A primer set and kit for amplifying feline STR marker loci

Abstract

The present invention provides a primer set and a kit for amplifying cat STR marker sites, which are composed of 23 pairs of STR marker site amplification primer sets consisting of 22 pairs of autosomal site primer sequences and 1 pair of sex site primer sequences. The amplification primer set of the present invention is applied to a cat STR test kit, and the test kit of the present invention can simultaneously detect 23 cat STR sites. The detection of multiple sites can provide richer data, increase the accuracy of individual identification, reduce errors, and accurately identify cats with close kinship, which is helpful for accurate individual identification and kinship identification, and helps to resolve complex parent-child disputes. The genome selected by the present invention is distributed on each chromosome as much as possible, and the selected sites do not have a linkage relationship, which helps to improve the recognition ability of the test kit. The amplification primer set and the test kit of the present invention have high sensitivity and accuracy as well as good species specificity, and have good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a primer set and a kit for amplifying STR marker sites for cat paternity testing and individual identification. Background Art

[0002] Short tandem repeats (STRs) are a class of DNA sequences with length polymorphism formed by tandem repeats of 2 - 6 base pairs as the core unit. Due to different numbers of repeat units, they have different lengths, and their distribution among individuals is different, constituting the genetic diversity of STRs. STRs are currently the most widely used genetic markers in the field of forensic science. In forensic medicine, different fluorescently labeled STR site primers are commonly used to identify individuals and conduct kinship identification on humans through polymerase chain reaction amplification combined with capillary electrophoresis technology. This method is also applicable to other species for individual identification and kinship identification. According to the "2022 China Pet Consumption Report Data", the number of cats reached 65.36 million in 2022, with a year-on-year increase of 12.6%, reaching a new high. It is particularly urgent to establish an accurate and effective method for cat individual identification and kinship identification. Summary of the Invention

[0003] In view of this, the present invention provides a primer set and a kit for amplifying cat STR marker sites.

[0004] The technical solution of the present invention is realized as follows: A primer set for amplifying cat STR marker sites consists of 22 pairs of autosomal site primer sequences and 1 pair of sex site primer sequences to form a 23 - pair STR marker site amplification primer set. The 23 - pair STR marker site amplification primer set includes the following marker sites and primer sequences, as shown in the following table:

[0005]

[0006]

[0007]

[0008] Among them, the sex site is ZFXY.

[0009] Furthermore, the 23 pairs of STR marker site amplification primer sets are located on 23 STR marker sites. The 23 STR marker sites are divided into 4 groups for labeling. The first group includes the following sites: FCA075, FCA201, FCA220, FCA310, FCA678, FCA229; the second group includes the following sites: FCA149, FCA649, D4: 74467019 - 74467254, F742, B4: 110849426 - 110849659, E1: 45460854 - 45461101; the third group includes the following sites: FCA453, FCA441, FCA391, B3: 122110521 - 122110768; the fourth group includes the following sites: FCA069, FCA026, FCA105, FCA293, ZFXY, A3: 37910472 - 37910715, B4: 101072586 - 101072823.

[0010] Furthermore, the first group uses FAM to label the sites, the second group uses HEX to label the sites, the third group uses TAMRA to label the sites, and the fourth group uses ROX to label the sites.

[0011] Furthermore, the STR marker site amplification primer sets are made into a primer mixture and applied to the feline STR kit.

[0012] Furthermore, the kit multiplex amplifies 23 feline loci. Polymerase chain reaction is used in the kit to form an amplification system. The polymerase chain reaction amplification system includes the following components: 1 μL of the DNA template to be tested, 4 μL of deionized water, 4 μL of reaction mixture, and 1 μL of primer mixture. The concentration range of the DNA template to be tested is 10 - 50 ng / μL.

[0013] Furthermore, the reaction mixture includes 0.2 μL of polymerase, 2.5 μL of polymerase chain reaction buffer, 1.0 μL of deoxynucleoside triphosphate, 0.1 μL of divalent magnesium ions, 0.1 μL of stabilizer, and 0.1 μL of enhancer.

[0014] Furthermore, the stabilizer is one of cetyltrimethylammonium bromide, formamide, and dimethyl sulfoxide.

[0015] Furthermore, the enhancer is one of betaine, ammonium sulfate, and sodium citrate.

[0016] Further, the kit includes the following detection steps: S1: Extract the DNA of the cat to be tested; S2: Prepare the polymerase chain reaction solution, mix it with the DNA extracted in S1, and label the primers with fluorescence; S3: Perform polymerase chain reaction amplification, and the amplification program is as follows: ① Pre-denature at 95°C for 2 min; ② Denature at 95°C for 10 s, anneal and extend at 60°C for 105 s, and cycle step ② 30 times; ③ Extend at 60°C for 30 min to obtain the polymerase chain reaction amplification product; ④ Store at low temperature of 4°C; S4: Detect the amplification product in S3 using a single-channel or multi-channel capillary gene sequencer.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: The amplification primer set of the present invention is composed of 22 pairs of autosomal marker site primer sequences and 1 pair of sex site primer sequences. The kit of the present invention can simultaneously detect 23 cat STR loci. The detection of multiple loci can provide richer data, increase the accuracy of individual identification, reduce errors, and can accurately identify even among cats with relatively close genetic relationships, which helps to accurately perform individual identification and kinship identification, and helps to solve complex parentage disputes. The selected genomes of the present invention are as evenly distributed on each chromosome as possible, and there is no linkage relationship between the selected loci, which helps to improve the recognition ability of the kit. Adding a stabilizer to the polymerase chain reaction mixture of the kit of the present invention can reduce the air pressure of the reaction solution and reduce water evaporation, thereby stabilizing the reaction system. Adding an enhancer to promote the binding of the primer to the template DNA can improve the efficiency and specificity of the polymerase chain reaction. Description of the Drawings

[0018] Figure 1 It is the offspring genotyping result diagram of the present invention;

[0019] Figure 2 It is the paternal genotyping result diagram of the present invention;

[0020] Figure 3 It is the maternal genotyping result diagram of the present invention. Detailed Embodiments

[0021] To better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0022] The experimental methods used in the embodiments of the present invention are all conventional methods unless otherwise specified.

[0023] The materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial channels unless otherwise specified.

[0024] 1. Sample Collection and Processing

[0025] 16 pairs of oral swab DNA samples were collected from the triplet family, i.e. 16 pairs of offspring, paternal, and maternal DNA samples. The DNA was extracted using an oral swab DNA extraction kit and magnetic bead method. The concentration and purity were determined using a micro-spectrophotometer, and the DNA concentration was diluted to ≥5 ng / ul, and the optical density value was controlled to 1.8-2.0.

[0026] 2. Locus screening and primer design

[0027] STR locus screening includes three approaches: a. selecting loci published by ISAG; b. cat STR loci published in the literature that can be used for individual identification and kinship testing; c. STR loci independently mined using NGS data.

[0028] Selection criteria: 1) The repeating unit is two bases, three bases or four bases; 2) The number of repeats is ≥5 times, and the repeat sequence is ≤500bp; 3) The number of alleles is >6; 4) The heterozygosity is greater than 0.5; 5) There is no linkage relationship between the selected loci. The 23 selected loci are shown in Table 1.

[0029] Primer5 software was used to design primers for the 23 selected loci, and the NCBI website was used to compare and check whether there was non-specific hybridization between the primers and the genome sequences of other species. The primer fragment size was designed to be 70-450 bp, and the Tm value was controlled at 58-62°C. The primer information is shown in Table 1.

[0030] For the 23 loci, after confirming that the primers could amplify specific bands by agarose gel electrophoresis, the primers were fluorescently labeled using FAM, HEX, TAMRA, and ROX as fluorescent markers.

[0031] Table 1. Sites and primer sequences

[0032]

[0033]

[0034]

[0035] The genome selected by the present invention is distributed on each chromosome as much as possible, and the selected sites have no linkage relationship, which helps to improve the recognition ability of the test kit.

[0036] 3. Establishing polymerase chain reaction conditions

[0037] 6) Amplify 23 STR loci individually, determine the fragment range of each locus, and the length range of the amplification products is between 70 - 450 bp; 7) Mix 23 pairs of primers in equal proportion for multiplex amplification, check the amplification effect and whether there is non-specific amplification; 8) Adjust reaction conditions such as the primer ratio, annealing temperature, number of PCR amplification cycles, DNA template concentration, etc., to make the amplification products of each locus basically meet the requirements of balance and specificity. The final determined reaction conditions are shown in Table 2 below.

[0038] Table 2. Polymerase Chain Reaction Conditions

[0039]

[0040]

[0041] 4. Electrophoresis and STR Typing

[0042] Take 1 μL of the diluted or undiluted polymerase chain reaction amplification product, 8.8 μL of formamide, and 0.2 μL of LIZ500 internal standard, and perform capillary electrophoresis detection using a 3730 sequencer. The detection data is analyzed by Genemapper 6.0 software to obtain 23 STR typing map information.

[0043] 5. Result Verification

[0044] 5.1 Species Specificity Test

[0045] Take 1 ng of human DNA standard 9947A or 9948 and DNA samples of 11 common animal species: monkey, dog, pig, cow, sheep, chicken, duck, mouse, rabbit, donkey, snake, apply the amplification primer set of the present invention for multiplex amplification and electrophoretic separation, verify its species specificity, and each sample is repeatedly detected 3 times.

[0046] In multiple repeated verifications, no non-specific amplification products are generated. It can be seen that the amplification primer set of the present invention has good species specificity, and when using this system for testing, it will not be affected by DNA of other species on the typing results.

[0047] 5.2 Identity Test

[0048] Randomly select 16 cat oral swab DNA samples, amplify the amplification primer set of the present invention separately 3 times, and perform capillary electrophoresis detection, and count whether the typing of each sample in the 3 detections is consistent.

[0049] The results show that the typing results of the same sample are consistent, indicating that the amplification primer set of the present invention has high accuracy.

[0050] 5.3 Sensitivity Test

[0051] After quantifying the cat DNA samples using a Qubit fluorometer, sensitivity tests were performed with template amounts of 10 ng, 5 ng, 2.5 ng, 1.25 ng, 0.625 ng, and 0.3125 ng, and each sample was tested three times repetitively.

[0052] The results showed that at 0.625 ng, complete genotyping of 23 loci could be stably detected. It can be seen that the amplification system of the present invention has high sensitivity.

[0053] Example 1

[0054] Using the primer set and kit for amplifying feline STR marker loci of the present invention to perform individual identification of cats, including the following steps: 1. Extract DNA of the cat to be tested: Use the Tiangen automated oral swab DNA extraction kit to collect oral swabs of the cat, and use the magnetic bead method to prepare DNA; 2. Configure the polymerase chain reaction amplification system: Vortex and mix the reaction mixture, primer mixture, and deionized water, and then configure the polymerase chain reaction amplification system according to the corresponding volumes. The composition of the reaction system is shown in Table 3 below, and it is dispensed into polymerase chain reaction tubes. Then, add 1 μL of DNA template to the reaction tubes, mix well, and perform fluorescence labeling. Among them, the reaction mixture includes 0.2 μL of polymerase, 2.5 μL of polymerase chain reaction buffer, 1.0 μL of deoxynucleoside triphosphate, 0.1 μL of divalent magnesium ions, 0.1 μL of cetyltrimethylammonium bromide, and 0.1 μL of betaine. 3. Perform polymerase chain reaction amplification: First step: Pre-denature at 95°C for 2 min; Second step: Denature at 95°C for 10 s, anneal and extend at 60°C for 105 s; Repeat the second step 30 times; Third step: Extend at 60°C for 30 min; Fourth step: Store at 4°C. 4. Capillary electrophoresis detection: Take 1 μL of the original solution or diluted product for amplification, add 8.8 μL of formamide and 0.2 μL of LIZ500 internal standard, mix and let stand for 5 min, then denature at 95°C for 3 min, take out and ice-bath for 3 min, centrifuge, and place it on an ABI3730 sequencer for detection. After the detection is completed, use the genemapper6.0 software to interpret the genotyping results of each locus. Set the detection sequence according to the product fragment size, and judge the genotyping of each locus based on the peak position and fluorescence color.

[0055] Table 3. Components of the polymerase chain reaction amplification system

[0056] Component Name Volume (uL) Reaction Mixture 4 Primer Mixture 1 Deionized Water 4 DNA Template 1 Total Volume 10

[0057] Adding stabilizers to the polymerase chain reaction mixture of the kit of the present invention can reduce the air pressure of the reaction solution, reduce water evaporation, and thus stabilize the reaction system. Adding enhancers can promote the binding of primers to template DNA, and can improve the efficiency and specificity of the polymerase chain reaction.

[0058] Example 2

[0059] Use the amplification primer set and kit for cat STR marker loci to conduct paternity testing on cats. 1. Extract DNA: Use the Tiangen automated oral swab DNA extraction kit to collect oral swabs from the cats whose paternity is to be identified, and use the magnetic bead method to prepare DNA; 2. Configure the reaction system according to Example 1; 3. Perform polymerase chain reaction amplification according to Example 1; 4. Perform capillary electrophoresis detection according to Example 1, analyze the data using genemapper6.0 software and generate a map. The data is shown in Table 4, and the filial generation genotyping results are shown in Figure 1 , the paternal generation genotyping results are shown in Figure 2 , and the maternal generation genotyping results are shown in Figure 3 .

[0060] Table 4. Genotyping data of the filial generation, paternal generation, and maternal generation cats to be identified

[0061] STR Locus Offspring Genotyping Paternal Genotyping Maternal Genotyping FCA075 95 / 97 95 / 97 157 / 179 FCA201 179 / 181 181 / 181 231 / 235 FCA220 233 / 235 233 / 235 279 / 279 FCA310 279 / 279 263 / 279 322 / 324 FCA678 324 / 324 324 / 324 366 / 366 FCA229 366 / 366 366 / 370 113 / 117 FCA149 113 / 117 117 / 117 186 / 186 FCA649 184 / 186 184 / 186 222 / 228 D4-7254 222 / 238 238 / 238 274 / 306 F742 274 / 318 294 / 318 345 / 345 B4-9659 345 / 351 351 / 351 396 / 396 E1-1101 396 / 396 396 / 408 125 / 133 FCA453 125 / 133 125 / 125 215 / 219 FCA441 215 / 219 219 / 219 255 / 271 FCA391 255 / 275 271 / 275 335 / 345 B3-0768 339 / 345 339 / 341 115 / 117 FCA069 103 / 117 103 / 103 166 / 170 FCA026 170 / 174 172 / 174 200 / 204 FCA105 204 / 204 200 / 204 252 / 252 FCA293 252 / 252 252 / 252 X / X ZFXY Y / X Y / X 337 / 339 A3-5732 337 / 337 337 / 337 391 / 392 B4-2823 381 / 391 381 / 392 157 / 179

[0062] From Table 4 and Figure 1 , Figure 2 , Figure 3 it can be seen that the triploid paternity testing conforms to Mendel's genetic law and has a paternity relationship.

[0063] In summary, the amplification primer set and kit of the present invention have high sensitivity, accuracy, and good species specificity, and can detect and analyze different types of cat DNA samples. The kit of the present invention can simultaneously detect 23 cat STR loci. The detection of multiple loci can provide richer data, increase the accuracy of individual identification, reduce errors, and can accurately identify among cats with close genetic relationships, which helps to accurately conduct individual identification and kinship identification, helps to solve complex paternity disputes, can meet the requirements of species identification, individual identification, and kinship identification of cats in identification practice, and has good application prospects.

[0064] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A primer set for amplifying cat STR marker loci, characterized in that: A 23-pair STR marker locus amplification primer set is composed of 22 pairs of autosomal locus primer sequences and 1 pair of sex locus primer sequences. The 23-pair STR marker locus amplification primer set includes the following marker loci and primer sequences as shown in the following table: Among them, the sex locus is ZFXY.

2. The primer set for amplifying feline STR marker loci according to claim 1, characterized in that: The 23-pair STR marker locus amplification primer set is located on 23 STR marker loci, and the 23 STR marker loci are divided into 4 groups for marking; The first group includes the following loci: FCA075, FCA201, FCA220, FCA310, FCA678, FCA229; the second group includes the following loci: FCA149, FCA649, D4: 74467019-74467254, F742, B4: 110849426-110849659, E1: 45460854-45461101; the third group includes the following loci: FCA453, FCA441, FCA391, B3: 122110521-122110768; The fourth group includes the following loci: FCA069, FCA026, FCA105, FCA293, ZFXY, A3: 37910472-37910715, B4: 101072586-101072823.

3. The primer set for amplifying cat STR marker loci according to claim 2, characterized in that: The first group uses FAM to mark the locus, the second group uses HEX to mark the locus, the third group uses TAMRA to mark the locus, and the fourth group uses ROX to mark the locus.

4. A primer set for amplifying feline STR marker loci according to any one of claims 1-3, characterized in that: The STR marker locus amplification primer set is made into a primer mixture and applied to a cat STR kit.

5. A feline STR kit comprising the primer mixture described in claim 4, characterized in that: For the kit, 23 cat gene loci are multiplex amplified. In the kit, polymerase chain reaction is used to form an amplification system. The polymerase chain reaction amplification system includes the following components: 1 μL of the DNA template to be tested, 4 μL of deionized water, 4 μL of reaction mixture, and 1 μL of primer mixture. The concentration range of the DNA template to be tested is 10-50 ng / μL.

6. The cat STR kit according to claim 5, wherein: The reaction mixture includes 0.2 μL of polymerase, 2.5 μL of polymerase chain reaction buffer, 1.0 μL of deoxyribonucleoside triphosphate, 0.1 μL of divalent magnesium ion, 0.1 μL of stabilizer, and 0.1 μL of enhancer.

7. A cat STR kit according to claim 6, characterized in that: The stabilizer is one of cetyltrimethylammonium bromide, formamide, and dimethyl sulfoxide.

8. A cat STR kit according to claim 6, characterized in that: The enhancer is one of betaine, ammonium sulfate, and sodium citrate.

9. A cat STR kit according to any one of claims 5-8, characterized in that: The kit includes the following detection steps: S1: Extract the DNA of the cat to be tested; S2: Prepare the polymerase chain reaction solution, mix it with the DNA extracted in S1, and use fluorescence to mark the primers; S3: Perform polymerase chain reaction amplification. The amplification program is as follows: ① Pre-denature at 95 °C for 2 min; ② Denature at 95 °C for 10 s, anneal and extend at 60 °C for 105 s, and cycle step ② 30 times; ③ Extend at 60 °C for 30 min to obtain the polymerase chain reaction amplification product; ④ Store at 4 °C at low temperature; S4: Detect the amplification product in S3 using a single-channel or multi-channel capillary gene sequencer.

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