Primers, kits for simultaneous identification of three rodent PCR molecular identification

Abstract

The application aims to provide a primer and kit for simultaneously identifying three kinds of murine PCR molecular identification. The primer sequence and amplified fragment length of the application are as follows: the specific amplification primer of the Cytb gene of the Indian civet includes primer 1 and primer 2, the upstream primer of primer 1 is 5'CTG CCC ATG TAT TTC AGA ATA TAC 3', the downstream primer is 5'GAT ATC CTT GGA GCA CTT ATT AT 3', the length of the amplified fragment is 300 bp, the upstream primer of primer 2 is 5'CAT TAG GAT AAG AAT CGA TAG AACA AGT 3', the downstream primer is 5'GC AGG AGT ACA CCT ACT ATT 3', and the length of the amplified fragment is 300 bp. The application is applied to the technical field of nucleic acid detection.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid detection technology, and in particular to a primer and kit for simultaneous identification of three rodent animals using PCR molecular identification. Background Technology

[0002] Species identification of rodents is a core component of vector-borne disease surveillance and epidemiological investigations. Accurate identification can be achieved through three dimensions: classification system analysis, morphological characteristic comparison, and professional technical assistance. However, rodents are diverse and numerous, and can carry and transmit various pathogens, including Yersinia pestis, Leptospira, Hantavirus, and Bartonella. Species identification of rodents has always been a challenge in rodent research. Currently, fieldwork primarily relies on morphological identification, which is not only time-consuming and labor-intensive but also requires complete specimens, typical morphological characteristics, and highly experienced personnel. Therefore, it significantly impacts the accuracy of identification results and the analysis of species diversity.

[0003] Commonly used animal testing methods both domestically and internationally include sensory identification techniques, spectral chromatography, enzyme-linked immunosorbent assay (ELISA), conventional PCR, real-time quantitative PCR, and digital PCR. Sensory identification techniques require personnel to have a strong understanding of the subtle morphology of various species. Near-infrared spectroscopy mainly detects hydrogen-containing groups, but its disadvantages include the need for a large number of samples to establish and analyze models, and its detection accuracy is not high. Chromatographic analysis is only suitable for fresh and frozen meat. During processing, proteins are easily denatured, rendering ELISA ineffective, so ELISA is only suitable for unprocessed products. Electronic noses and electronic tongues still have shortcomings in terms of hardware structure and recognition algorithms, resulting in poor biomimicry. Currently, national and industry standards for the detection of animal-derived components mainly adopt PCR. Real-time quantitative PCR and digital PCR can simultaneously perform specific identification and quantification, providing more accurate and reliable quantitative data. However, the instruments and reagents used are expensive, the process is time-consuming, and it requires highly skilled testing personnel and is difficult to operate, making it unsuitable for rapid batch testing. For example, Chinese patent CN122128444A discloses the application of a Pitx2 gene haplotype marker in identifying the photothermosensitive trait in brown rats. However, it requires a large number of samples to establish and analyze the model, and the detection accuracy is not high. Therefore, it is necessary to provide a primer and kit for PCR molecular identification of three rodents that can simultaneously identify three rodents with short detection time, good specificity, high sensitivity, and the ability to identify three rodents at the same time. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a primer and kit for PCR molecular identification of three rodents simultaneously. The detection time is short, the specificity is good, the sensitivity is high, and it can identify three rodents simultaneously.

[0005] The technical solution adopted in this invention is as follows: The primer sequence and amplification fragment length of this invention are as follows: A. The specific amplification primers for the Cytb gene of the shrew include primer 1 and primer 2. The upstream primer of primer 1 is 5'CTG CCA CTA GTA TTC AGA ATA TAC 3'; the downstream primer is 5'GATATC CTT GGA GCA CTT ATT AT3'; the amplified fragment length is 300bp. The upstream primer of primer 2 is 5'CA TTA GGA TAA GAA TCG ATA GAA CAA GT 3'; the downstream primer is 5'GC AGG AGT ACA CCT ACT ATT 3'; the amplified fragment length is 300bp; B. The specific amplification primers for the Cytb gene in brown rats include primer 3, with upstream primer: 5'TTG TCT TCAACA ATT CCA GAG 3'; downstream primer: 5'CCTAAT CTT RTC AAT CCT AAT 3'; the amplified fragment length is 230bp; C. The specific amplification primers for the Cytb gene in mice include primer 4, upstream primer: 5'GAG AAT TAAGAT GAT TGA GAA GTA TG 3'; downstream primer: 5'AGC AAT CGT TCA CCT TCT T 3'; the amplified fragment length is 500bp.

[0006] As can be seen from the above scheme, this application can accurately identify the specific molecular markers of three rodent species. The kit, as a convenient PCR detection kit for shrews, brown rats, and house mice, can save detection time, reduce costs, and improve efficiency. The core principle of the primers for identifying the three rodent species relies on the sequence-specific complementary binding characteristics of molecular biology, combined with the differences in gene sequences between species to achieve accurate species differentiation. The core logic revolves around three aspects: target gene selection, primer-specific design, and PCR amplification verification.

[0007] The primers themselves are oligonucleotide chains that are completely complementary to both ends of the target sequence. The uniqueness of the sequence match ensures the specificity of the identification: the primer sequences perfectly match the conserved regions at both ends of the target gene in the target mouse species, and can only bind precisely to the DNA template of the corresponding species, avoiding non-specific binding to non-target sequences of other species. Primers are designed for the 3' ends of specific SNP sites in different mouse species, ensuring that only completely matching templates can initiate subsequent PCR amplification, thus avoiding interference from cross-amplification at the source. The GC content and Tm value of the primers are uniformly controlled within the range of 40%-60%, ensuring that multiple primer pairs can work stably under the same amplification program, adapting to batch identification scenarios with multiple samples. The principle of PCR amplification and identification relies on the denaturation-annealing-extension cycle of PCR to achieve exponential amplification of trace templates, ultimately determining the species through product characteristics: the denaturation stage at approximately 95℃ unwinds the double strands of the mouse genomic DNA; the annealing stage cools to 50-60℃, allowing the primers to specifically bind to the complementary target sequence; and the extension stage synthesizes new DNA chains under the action of Taq enzyme. If the template DNA and primers are a perfect match, the target band of the expected size can be amplified. The position and number of bands can be directly distinguished between different mouse species by gel electrophoresis. If the template does not match, no amplified band will be produced, ruling out the possibility of that species. After sequencing, the amplified products are compared with nucleic acid databases. The species classification is determined by sequence similarity, further verifying the accuracy of the identification results. Even trace amounts of degraded samples such as hair and feces can be effectively identified.

[0008] In a preferred embodiment, the kit includes a genomic DNA extraction system comprising 30 mL of solution P1, 30 mL of solution P2, 20 mL of solution P3, 50 mL of solution P4, 50 mL of solution P5, and 80 mL of solution P6.

[0009] In a preferred embodiment, the kit further includes a PCR reaction system comprising an identification reaction system, a positive control reaction system, and sterile distilled water; wherein the total volume of the identification reaction system is 50 μL, containing 5 μL of reaction buffer, 2-5 μL of dNTPs, 3 μL of MgCl2, 1-5 μL of Taq DNA polymerase, 2.25 μL of primers for shrews, 3 μL of primers for brown rats, 2 μL of primers for house mice, 2-6 μL of DNA from the sample to be tested, and the remainder being double-distilled water.

[0010] In a preferred embodiment, the kit further includes a positive control reaction system, comprising 44 μL of positive plasmids from shrews, brown rats, and house mice, and 2 μL of positive plasmids from each of these species, in an identification reaction system tube.

[0011] In a preferred embodiment, the kit further includes a negative control reaction system, comprising 44 μL of the negative control reaction system and 6 μL of sterile double-distilled water in the identification reaction system tube. Attached Figure Description

[0012] Figure 1 This is the electrophoresis image of primer 1; Figure 2 This is the electrophoresis image of primer 2; Figure 3 This is the electrophoresis image of primer 3; Figure 4 This is the electrophoresis diagram of primer 4. Detailed Implementation

[0013] In this embodiment, the primer sequence and amplification fragment length of the present invention are as follows: A. The specific amplification primers for the Cytb gene of the shrew include primer 1 and primer 2. The upstream primer of primer 1 is 5'CTG CCA CTA GTA TTC AGA ATA TAC 3'; the downstream primer is 5'GAT ATC CTT GGA GCA CTT ATT AT3'; the amplified fragment length is 300bp. The upstream primer of primer 2 is 5'CA TTA GGA TAA GAA TCG ATA GAA CAA GT 3'; the downstream primer is 5'GC AGG AGT ACA CCT ACT ATT 3'; the amplified fragment length is 300bp; B. The specific amplification primers for the Cytb gene in brown rats include primer 3, upstream primer: 5'TTG TCT TCAACA ATT CCA GAG 3'; downstream primer: 5'CCT AAT CTT RTC AATCCT AAT 3'; the amplified fragment length is 230bp; C. The specific amplification primers for the Cytb gene in mice include primer 4, upstream primer: 5'GAG AAT TAAGAT GAT TGA GAA GTA TG 3'; downstream primer: 5'AGC AAT CGT TCA CCT TCT T 3'; the amplified fragment length is 500bp.

[0014] In this embodiment, the kit includes a genomic DNA extraction system, which includes 30 mL of P1 solution, 30 mL of P2 solution, 20 mL of P3 solution, 50 mL of P4 solution, 50 mL of P5 solution, and 80 mL of P6 solution.

[0015] In this embodiment, the kit further includes a PCR reaction system, which includes an identification reaction system, a positive control reaction system, and sterile distilled water. The total volume of the identification reaction system is 50 μL, containing 5 μL of reaction buffer, 2-5 μL of dNTPs, 3 μL of MgCl2, 1-5 μL of Taq DNA polymerase, 2.25 μL of primers for shrews, 3 μL of primers for brown rats, 2 μL of primers for house mice, 2-6 μL of DNA from the sample to be tested, and the remainder being double-distilled water.

[0016] In this embodiment, the kit also includes a positive control reaction system, in which 44 μL of positive plasmids from shrews, brown rats, and house mice are aspirated into the identification reaction system tube, along with 2 μL of positive plasmids from each of the shrew, brown rat, and house mouse.

[0017] In this embodiment, the kit also includes a negative control reaction system, wherein 44 μL of the negative control reaction system and 6 μL of sterile double-distilled water are drawn into the identification reaction system tube.

[0018] In this embodiment, the PCR amplification system and PCR amplification procedure are as follows: 1. PCR amplification system Using total DNA as a template, PCR amplification was performed. The 25 μL reaction system contained: 12.5 μL of 2xTaq PCR MaterMix, 1 μL of 10 μM upstream primer, 1 μL of 10 μM downstream primer, 1-2 μL of DNA sample, and the remainder was made up with water.

[0019] 2. PCR amplification reaction conditions The PCR amplification reaction conditions were as follows: pre-denaturation at 94℃ for 5 min, followed by denaturation at 94℃ for 30 sec, annealing at 52℃ for 30 sec, extension at 72℃ for 30 sec, for 40 cycles, and a final extension at 72℃ for 7 min. After the reaction, the amplification products were identified by agarose gel electrophoresis.

[0020] 3. Electrophoresis 3.1 Preparation of agarose gel: Weigh 1g of agarose into an Erlenmeyer flask, add 100ml of 1×TBE buffer, heat in a microwave oven until completely dissolved, cool to about 60℃, add an appropriate amount of Goidview, shake gently, and slowly pour into an electrophoresis plate with a comb, being careful not to create air bubbles. Let it stand and cool for at least 30 minutes, then place it in the electrophoresis tank to load the sample. The electrophoresis buffer should have the same concentration as the buffer used to prepare the gel.

[0021] 3.2 Sample application and gel running Add 1 / 6 volume of 6× Loading buffer to the electrophoresis sample, mix well, and then load the sample. Perform electrophoresis at 10 V / cm for 30 min. After electrophoresis, remove the gel, observe it under UV light, and take photographs.

[0022] like Figure 1 As shown, 1: DNA Marker; 2-4: Stink Shredder 1-3 (300bp); 5-7: Brown Rat; 8-10: House Mouse; 11-13: Yellow-breasted Rat; 14: Negative Control; 15: DNA Marker.

[0023] like Figure 2 As shown, 1: DNA Marker; 2-4: Stink Shredder 1-3 (300bp); 5-7: Brown Rat; 8-10: House Mouse; 11-13: Yellow-breasted Rat; 14: Negative Control; 15: DNA Marker.

[0024] like Figure 3 As shown, 1: DNA Marker; 2-4: Stink Shredder 1-3; 5-7: Brown Rat (230bp); 8-10: House Mouse; 11-13: Yellow-breasted Rat; 14: Negative Control; 15: DNA Marker.

[0025] like Figure 4 As shown, 1: DNA Marker; 2-4: Stink Shredder 1-3; 5-7: Brown Rat; 8-10: House Mouse (500bp); 11-13: Yellow-breasted Rat; 14: Negative Control; 15: DNA Marker.

[0026] In this embodiment, the primer sequence listing for PCR identification of mouse-like animals is as follows: Although the embodiments of the present invention are described with reference to actual solutions, they do not constitute a limitation on the meaning of the present invention. Modifications to the embodiments and combinations with other solutions based on this specification will be obvious to those skilled in the art.

Claims

1. A primer for simultaneous identification of three rodent species using PCR molecular identification, characterized in that, The primer sequences and amplified fragment lengths are as follows: A. The specific amplification primers for the Cytb gene of the shrew include primer 1 and primer 2. The upstream primer of primer 1 is: 5'CTGCCA CTA GTA TTC AGA ATA TAC 3'; the downstream primer is: 5'GAT ATC CTT GGA GCA CTT ATT AT 3'; the amplified fragment length is 300bp. The upstream primer of primer 2 is 5'CA TTA GGA TAA GAA TCG ATA GAA CAA GT 3'; the downstream primer is 5'GCAGG AGT ACA CCT ACT ATT 3'; the amplified fragment length is 300bp; B. The specific amplification primers for the Cytb gene in brown rats include primer 3, with the upstream primer being 5'TTG TCT TCA ACAATT CCA GAG 3' and the downstream primer being 5'CCT AAT CTT RTC AAT CCT AAT 3'; the amplified fragment length is 230bp. C. The specific amplification primers for the Cytb gene in mice include primer 4, upstream primer: 5'GAG AAT TAA GATGAT TGA GAA GTA TG 3'; downstream primer: 5'AGC AAT CGT TCA CCT TCT T 3'; the amplified fragment length is 500bp.

2. A kit comprising primers for simultaneous identification of three rodent species using PCR molecular identification as described in claim 1, characterized in that: The kit includes a genomic DNA extraction system, which comprises 30 mL of solution P1, 30 mL of solution P2, 20 mL of solution P3, 50 mL of solution P4, 50 mL of solution P5, and 80 mL of solution P6.

3. The reagent kit according to claim 2, characterized in that: The kit also includes a PCR reaction system, which comprises an identification reaction system, a positive control reaction system, and sterile distilled water. The total volume of the identification reaction system is 50 μL, containing 5 μL of reaction buffer, 2-5 μL of dNTPs, 3 μL of MgCl2, 1-5 μL of Taq DNA polymerase, 2.25 μL of primers for shrews, 3 μL of primers for brown rats, 2 μL of primers for house mice, 2-6 μL of DNA from the sample to be tested, and the remainder being double-distilled water.

4. The reagent kit according to claim 3, characterized in that: The kit also includes a positive control reaction system, in which 44 μL of positive plasmids from shrews, brown rats, and house mice are aspirated into the identification reaction system tube.

5. The reagent kit according to claim 3, characterized in that: The kit also includes a negative control reaction system, in which 44 μL of the negative control reaction system and 6 μL of sterile double-distilled water are drawn into the identification reaction system tube.

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