A composite amplification kit for simultaneously detecting 12 loci of giant pandas and its use method and application

Abstract

The present invention relates to the field of DNA detection and identification technology, and in particular to a composite amplification kit for simultaneously detecting 12 loci of giant pandas, and its use method and application. A composite amplification kit for simultaneously detecting 12 loci of giant pandas, wherein the 12 loci are: gpz-054, GPL-047, gpz-020, SRY, gpz-006, GPL-031, GPL-029, GPL-053, gpz-051, ZF, gpz-047 and GPL-060; the primer sequences corresponding to the 12 loci are shown in SEQ ID NOs: 1 to 24. The fluorescence composite amplification system of the kit of the present invention has high sensitivity. Under the condition that the DNA template amount is 0.1 ng, all 12 loci can be detected, and individual identification, sex identification and paternity identification of giant pandas can be quickly achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of DNA detection and identification, and in particular to a composite amplification kit for simultaneously detecting 12 gene loci of a giant panda, and a use method and application thereof. Background Art

[0002] Giant pandas have been profoundly impacted by climate change and human activities, and are now confined to six isolated, patchy distributions in high mountain valleys on the edge of the Qinghai-Tibet Plateau. Giant pandas are naturally wary, making direct observation difficult. The rapid development of modern molecular biology has led to breakthroughs in giant panda ecological research. In particular, the combined use of microsatellite markers and non-invasive genetic sampling techniques has enabled population-level studies of giant pandas without disturbing or harming them. This has made significant contributions to giant panda conservation over the past 20 years.

[0003] Microsatellite markers, also known as short tandem repeats (STRs), are composed of short tandem repeats of 3 to 7 base pairs in length. These repeats are widely present in the giant panda genome. Due to their high polymorphism and stability, adherence to Mendelian inheritance, and co-dominant inheritance, STR genotyping methods offer advantages such as short amplified products and low template quality requirements, allowing analysis even of degraded DNA templates. STR loci are now widely used in a variety of fields, including giant panda paternity testing, population surveys, genetic diversity assessment, population genetic structure analysis, and population genetic management.

[0004] Currently used giant panda STR loci are mostly two-base repeat loci. During PCR amplification, strand slipping can easily produce stuttering bands, leading to typing errors. Four-base repeat STR loci produce far fewer stutter products than two-base repeat STR loci and are easier to type by electrophoresis. Therefore, four-base repeat STR loci are currently the most commonly used genetic markers in human forensics. For example, the 13 core STR loci selected by the US CODIS (Combined DNA Index System) national DNA database are all four-base repeats. Therefore, the use and promotion of more stable four-base repeat STR markers should be increased in future giant panda research.

[0005] There are no discernible differences in appearance between male and female giant pandas, and the sex of adult pandas can be determined by the appearance of their genitals. However, the reproductive organs of newborn giant panda cubs are not readily apparent, and even experienced keepers can occasionally misjudge them, necessitating DNA-based sex determination. In giant pandas, sex is typically indicated using the sex determining region Y (SRY) and the Zinc-finger protein gene (ZF). SRY is located on the Y chromosome, with male giant pandas displaying a single band and females exhibiting no band. The ZF gene is located on both the X and Y chromosomes, and the ZF-X band in the target region is 7 bp shorter than the ZF-Y band. Sex can be determined by amplifying the peaks: a single peak indicates female, and double peaks indicate male. Currently, in giant panda STR testing, autosomal typing and sex chromosome testing are performed separately. Simultaneously testing autosomal STR loci and sex chromosome loci could reduce workload and improve testing efficiency.

[0006] Therefore, how to provide a kit that can be used for both autosomal typing and sex chromosome loci of giant pandas to solve the technical problem of the lack of relevant detection methods in the existing technology is an urgent problem that needs to be solved by those skilled in the art. Summary of the Invention

[0007] The present invention aims to provide a composite amplification kit for simultaneously detecting 12 loci of giant pandas, as well as its use method and application. Specifically, the present invention relates to a five-color fluorescent-labeled composite amplification kit for simultaneously analyzing 10 autosomal loci and 2 sex chromosome loci of giant pandas. The kit contains allele typing standards and can accurately perform STR typing. The kit can be used for individual identification, sex determination, and paternity testing.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a composite amplification kit for simultaneously detecting 12 gene loci of giant pandas, wherein the 12 gene loci are: gpz-054, GPL-047, gpz-020, SRY, gpz-006, GPL-031, GPL-029, GPL-053, gpz-051, ZF, gpz-047 and GPL-060;

[0010] The primer sequences corresponding to the 12 loci are:

[0011] gpz-054 is shown in SEQ ID NOs: 1 and 2;

[0012] GPL-047 is shown in SEQ ID NOs: 3 and 4;

[0013] gpz-020 is shown in SEQ ID NOs: 5 and 6;

[0014] SRY is shown in SEQ ID NOs: 7 and 8;

[0015] gpz-006 is shown in SEQ ID NOs: 9 and 10;

[0016] GPL-031 is shown in SEQ ID NOs: 11 and 12;

[0017] GPL-029 is shown in SEQ ID NOs: 13 and 14;

[0018] GPL-053 is shown in SEQ ID NOs: 15 and 16;

[0019] gpz-051 is shown in SEQ ID NOs: 17 and 18;

[0020] ZFs are shown in SEQ ID NOs: 19 and 20;

[0021] gpz-047 is shown in SEQ ID NOs: 21 and 22;

[0022] GPL-060 is shown in SEQ ID NOs: 23 and 24.

[0023] Preferably, the 5' end of at least one primer corresponding to each locus is labeled with a fluorescent dye marker.

[0024] Preferably, the 12 loci are divided into 4 groups; the first group: gpz-054, GPL-047, gpz-020, and the corresponding primer labeling fluorescent dye marker is FAM; the second group: SRY, gpz-006, GPL-031, and the corresponding primer labeling fluorescent dye marker is HEX; the third group: GPL-029, GPL-053, gpz-051, and the corresponding primer labeling fluorescent dye marker is L-552; the fourth group: ZF, gpz-047, GPL-060, and the corresponding primer labeling fluorescent dye marker is LR600.

[0025] Preferably, the kit further comprises a PCR amplification reagent; the PCR amplification reagent comprises deionized water and a reaction buffer.

[0026] Preferably, the reaction buffer comprises: 2U of hot start Taq enzyme, 30mM KCl, 50mM Tris-HCl buffer, 3.0mM MgCl2, 0.5mg / ml BSA and 0.4mM dNTPs;

[0027] The pH of the Tris-HCl buffer is 8.2-8.4.

[0028] The present invention also provides a method for using the kit, using giant panda genomic DNA as a template and performing PCR amplification using the kit.

[0029] The amplification system of the kit during PCR amplification includes the following components based on 25.0 μL: 14.0 μL of deionized water; 7.5 μL of reaction buffer; 2.5 μL of primer mixture; and 1.0 μL of template DNA. The concentration of the template DNA is 0.1 to 0.3 ng / μL.

[0030] Preferably, the amplification program of the kit during PCR amplification includes the following steps: 95°C for 1 min; 94°C for 30 s, 59°C for 35 s, for 30 cycles; and 4°C for PCR amplification.

[0031] The present invention also provides the use of the composite amplification kit for simultaneously detecting 12 gene loci of giant pandas in individual identification of giant pandas.

[0032] The present invention further provides the use of the composite amplification kit for simultaneously detecting 12 gene loci of giant pandas in sex identification or paternity testing of giant pandas.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] 1. The kit of the present invention is a multiplex amplification system comprising 12 chromosomal loci, including 10 autosomal STR loci and two sex chromosomal loci, all of which are four-base microsatellite loci. ZF and SRY are sex chromosomal loci. The kit provided by the present invention can simultaneously amplify all 12 chromosomal loci in a single multiplex amplification reaction system. Using these STR loci, individual identification, sex determination, and paternity testing of giant pandas can be achieved with high individual identification capabilities.

[0035] 2. The kit of the present invention contains allelic typing standards, which can accurately perform STR typing.

[0036] 3. The fluorescence multiplex amplification system of the present invention has high sensitivity and can detect all 12 loci when the DNA template amount is 0.1 ng.

[0037] 4. The 10 autosomal loci selected in the present invention are all four-base microsatellite loci, which have high polymorphism and stability and strong resolution ability.

[0038] 5. The present invention amplifies template DNA in the above-described reaction buffer system according to a specified reaction schedule to obtain mixed amplification products from various loci. Because fluorescently labeled primers are used in the present invention, the amplification products also carry fluorescent markers. Furthermore, the markers can emit optical signals under laser excitation that can be recognized by sequencers (e.g., ABI 3730, 3500). Therefore, the amplification products can be subjected to electrophoresis and detection analysis on the sequencer.

[0039] 6. During sequencing on a sequencer, the amplified product is mixed with a molecular weight internal standard and formamide in a specific ratio and then introduced into the instrument capillary. The molecular weight internal standard, composed of multiple fluorescently labeled DNA fragments of known length, is used to calculate the length of the PCR amplified product fragments, thereby determining genotyping and comparing it with allelic typing standards. The electrophoresis data can be analyzed using data analysis software such as Gene Mapper and Gene Marker to obtain STR genotyping patterns and data. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0041] Figure 1 : STR typing pattern of giant panda male standard sample 563 after PCR amplification using a multiplex amplification kit;

[0042] Figure 2 : Allelic Ladder analysis profile of allelic markers.

[0043] Figure 3 : STR typing pattern of a giant panda cub sample after PCR amplification using a multiplex amplification kit;

[0044] Figure 4 : STR typing pattern of a giant panda female sample after PCR amplification using a multiplex amplification kit;

[0045] Figure 5 : STR typing pattern of a suspected paternal sample of giant panda after PCR amplification using a multiplex amplification kit. DETAILED DESCRIPTION

[0046] The present invention provides a composite amplification kit for simultaneously detecting 12 gene loci of giant pandas, wherein the 12 gene loci are: gpz-054, GPL-047, gpz-020, SRY, gpz-006, GPL-031, GPL-029, GPL-053, gpz-051, ZF, gpz-047 and GPL-060;

[0047] The primer sequences corresponding to the 12 loci are:

[0048] gpz-054 is shown in SEQ ID NOs: 1 and 2;

[0049] GPL-047 is shown in SEQ ID NOs: 3 and 4;

[0050] gpz-020 is shown in SEQ ID NOs: 5 and 6;

[0051] SRY is shown in SEQ ID NOs: 7 and 8;

[0052] gpz-006 is shown in SEQ ID NOs: 9 and 10;

[0053] GPL-031 is shown in SEQ ID NOs: 11 and 12;

[0054] GPL-029 is shown in SEQ ID NOs: 13 and 14;

[0055] GPL-053 is shown in SEQ ID NOs: 15 and 16;

[0056] gpz-051 is shown in SEQ ID NOs: 17 and 18;

[0057] ZFs are shown in SEQ ID NOs: 19 and 20;

[0058] gpz-047 is shown in SEQ ID NOs: 21 and 22;

[0059] GPL-060 is shown in SEQ ID NOs: 23 and 24.

[0060] In the present invention, the 5' end of at least one primer corresponding to each locus is labeled with a fluorescent dye marker.

[0061] In the present invention, the 12 loci are divided into 4 groups; the first group: gpz-054, GPL-047, gpz-020, and the corresponding primer labeling fluorescent dye marker is FAM; the second group: SRY, gpz-006, GPL-031, and the corresponding primer labeling fluorescent dye marker is HEX; the third group: GPL-029, GPL-053, gpz-051, and the corresponding primer labeling fluorescent dye marker is L-552; the fourth group: ZF, gpz-047, GPL-060, and the corresponding primer labeling fluorescent dye marker is LR600.

[0062] In the present invention, the kit further comprises a PCR amplification reagent; the PCR amplification reagent comprises deionized water and a reaction buffer.

[0063] In the present invention, the reaction buffer comprises: hot start Taq enzyme 2U, KCl 30mM, Tris-HCl buffer 50mM, MgCl2 3.0mM, BSA 0.5mg / ml and dNTPs 0.4mM;

[0064] In the present invention, the pH of the Tris-HCl buffer is 8.2-8.4, preferably 8.3.

[0065] The present invention also provides a method for using the kit, using giant panda genomic DNA as a template and performing PCR amplification using the kit.

[0066] In the present invention, the amplification system of the kit during PCR amplification comprises the following components in 25.0 μL: 14.0 μL of deionized water; 7.5 μL of reaction buffer; 2.5 μL of primer mixture; 1.0 μL of template DNA;

[0067] In the present invention, the concentration of the template DNA is 0.1-0.3 ng / μL, preferably 0.2 ng / μL.

[0068] In the present invention, the amplification program of the kit during PCR amplification includes the following steps: 95°C for 1 min; 94°C for 30 s, 59°C for 35 s, and 30 cycles; and 4°C for PCR amplification.

[0069] The present invention also provides the use of the composite amplification kit for simultaneously detecting 12 gene loci of giant pandas in individual identification of giant pandas.

[0070] The present invention further provides the use of the composite amplification kit for simultaneously detecting 12 gene loci of giant pandas in sex identification or paternity testing of giant pandas.

[0071] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0072] The amplification reaction in the examples was performed on an ABI 9700 thermal cycler, electrophoresis and detection were performed on an ABI 3130xl genetic analyzer, and data analysis was performed using GeneMapper IDX v1.5 software. The reagents and materials used in the examples, such as the allele ladder, were all conventional materials commonly used by those skilled in the art.

[0073] Example 1 Individual Identification and Sex Identification of Giant Panda Standard No. 563

[0074] 1. Primer design

[0075] The 12 giant panda loci are: gpz-054, GPL-047, gpz-020, sex-determining region Y (SRY), gpz-006, GPL-031, GPL-029, GPL-053, gpz-051, Zinc-finger protein gene (ZF), gpz-047, and GPL-060. gpz-054, GPL-047, gpz-020, gpz-006, GPL-031, GPL-029, GPL-053, gpz-051, gpz-047, and GPL-060 are autosomal STR loci, while ZF and SRY are sex-determining loci. Specific primers were designed for each of the 12 loci, flanking their repeat sequences. The primer sequences and concentrations are shown in the table below:

[0076] Table 1 Loci and their corresponding primers

[0077]

[0078]

[0079] According to the different fluorescent dye markers of the primers, the 12 loci are grouped as follows: the first group: gpz-054, GPL-047, gpz-020, the fluorescent dye marker of the primers in this group is FAM; the second group: SRY, gpz-006, GPL-031, the fluorescent dye marker of the primers in this group is HEX; the third group: GPL-029, GPL-053, gpz-051, the fluorescent dye marker of the primers in this group is L-552; the fourth group: ZF, gpz-047, GPL-060, the fluorescent dye marker of the primers in this group is LR600, and one primer in each pair of primers is labeled with a fluorescent dye at the 5′ end.

[0080] 2. PCR amplification

[0081] Blood samples were collected from 563 giant pandas. DNA was extracted using the TIANGEN Blood Genomic DNA Extraction Kit (spin column format) following the instructions. Alternatively, direct DNA testing using blood samples can be performed using a 1.2 mm pore size FTA card.

[0082] The DNA extracted from the blood of the giant panda was used as a template for PCR amplification. PCR amplification was performed using a mixed primer kit obtained by mixing the primers in Table 1. The PCR amplification reaction system is shown in Table 2:

[0083] Table 2 PCR amplification reaction system

[0084]

[0085]

[0086] The reaction buffer contains 2 U of hot-start Taq enzyme, 30 mM KCl, 50 mM Tris-HCl buffer (pH 8.3), 3.0 mM MgCl2, 0.5 mg / ml BSA (bovine serum albumin), and 0.4 mM dNTPs. dNTPs are an equimolar mixture of four deoxyribonucleotides (dATP, dTTP, dCTP, and dGTP).

[0087] The PCR amplification reaction was performed on a thermal cycler (ABI9700 PCR instrument) according to the following reaction conditions: 95°C for 1 minute; 94°C for 30 seconds, 59°C for 35 seconds, and 30 cycles; and 4°C for continuous incubation. After the PCR amplification was completed, the sample was removed.

[0088] 3. Electrophoresis and detection

[0089] a. Prepare a mixture of (0.5 μL fluorescent molecular weight internal standard (select orange fluorescent marker, fluorescent marker is L635) + 10 μL deionized formamide) × (number of samples);

[0090] b. Mix thoroughly and aliquot 10 μL per tube. Add 1 μL of amplified product and allelic typing ladder, respectively. Centrifuge briefly to collect the liquid at the bottom of the tube.

[0091] c. Denature the sample at 95°C for 3 minutes, then quickly cool on ice for 3 minutes to completely denature the DNA and maintain its denatured state;

[0092] d. Place the sample into the sample tray of the genetic analyzer and start electrophoresis detection;

[0093] e. After about 40 minutes, the electrophoresis was completed and the experimental data were analyzed using Gene Mapper software to obtain the map and typing results. The results are as follows: Figure 1 As shown by Figure 1 The map shows four fluorescent colors. The first, blue fluorescence, detects the gpz-054, GPL-047, and gpz-020 loci; the second, green fluorescence, detects the SRY, gpz-006, and GPL-031 loci; the third, yellow fluorescence, detects the GPL-029, GPL-053, and gpz-051 loci; and the fourth, red fluorescence, detects the ZF, gpz-047, and GPL-060 loci, for a total of 12 detectable loci. The first 102-bp peak in the second row of green fluorescence is SRY. Because SRY has only one allele, allelic typing standards cannot be used for calibration in Gene Mapper analysis, so the panel cannot be annotated. The fourth row of red fluorescence, the ZF panel, detects two peaks, X and Y. Combining the results from the SRY and ZF sexing loci, it is determined that giant panda 563 has a Y chromosome and is male. The typing results of the 10 autosomes are shown in Table 3. Different giant panda individuals have different typing results, which can be used for individual identification.

[0094] Table 3 Test results of giant panda standard specimen No. 563

[0095] Detection loci Test results GPL-029 6 / 9 GPL-031 9 / 9 GPL-047 11 / 15.3 GPL-053 10 / 11.3 GPL-060 12 / 14 gpz-006 13 / 13 gpz-020 17 / 18 gpz-047 11 / 14 gpz-051 8 / 10 gpz-054 10.1 / 12

[0096] Implementation Case 2

[0097] Application of 12-locus multiplex amplification kit for parentage testing

[0098] Blood samples were collected from giant panda mothers, cubs, and suspected fathers.

[0099] 1. DNA extraction reference case 1; 2. PCR amplification reference case 1

[0100] 3. Electrophoresis and detection refer to case 1; 4. Analysis of test results

[0101] See the pup detection chart Figure 3 , maternal detection spectrum see Figure 4 , suspected paternal test pattern see Figure 5 The test results showed (see Table 4) that the father and the cub met the genetic rules at the 10 autosomal loci tested, confirming the father-son relationship.

[0102] Table 4. Results of paternity testing

[0103]

[0104]

[0105] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A multiplex amplification kit for simultaneously detecting 12 loci of giant pandas, characterized in that: The 12 loci are: gpz-054, GPL-047, gpz-020, SRY, gpz-006, GPL-031, GPL-029, GPL-053, gpz-051, ZF, gpz-047 and GPL-060; The primer sequences corresponding to the 12 loci are: gpz-054 is shown in SEQ ID NOs: 1 and 2; GPL-047 is shown in SEQ ID NOs: 3 and 4; gpz-020 is shown in SEQ ID NOs: 5 and 6; SRY is shown in SEQ ID NOs: 7 and 8; gpz-006 is shown in SEQ ID NOs: 9 and 10; GPL-031 is shown in SEQ ID NOs: 11 and 12; GPL-029 is shown in SEQ ID NOs: 13 and 14; GPL-053 is shown in SEQ ID NOs: 15 and 16; gpz-051 is shown in SEQ ID NOs: 17 and 18; ZFs are shown in SEQ ID NOs: 19 and 20; gpz-047 is shown in SEQ ID NOs: 21 and 22; GPL-060 is shown in SEQ ID NOs: 23 and 24.

2. A composite amplification kit for simultaneously detecting 12 loci of giant pandas according to claim 1, characterized in that: The 5' end of at least one primer corresponding to each locus is labeled with a fluorescent dye.

3. A composite amplification kit for simultaneously detecting 12 loci of giant pandas according to claim 2, characterized in that, The 12 loci are divided into 4 groups; the first group: gpz-054, GPL-047, gpz-020, the corresponding primers are labeled with FAM fluorescent dye; The second group: SRY, gpz-006, GPL-031, the corresponding primer labeling fluorescent dye marker is HEX; the third group: GPL-029, GPL-053, gpz-051, the corresponding primer labeling fluorescent dye marker is L-552; the fourth group: ZF, gpz-047, GPL-060, the corresponding primer labeling fluorescent dye marker is LR600.

4. A composite amplification kit for simultaneously detecting 12 loci of giant pandas according to claim 1, characterized in that, The kit further comprises a PCR amplification reagent; the PCR amplification reagent comprises deionized water and a reaction buffer.

5. A composite amplification kit for simultaneously detecting 12 loci of giant pandas according to claim 4, characterized in that, The reaction buffer includes: hot start Taq enzyme 2U, KCl 30mM, Tris-HCl buffer 50mM, MgCl2 3.0mM, BSA 0.5mg / ml and dNTPs 0.4mM; The pH of the Tris-HCl buffer is 8.2-8.

4.

6. The method for using the kit according to any one of claims 1 to 5, characterized in that: The giant panda genomic DNA was used as a template and PCR amplification was performed using the kit.

7. The method of use according to claim 6, characterized in that: The amplification system of the kit during PCR amplification includes the following components based on 25.0 μL: 14.0 μL of deionized water; 7.5 μL of reaction buffer; 2.5 μL of primer mixture; and 1.0 μL of template DNA. The concentration of the template DNA is 0.1 to 0.3 ng / μL.

8. The method of use according to claim 6, characterized in that: The amplification program of the kit during PCR amplification includes the following steps: 95°C for 1 minute; 94°C for 30 seconds, 59°C for 35 seconds, and 30 cycles; and 4°C for 30 seconds.

9. Use of the composite amplification kit for simultaneously detecting 12 loci of giant pandas according to claim 1 in individual identification of giant pandas.

10. Use of the multiplex amplification kit for simultaneously detecting 12 loci of giant pandas according to claim 1 in sex identification or paternity testing of giant pandas.

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