals are often collected. A mixed stain refers to a stain formed by the mixture of blood, body fluids or secretions of two or more individuals. When these samples are detected by capillary electrophoresis of traditional short tandem repeats (STRs), due to the unbalanced mixture of DNA, trace minor components present in such mixed DNA samples are usually missed, posing great challenges to the discovery and further detection of evidence.

[0003] The research on mixed stains mainly focuses on two aspects. One is to separate the components of the mixed stain through experimental techniques to make the mixed sample a single sample for detection and analysis. The other is to compare with reference samples and use means such as mathematical calculation and statistical analysis to obtain as much valuable information as possible to achieve the purpose of identifying the source of mixed DNA that cannot be obtained from a single sample. However, it is still challenging to detect low-abundance DNA fragments in unbalanced mixed DNA using these methods. To solve the problem of detecting unbalanced mixed samples, researchers have successively proposed new genetic markers such as deletion-insertion polymorphism-short tandem repeat (DIP-STR), single nucleotide polymorphism-short tandem repeat (SNP-STR), and microhaplotype (MH) that can more sensitively detect the low-abundance part in unbalanced mixed samples. These genetic markers have been proven to be an effective tool for detecting unbalanced forensic samples. However, the combination of the above genetic markers and traditional detection techniques is only applicable to the detection of unbalanced mixed samples within 1:1000 and cannot detect extremely unbalanced mixed samples. Moreover, due to the cumbersome operation, these methods are not suitable for discovering minor components in mixed samples and determining the mixing ratio of extremely unbalanced mixed samples. Summary of the Invention

[0004] The object of the present invention is to provide a primer and probe combination for detecting extremely imbalanced mixed samples by using non-recombinant locus of X and Y chromosomes, and its application, so as to solve the problems existing in the above-mentioned prior art. The present invention develops a primer and probe combination for non-recombinant regions of X and Y chromosomes, and establishes a simple, efficient and sensitive digital PCR detection method, providing a new technical means for the detection of extremely imbalanced mixed samples in forensic medicine.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a primer and probe combination for detecting extremely imbalanced mixed samples by using non-recombinant locus of X and Y chromosomes, and the primer and probe combination includes primers and probes for amplifying X chromosome, and primers and probes for amplifying Y chromosome;

[0007] The primers and probes for amplifying X chromosome include Homochr-X1-230817-F, Homochr-X1-230817-R and Homochr-X1-230817-P whose sequences are shown in SEQ ID NO.1-3 respectively;

[0008] The primers and probes for amplifying Y chromosome include Homochr-Y1-230817-F, Homochr-Y1-230817-R and Homochr-Y1-230817-P whose sequences are shown in SEQ ID NO.4-6 respectively.

[0009] Preferably, the 5' end and 3' end of Homochr-X1-230817-P are respectively provided with a VIC fluorescent group and a BHQ1 quenching group; the 5' end and 3' end of Homochr-Y1-230817-P are respectively provided with a FAM fluorescent group and a BHQ1 quenching group.

[0010] Preferably, the sequence region amplified by the primers and probes for amplifying X chromosome is 64456831-64456851, and the accession number in GeneBank is NC_000023.11; the sequence region amplified by the primers and probes for amplifying Y chromosome is 15125761-15125785, and the accession number in GeneBank is NC_000024.10.

[0011] The present invention provides a detection kit for detecting extremely imbalanced mixed samples by using non-recombinant locus of X and Y chromosomes, and the detection kit contains the above-mentioned primer and probe combination.

[0012] Preferably, the detection kit further contains a DNA standard product and a PCR reaction mixture.

[0013] The present invention provides an application of the above primer and probe combination or the above detection kit in the detection of extremely unbalanced mixed forensic samples, and the extremely unbalanced mixed samples include samples with a male-female ratio of (1-10):(1-10000).

[0014] The present invention provides a method for detecting extremely unbalanced mixed samples by using non-recombinant locus of X and Y chromosomes, and uses the above detection kit for detection.

[0015] Preferably, the detection includes the following steps:

[0016] (1) Extract DNA of the sample to be tested;

[0017] (2) Using the DNA in step (1) as a template, perform droplet digital PCR with the above primer and probe combination;

[0018] (3) After the reaction, determine the result according to the fluorescence of the droplets.

[0019] Preferably, in step (2), the reaction system of the droplet digital PCR is: 10.0 μL of PCR reaction mixture, 1.8 μL of primer mixture for amplifying X chromosome, 1.8 μL of primer mixture for amplifying Y chromosome, 0.5 μL of 10 μM Homochr-X1-230817-P, 0.5 μL of 10 μM Homochr-Y1-230817-P, 1.0 μL of template, and make up to 20 μL with deionized water;

[0020] In the primer mixture for amplifying X chromosome, the concentrations of Homochr-X1-230817-F and Homochr-X1-230817-R are both 10 μM; in the primer mixture for amplifying Y chromosome, the concentrations of Homochr-Y1-230817-F and Homochr-Y1-230817-R are both 10 μM.

[0021] Preferably, in step (2), the reaction program of the droplet digital PCR is: 95°C for 10 minutes; 95°C for 30 seconds, 58°C for 60 seconds, 45 cycles; then 98°C for 10 minutes; store at 16°C.

[0022] The present invention discloses the following technical effects:

[0023] The present invention uses digital PCR technology to detect the non-recombinant locus sites of X and Y chromosomes in a highly imbalanced male-female mixed sample, and evaluate the sample to be tested. The primer pairs and probes provided by the present invention can specifically detect the non-recombinant locus sites of X or Y chromosomes, with good specificity, capable of specifically amplifying X or Y chromosomes, but not specifically amplifying other components outside the sex chromosomes; moreover, the primer pairs and probes have good reproducibility, and the results are stable and reliable.

[0024] The primer pairs and probes of the present invention can be used to identify low-abundance DNA in a mixed sample. When male DNA and female DNA are mixed at a ratio of 1:10000, even if there are only 2 copies of the Y chromosome in the mixture, the mixing ratio of the imbalanced mixture can be accurately determined. When male DNA and female DNA are mixed at a highly imbalanced ratio exceeding 1:10000, the system can still very sensitively detect as low as 31 pg of target DNA in the presence of a large amount of background DNA interference. And in the presence of higher-concentration background DNA interference, it is still possible to detect trace amounts of target DNA of about 15 pg.

[0025] The method for detecting highly imbalanced mixed samples using the non-recombinant locus sites of X and Y chromosomes according to the present invention is simple, economical, fast and accurate, and is easy to be popularized and applied in forensic practice. At the same time, the primer pairs and probes included in the kit have the characteristics of short amplified fragments and good specificity, meeting the detection requirements for highly imbalanced samples, providing a new simple, economical, fast and effective technical means for the detection of highly imbalanced samples in the field of forensic medicine, and having broad application prospects in the field of forensic medicine. Description of the Drawings

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0027] Figure 1 It is the detection result of male sample M-3 in Example 2;

[0028] Figure 2 It is the detection result of female sample F-155 in Example 2;

[0029] Figure 3 It is the detection result of the sample with a male-female ratio of 1:100 in Example 3;

[0030] Figure 4 It is the detection result of the sample with a male-female ratio of 1:10000 in Example 4. Detailed Embodiments

[0031] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation on the present invention, but rather as a more detailed description of certain aspects, features, and implementation schemes of the present invention.

[0032] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0033] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0034] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0035] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0036] The experimental methods in the embodiments of the present invention are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0037] The sources of DNA samples in the embodiments of the present invention are as follows:

[0038] The male DNA sample M-3 was collected from a volunteer on June 15, 2023;

[0039] The female DNA sample F-1 was collected from a volunteer on June 15, 2023;

[0040] The female DNA sample F-155 was collected from a volunteer on June 15, 2023.

[0041] Example 1

[0042] In this example, a detection kit for detecting extremely unbalanced mixed samples using non-recombinant locus of X and Y chromosomes was prepared. The detection kit contains the following reagents:

[0043] a) Primers and probes for amplifying the X chromosome, whose names and sequences are shown in Table 1. The amplified sequence region of these primers and probes is 64456831 - 64456851, and the accession number in GeneBank is NC_000023.11. The primers and probes were synthesized by Aikery Biotech Co., Ltd. The synthesized primers and probes were configured with ultrapure water to 100 μM, and then the upstream and downstream primers were configured with ultrapure water into primer mixtures with final concentrations of 10 μM respectively, and the probe was configured to a final concentration of 10 μM, with green VIC fluorescence labeling.

[0044] b) Primer pairs and probes for amplifying the Y chromosome, whose names and sequences are shown in Table 1. The amplified sequence region of these primers and probes is 15125761 - 15125785, and the accession number in GeneBank is NC_000024.10. The primers and probes were synthesized by Aikery Biotech Co., Ltd. The synthesized primers and probes were configured with ultrapure water to 100 μM, and then the upstream and downstream primers were configured with ultrapure water into primer mixtures with final concentrations of 10 μM respectively, and the probe was configured to a final concentration of 10 μM, with blue FAM fluorescence labeling.

[0045] c) PCR reaction mixture. In this example and other examples, the ddPCR PCR reaction mixture of Yongnuo Company was used. TM Supermix.

[0046] d) DNA standard. The DNA standards used were standard DNA9948 and DNA9947A.

[0047] Table 1 Sequence information of primers and probes

[0048]

[0049]

[0050] After packaging the above reagents according to their respective conventional requirements, a detection kit for detecting extremely unbalanced mixed samples using non-recombinant locus of X and Y chromosomes was prepared for subsequent experiments.

[0051] Example 2

[0052] The detection kit prepared in Example 1 was used to detect one saliva sample of a Han male from Hunan (numbered M-3) and one saliva sample of a Han female from Hunan (numbered F-155). The specific detection process is as follows:

[0053] (1) Extract the DNA of the sample by salting-out method;

[0054] (2) Using the DNA in step (1) as a template, prepare the reaction system shown in Table 2 with the reagents in the detection kit of Example 1:

[0055] Table 2 Reaction System

[0056]

[0057] The above system was used to generate droplets with the microdroplet generation chip and microdroplet generator of China Yongnuo Company for PCR amplification; the PCR amplification program was 95°C for 10 minutes; 95°C for 30 seconds, 58°C for 60 seconds, for 45 cycles; then 98°C for 10 minutes; and stored at 16°C.

[0058] (3) Detect with the MicroDrop-20A biochip analyzer of China Yongnuo Company. Analyze the data using QuantaSoft 1.7 software (Bio-Rad Laboratories). The results are as Figure 1 and 2 shown.

[0059] Figure 1 This is the detection result of the male sample. Among them, the abscissa value is the fluorescence intensity of the VIC channel, the ordinate value represents the fluorescence signal intensity of the FAM channel, the dots in box No. 3 indicate the droplets that detected FAM fluorescence, the dots in box No. 2 indicate the droplets that detected VIC fluorescence, and the dots in box No. 1 indicate the negative droplets that did not detect fluorescence. It can be seen that the positive droplets of the X and Y chromosomes are clearly separated, and the positive result of this sample can be clearly identified, and the ratio of the X and Y chromosomes is 1:1.

[0060] Figure 2 This is the detection result of the female sample. Among them, the abscissa value is the fluorescence intensity of the VIC channel, the ordinate value represents the fluorescence signal intensity of the FAM channel, the dots in box No. 2 indicate the droplets that detected VIC fluorescence, and the dots in box No. 1 indicate the negative droplets that did not detect fluorescence. It can be seen that the positive droplets of the X chromosome are clearly separated, and the positive result of this sample can be clearly identified, and only the X chromosome is detected.

[0061] Example 3

[0062] The detection kit prepared in Example 1 was used to detect an artificially mixed highly unbalanced mixed sample. The specific process is as follows:

[0063] Select a female DNA sample (numbered F-1) at 5 ng / μL and a male DNA sample (numbered M-3) at 5 ng / μL as the initial DNA samples. Dilute and mix the initial DNA samples in a geometric progression to obtain highly imbalanced mixed samples with male-female ratios of 1:1, 1:10, 1:100, and 10:1. Conduct the detection according to the detection process described in Example 2, and the detection results are shown in Table 3.

[0064] Table 3 Detection Results of Samples with Different Mixing Ratios

[0065]

[0066] Figure 3 For the detection results of the sample with a male-female ratio of 1:100, where the horizontal axis value is the fluorescence intensity of the VIC channel, the vertical axis value represents the fluorescence signal intensity of the FAM channel. The dots in box No. 3 indicate the droplets with FAM fluorescence detected, the dots in box No. 2 indicate the droplets with VIC fluorescence detected, and the dots in box No. 1 indicate the negative droplets without fluorescence detected. It can be seen that the present invention has a significant effect in detecting highly imbalanced mixed specimens and obtains accurate mixing ratios.

[0067] Example 4

[0068] Use the detection kit prepared in Example 1 to detect artificially mixed extremely imbalanced mixed samples. The specific process is as follows:

[0069] Select a female DNA sample (numbered F-155) at 400 ng / μL and a male DNA sample (numbered M-3) at 5 ng / μL as the initial DNA samples. Dilute and mix the initial DNA samples in a geometric progression to obtain extremely imbalanced mixed samples with male-female ratios of 1:1000, 1:5000, 1:10000, and 1:25000. Conduct the detection according to the detection process described in Example 2, and the detection results are shown in Tables 4 and 5.

[0070] Table 4 Quantitative Detection Results of Samples with Different Mixing Ratios

[0071]

[0072] Table 5 Ratio Detection Results of Samples with Different Mixing Ratios

[0073]

[0074] Figure 4The detection results of a sample with a male-female ratio of 1:10000 are shown. Here, the horizontal axis represents the fluorescence intensity of the VIC channel, and the vertical axis represents the fluorescence signal intensity of the FAM channel. The points in box No. 4 indicate the droplets that detected both FAM and VIC fluorescence. The points in box No. 2 indicate the droplets that detected VIC fluorescence. The points in box No. 1 indicate the negative droplets that did not detect fluorescence. It can be seen that the present invention has a remarkable effect in detecting extremely unbalanced mixed samples, can sensitively detect only trace amounts of target DNA under the interference of a large amount of background DNA, and can sensitively detect only 2 copies of the Y chromosome.

[0075] Comparative Example 1

[0076] Use a qPCR kit The Pro Kit was used to detect an extremely unbalanced mixed sample prepared artificially. The specific process is as follows:

[0077] A female DNA sample with a concentration of 400 ng / μL (sample number F-155) and a male DNA sample with a concentration of 5 ng / μL (sample number M-3) were selected as the initial DNA samples. The initial DNA samples were diluted and mixed in equal ratios to obtain extremely unbalanced mixed samples with male-female ratios of 1:1000, 1:5000, and 1:10500. The detection results are shown in Table 6.

[0078] Table 6 Detection results of extremely unbalanced mixed samples based on the qPCR system

[0079]

[0080] Table 6 shows that traditional detection techniques are greatly interfered by background DNA when detecting extremely unbalanced mixed samples, have relatively low sensitivity, and the quantitative results obtained have a large error compared with the mixing ratio.

[0081] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the spirit of the present invention's design, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. A primer and probe combination for detecting extremely unbalanced mixed samples using non-recombination region sites of chromosomes X and Y, characterized in that: The primer and probe combination includes a primer and a probe for amplifying chromosome X, and a primer and a probe for amplifying chromosome Y; The primers and probes for amplifying chromosome X include Homochr-X1-230817-F, Homochr-X1-230817-R and Homochr-X1-230817-P, whose nucleotide sequences are shown in SEQ ID NOs. 1 to 3 respectively; The primers and probes for amplifying chromosome Y include Homochr-Y1-230817-F, Homochr-Y1-230817-R and Homochr-Y1-230817-P, whose nucleotide sequences are shown in SEQ ID NOs. 4 to 6, respectively.

2. The primer and probe combination according to claim 1, characterized in that: The 5' end and 3' end of the Homochr-X1-230817-P respectively carry a VIC fluorescent group and a BHQ1 quenching group; the 5' end and 3' end of the Homochr-Y1-230817-P respectively carry a FAM fluorescent group and a BHQ1 quenching group.

3. A detection kit for detecting extremely unbalanced mixed samples using non-recombination region sites of X and Y chromosomes, characterized in that: The detection kit comprises the primer and probe combination according to claim 1 or 2.

4. The detection kit according to claim 3, characterized in that The detection kit also includes a DNA standard and a PCR reaction mixture.

5. Use of the primer and probe combination according to claim 1 or 2 or the detection kit according to claim 3 or 4 in the detection of extremely unbalanced mixed samples in forensic medicine, characterized in that: The extremely unbalanced mixed samples include samples with a male-female ratio of (1-10):(1-10000).

6. A method for detecting extremely unbalanced mixed samples using non-recombination region sites of X and Y chromosomes, characterized in that: Using the detection kit according to claim 3 or 4 for detection; The detection comprises the following steps: (1) extracting DNA from the sample to be tested; (2) using the DNA of step (1) as a template and using the primer and probe combination of claim 1 or 2 to perform droplet digital PCR; (3) After the reaction is completed, the result is determined based on the fluorescence of the droplets.

7. The method according to claim 6, characterized in that In step (2), the reaction system of the droplet digital PCR is: 10.0 μL of PCR reaction mixture, 1.8 μL of primer mixture for amplifying chromosome X, 1.8 μL of primer mixture for amplifying chromosome Y, 0.5 μL of 10 μM omochr-X1-230817-P, 0.5 μL of 10 μM Homochr-Y1-230817-P, 1.0 μL of template, and deionized water to 20.0 μL; The concentrations of Homochr-X1-230817-F and Homochr-X1-230817-R in the primer mixture for amplifying chromosome X are both 10 μM; the concentrations of Homochr-Y1-230817-F and Homochr-Y1-230817-R in the primer mixture for amplifying chromosome Y are both 10 μM.

8. The method according to claim 6, characterized in that In step (2), the reaction procedure of the droplet digital PCR is: 95°C, 10 minutes; 95°C, 30 seconds, 58°C, 60 seconds, 45 cycles; then 98°C, 10 minutes; and stored at 16°C.

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