PCR (Polymerase Chain Reaction) primer group and kit for mouse-shaped animal species detection and application of PCR primer group and kit
By using a specific PCR primer set to perform two PCR amplifications in murine animal species detection, the problem of insufficient Cytb gene concentration was solved, efficient gene amplification and simplified operation process were achieved, and detection efficiency and sensitivity were improved.
Patent Information
- Application Number
- CN202411786017.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-06
- Publication Date
- 2025-04-11
AI Technical Summary
In the process of using the Cytb gene to identify murine animal species, the concentration of Cytb gene in the amplified products of conventional PCR technology is low, which is difficult to meet the sequencing requirements, and the operation is complex, time-consuming and labor-intensive, and difficult.
It provides a PCR primer set for mouse-shaped animal species detection, including a primary PCR primer set and a secondary PCR primer set. Through two PCR amplification, the concentration of the entire Cytb gene sequence can be effectively increased, meet the sequencing requirements, and simplified the operation process.
The concentration of target gene fragments in the PCR amplification product was improved, the operation process was simplified, the operation difficulty and detection time were reduced, from a few days to about 2 hours, and the efficiency of rat-shaped animal species detection was improved.
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Figure CN119799908A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of biological species detection, and in particular to a PCR primer set, a kit and applications thereof for detecting species of mouse-shaped animals. Background Art
[0002] Rodents include the family Muridae of the order Rodentia and the family Shrewidae of the order Insectivora. The identification of their species is of great significance for the study of rodent populations and communities, the control of rodent pests, and the prevention and control of rodent-borne diseases. DNA barcoding is the use of a standard, sufficiently variable, easily amplified, and short DNA fragment in the genome of an organism to quickly identify species. Compared with morphological classification and identification methods, it has the advantages of being simple and convenient, highly accurate, and having low requirements for the integrity of the specimen. It has become a commonly used species identification method in biodiversity, animal ecology, and animal epidemiology surveys and studies.
[0003] Mitochondrial cytochrome C, oxidase subunit I (COI), mitochondrial cytochrome B (Cytb), 16S rRNA and mitochondrial DNA control region (D-loop) gene sequences are often used as DNA barcodes for species identification of rodents. Among them, the Cytb gene is the most effective indicator for species analysis and identification. It has a suitable fragment size and is a target gene rich in biological information. By amplifying and sequencing the Cytb gene, species identification of rodents can be achieved.
[0004] However, at present, the following problems exist in the process of using the Cytb gene to identify rodent species: when conventional PCR technology is used to amplify the Cytb gene, it is easy for the Cytb gene concentration in the product to be low and unable to meet the sequencing requirements (especially when the concentration of the extracted DNA sample is low). If the Cytb gene concentration is to meet the sequencing requirements, the product must be connected to the plasmid after PCR amplification, and then transformed into the host cell. The Cytb gene is further amplified by culturing the host cell. The operation is relatively complicated. The process of plasmid construction, transformation and culturing is time-consuming and labor-intensive, and the operation is difficult, which requires a high level of professionalism from the experimenter. Summary of the invention
[0005] In order to solve the above technical problem, that is, in the process of using Cytb gene to detect the species of murine animals, it is easy for the PCR product to be difficult to reach the concentration required for sequencing, the present invention provides a PCR primer set for detecting the species of murine animals. The PCR primer set of the present invention can effectively increase the concentration of the target gene fragment (including the full gene sequence of Cytb) in the product, so that it meets the requirements of subsequent sequencing, and can work on a variety of murine animals.
[0006] The specific technical scheme of the present invention is: In the first aspect, the present invention provides a PCR primer set for detecting species of rodents, comprising a primary PCR primer set and a secondary PCR primer set; the primary PCR primer set comprises upstream and downstream primers as shown in SEQ ID NO.1 and SEQ ID NO.2, respectively, and also comprises upstream and downstream primers as shown in SEQ ID NO.3 and SEQ ID NO.4, respectively; the secondary PCR primer set comprises upstream and downstream primers as shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.
[0007] The above-mentioned PCR primer set includes primers for performing two PCR amplifications. When used, after the first PCR amplification is performed using a primary PCR primer set with a specific sequence, the second PCR amplification is performed using a secondary PCR primer set with a specific sequence. The full Cytb gene sequence of rodents can be amplified, and the concentration of the target gene fragment (including the full Cytb gene sequence) in the product can be effectively increased. Without performing plasmid construction, transformation, and bacterial culture, only PCR is required to ensure that the concentration of the target gene fragment meets the requirements of subsequent sequencing, which helps to improve the efficiency of rodent species detection, reduce the difficulty of operation, and reduce the detection time (from several days to about 2 hours).
[0008] Moreover, the mouse-shaped animals include the rodent family Muridae and the Insectivora family Shrew family, and there are large differences in the gene sequences of different species, which brings great difficulty to the PCR primer set to take into account the species detection requirements of different mouse-shaped animals. In the PCR primer set of the present invention, two pairs of primary PCR primers (primer pairs shown in SEQ ID NO.1 and SEQ ID NO.2, and primer pairs shown in SEQ ID NO.3 and SEQ ID NO.4) with specific sequences are used in conjunction with a pair of secondary PCR primers (primer pairs shown in SEQ ID NO.5 and SEQ ID NO.6), which can better amplify the Cytb full gene sequence of a variety of mouse-shaped animals (including common brown rats, yellow-haired rats, yellow-breasted rats, black-lined field mice, community mice, small house mice, black house mice, Chinese field mice and Burmese mice in the rodent family Muridae, and common stinky shrews, large musk shrews and small musk shrews in the Insectivora family) to achieve the concentration required for sequencing.
[0009] In a second aspect, the present invention provides a kit for detecting species differentiation of rodents, the kit comprising the PCR primer set.
[0010] Preferably, the kit also includes DNA polymerase and Mg 2+ and dNTP buffer.
[0011] Furthermore, the DNA polymerase is Taq enzyme.
[0012] In a third aspect, the present invention provides the use of the PCR primer set or the kit in the detection of species of rodents.
[0013] Preferably, the mouse-like animal is a rodent of the family Muridae or an Insectivora of the family Shrew.
[0014] Furthermore, the rodent of the family Muridae is Rattus norvegicus, Rattus norvegicus, Rattus flavoche, Apodemus agrarius, Rattus musculus, Rattus house mouse, Rattus house mouse, Apodemus chinensis or Rattus burmese; the Insectivora of the family Shrew is Rattus norvegicus, Rattus dasyphylla or Rattus serratus.
[0015] Preferably, the application comprises the following steps: S1: extracting DNA from the mouse-shaped animal to be tested to obtain a DNA sample to be tested; S2: preparing a primary PCR system containing the primary PCR primer set and the DNA sample to be tested, performing a first PCR reaction, and obtaining a primary PCR product; S3: preparing a secondary PCR system containing the secondary PCR primer set and the primary PCR product, and performing a second PCR reaction to obtain a secondary PCR product; S4: Sequence the secondary PCR product, and determine the species of the mouse-like animal to be tested according to the sequencing results.
[0016] Preferably, after step S2, the primary PCR product is subjected to electrophoresis detection. When the result is negative (i.e., no band appears in the electrophoresis pattern of the primary PCR product), the primary PCR product is directly used to prepare the secondary PCR system in step S3; when the result is positive (i.e., a band appears in the electrophoresis pattern of the secondary PCR product), the primary PCR product is diluted 10 to 100 times and then used to prepare the secondary PCR system in step S3.
[0017] When the electrophoresis result of the primary PCR product is negative, it indicates that the concentration of the target gene fragment in the first PCR reaction is low. Therefore, the primary PCR product is directly prepared into a secondary PCR system for a second PCR reaction. When the electrophoresis result of the primary PCR product is positive, it indicates that the concentration of the target gene fragment in the first PCR reaction is high. In order to avoid affecting the amplification effect of the second PCR, it is diluted and then prepared into a secondary PCR system.
[0018] Preferably, in step S2, the first PCR reaction is performed according to the following steps: 1) React at 94-95°C for 4-6 minutes; 2) Perform 35-40 cycles according to the following process: 94-95°C for 60-90 s → 55-57°C for 60-90 s → 70-73°C for 90-100 s; 3) React at 71-73°C for 10-15 minutes.
[0019] Preferably, in step S3, the second PCR reaction is performed according to the following steps: 1) React at 94-95°C for 4-6 minutes; 2) Perform 35-40 cycles according to the following process: 94-95°C for 60-90 s → 60-62°C for 60-90 s → 70-73°C for 90-100 s; 3) React at 71-73°C for 10-15 minutes.
[0020] Preferably, in step S2, the primary PCR system contains DNA polymerase, Mg 2+ and dNTPs.
[0021] Preferably, in step S1, the DNA sample to be tested is extracted from the liver tissue of the mouse-like animal to be tested.
[0022] Preferably, in step S4, the process of determining the species of the mouse to be tested according to the sequencing results is as follows: the sequencing results are compared with the gene library, and the species corresponding to the reference sequence with the highest homology in the gene library is taken as the species of the mouse to be tested.
[0023] Compared with the prior art, the present invention has the following advantages: (1) The present invention can effectively increase the concentration of the target gene fragment (including the full gene sequence of Cytb) in the PCR amplification product, so that without plasmid construction, transformation and bacterial culture, only PCR can be performed to ensure that the concentration of the target gene fragment meets the requirements of subsequent sequencing, which helps to improve the efficiency of rodent species detection, reduce the difficulty of operation, and shorten the detection time.
[0024] (2) The present invention has good universality for rodents, and can play a good role in the species detection of various rodents (including the common brown rat, yellow-haired rat, yellow-breasted rat, black-striped field mouse, social mouse, house mouse, black house mouse, Chinese field mouse and Burmese mouse in the family Muridae of the order Rodentia, and the common stinking shrew, big musk shrew and small musk shrew in the family Shrewidae of the order Insectivora), and can effectively increase the concentration of the target gene fragment in the PCR amplification product, so as to facilitate the subsequent determination of the species of the rodent by sequencing the PCR amplification product. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1It is the electrophoresis pattern of the secondary PCR product in Example 1. Among them: electrophoresis lane M is a DNA molecular weight reference, and the bands from top to bottom represent molecular weights of 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp respectively; electrophoresis lanes 1 to 12 are Rattus flavipectus, Rattus norvegicus, Rattus truncatus, Apodemus agrarius, Rattus musculus, Rattus sibiricus, Rattus nigromaculus, Apodemus chinensis, Rattus burmese, Scolopendra subspinipes ...
[0026] Figure 2 It is the electrophoresis pattern of the PCR product in comparative example 1. Among them: electrophoresis lane M is a DNA molecular weight reference, and the bands from top to bottom represent molecular weights of 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp respectively; electrophoresis lanes 1 to 12 are Rattus flavipectus, Rattus norvegicus, Rattus truncatus, Apodemus agrarius, Rattus musculus, Rattus sibiricus, Rattus nigromaculus, Apodemus chinensis, Rattus burmese, Scolopendra shrew, Scolopendra shrew, and Scolopendra shrew. DETAILED DESCRIPTION
[0027] The present invention will be further described below in conjunction with the embodiments.
[0028] Overall embodiment First, the present invention relates to a PCR primer set for detecting species of rodents, including a primary PCR primer set and a secondary PCR primer set; the primary PCR primer set includes upstream and downstream primers as shown in SEQ ID NO.1 and SEQ ID NO.2, respectively, and also includes upstream and downstream primers as shown in SEQ ID NO.3 and SEQ ID NO.4, respectively; the secondary PCR primer set includes upstream and downstream primers as shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.
[0029] In the above-mentioned PCR primer set, two pairs of primary PCR primers with specific sequences are used in combination with a pair of secondary PCR primers. In the species detection of various rodent-like animals (including the common brown rat, yellow-haired rat, yellow-breasted rat, black-lined field mouse, social mouse, house mouse, black house mouse, Chinese field mouse and Burmese mouse in the family Muridae of the order Rodentia, and the common stinking shrew, big musk shrew and small musk shrew in the family Shrewidae of the order Insectivora), the concentration of the target gene fragment (including the full gene sequence of Cytb) in the PCR amplification product can be effectively increased. Therefore, without plasmid construction, transformation and culture enrichment, only PCR is required to ensure that the concentration of the target gene fragment meets the requirements of subsequent sequencing, which is helpful to improve the efficiency of species detection of rodent-like animals and reduce the difficulty of operation.
[0030] Second, the present invention relates to a kit for detecting species differentiation of rodents, the kit comprising the aforementioned PCR primer set.
[0031] In some embodiments, the kit further comprises a DNA polymerase and a Mg 2+ and a buffer containing dNTPs; and the DNA polymerase is Taq enzyme.
[0032] Thirdly, the present invention relates to the use of the aforementioned PCR primer set or the aforementioned kit in the detection of rodent species.
[0033] In some specific embodiments, the rodent is a rodent of the family Muridae or an Insectivora of the family Shrew; the rodent is Rattus norvegicus, Rattus truncatus, Rattus flavipectus, Apodemus agrarius, Rattus musculus, Rattus house mouse, Rattus house mouse, Apodemus chinensis or Rattus burmese; the Insectivora of the family Shrew is Rattus odoratus, Rattus dasyphyllus or Rattus serratus.
[0034] In some specific embodiments, the application comprises the following steps: S1: extracting DNA from the mouse-shaped animal to be tested to obtain a DNA sample to be tested; S2: preparing a primary PCR system containing the primary PCR primer set and the DNA sample to be tested, performing a first PCR reaction, and obtaining a primary PCR product; S3: preparing a secondary PCR system containing the secondary PCR primer set and the primary PCR product, and performing a second PCR reaction to obtain a secondary PCR product; S4: Sequence the secondary PCR product, and determine the species of the mouse-like animal to be tested according to the sequencing results.
[0035] In the above specific implementation, in step S1, the DNA sample to be tested is extracted from the liver tissue of the mouse-shaped animal to be tested.
[0036] In the above specific implementation, after step S2, the primary PCR product is subjected to electrophoresis detection. When the result is negative (i.e., no band appears in the electrophoresis pattern of the primary PCR product), the primary PCR product is directly used to prepare the secondary PCR system in step S3; when the result is positive (i.e., a band appears in the electrophoresis pattern of the secondary PCR product), the primary PCR product is diluted 10 to 100 times and then used to prepare the secondary PCR system in step S3.
[0037] In the above specific implementation, in step S2, the primary PCR system contains DNA polymerase, Mg 2+ and dNTPs.
[0038] In the above specific implementation, in step S2, the first PCR reaction is performed according to the following steps: 1) React at 94-95°C for 4-6 minutes; 2) Perform 35-40 cycles according to the following process: 94-95°C for 60-90 s → 55-57°C for 60-90 s → 70-73°C for 90-100 s; 3) React at 71-73°C for 10-15 minutes.
[0039] In the above specific implementation, in step S3, the second PCR reaction is performed according to the following steps: 1) React at 94-95°C for 4-6 minutes; 2) Perform 35-40 cycles according to the following process: 94-95°C for 60-90 s → 60-62°C for 60-90 s → 70-73°C for 90-100 s; 3) React at 71-73°C for 10-15 minutes.
[0040] In the above specific implementation, in step S4, the process of determining the species of the mouse to be tested based on the sequencing results is as follows: the sequencing results are compared with the gene library, and the species corresponding to the reference sequence with the highest homology in the gene library is taken as the species of the mouse to be tested. Specific embodiments The present invention is described below by specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention, and the attached claims and any equivalents thereof are the protection scope of the present invention.
[0042] Unless otherwise defined, all technical terms and scientific terms used in the present invention have the same meanings as those commonly understood by ordinary technicians in the field to which the present disclosure belongs. Unless otherwise specified, the raw materials and equipment used in the present invention are conventional raw materials and equipment in the field and can be obtained from conventional commercial channels; unless otherwise specified, the methods used in the present invention are conventional methods in the field.
[0043] Example 1 A kit for detecting species of rodents, comprising the following reagents: (1) 2× Multiplex PCR Buffer (provided by TaKaRa's RR062A and RR902A kits, containing Mg 2+ and dNTPs); (2) Premix Taq Buffer (provided by TaKaRa RR902A kit) (3) Taq enzyme (provided by RR062A kit of TaKaRa Company); (4) 20 μmol / L forward primer WF1 (sequence see Table 1); (5) 20 μmol / L reverse primer WR1 (sequence see Table 1); (6) 20 μmol / L forward primer WF2 (sequence see Table 1); (7) 20 μmol / L reverse primer WR2 (sequence see Table 1); (8) 20 μmol / L forward primer IF1 (sequence see Table 1); (9) 20 μmol / L reverse primer IR1 (sequence see Table 1).
[0044] Table 1 Primer sequences in the kit of Example 1 Primer name sequence Sequence number WF1 5'-CCT CRG TAG CCATAG CWG TYG-3' SEQ ID NO.1 WR1 5'-TTG RTT TCACGG AGG ATG GTA-3' SEQ ID NO.2 WF2 5'-AGATAY CAATCACCC TCY CCA-3' SEQ ID NO.3 WR2 5'-TTG RCC TCC AAT TCARGT TAR-3' SEQ ID NO.4 IF1 5'-ACC AAT GAC ATG AAAAAT CAT CGT T-3' SEQ ID NO.5 IR1 5'-TCT CCATTT CTG GTT TAC AAG AC-3' SEQ ID NO.6 The kit of this embodiment is used to perform species detection of murine animals, and the steps are as follows: S1: Adoption The Blood&Tissus kit is used to extract DNA from the liver tissue of the mouse to be tested. The specific steps are as follows: S1.1: Take 25 mg of liver tissue and place it in a 1.5 mL centrifuge tube, then add 180 μL of Buffer ALT.
[0045] S1.2: Add 20 μL of proteinase K, vortex and incubate at 56°C until the tissue is completely digested, shaking occasionally.
[0046] S1.3: Vortex for 15 seconds, add 200 μL Buffer ALT, vortex to mix thoroughly, add 200 μL alcohol, vortex to mix thoroughly.
[0047] S1.4: Transfer all the mixed solutions in step S1.3 to a DNeasy Mini spin column, place the column in a 2 mL collection tube, centrifuge at 8000 rpm for 1 min, and discard the collection solution.
[0048] S1.5: Place the centrifuge column in a new 2 mL collection tube, add 500 μL of buffer AW1, centrifuge at 8000 rpm for 1 min, and discard the collection solution.
[0049] S1.6: Place the centrifuge column in a new 2 mL collection tube, add 500 μL of buffer AW2, centrifuge at 14,000 rpm for 3 min, and discard the collection solution.
[0050] S1.7: Place the centrifuge column in a new 1.5 mL centrifuge tube, pipette 200 μL of buffer AE onto the adsorption membrane, centrifuge at room temperature for 1 min, and centrifuge at 8000 rpm for 1 min to obtain the DNA sample to be tested.
[0051] S2: First PCR reaction: S2.1: Prepare a PCR reaction system according to the formula in Table 2.
[0052] Table 2: Formula of the primary PCR reaction system of Example 1 Reagents Dosage 2×Multiplex PCR Buffer 25μL Taq enzyme 0.25μL 20 μmol / L forward primer WF1 0.5μL 20 μmol / L reverse primer WR1 0.5μL 20 μmol / L forward primer WF2 0.5μL 20 μmol / L reverse primer WR2 0.5μL DNA sample to be tested 5.0μL Sterile water 17.75μL S2.2: Using the primary PCR reaction system prepared in step S2.1, perform the first PCR reaction according to the PCR reaction program in Table 3 to obtain a primary PCR product, which is stored at 4°C.
[0053] Table 3: Primary PCR reaction procedure of Example 1 S2.3: The primary PCR product is subjected to agarose gel electrophoresis. When the electrophoresis result shows a negative result (no band appears in the electrophoresis pattern of the primary PCR product), the primary PCR product is directly used to prepare the secondary PCR reaction system in step S3; when the electrophoresis result shows a positive result (a band appears in the electrophoresis pattern of the primary PCR product), the primary PCR product is diluted 20 times and then used to prepare the secondary PCR system in step S3.
[0054] S3: Second PCR reaction: S3.1: Prepare the secondary PCR reaction system according to the formula in Table 4.
[0055] Table 4 Secondary PCR reaction system formula of Example 1 Reagents Dosage Premix Taq Buffer 25μL 20 μmol / L forward primer IF1 1μL 20 μmol / L reverse primer IR1 1μL One PCR product 5.0μL Sterile water 18μL S2.2: Using the secondary PCR reaction system prepared in step S2.1, perform a second PCR reaction according to the PCR reaction program in Table 5 to obtain a secondary PCR product, which is stored at 4°C.
[0056] Table 5 Secondary PCR reaction program of Example 1 S4: Sequencing to confirm species: The secondary PCR product was sequenced (performed by Sangon Biotech (Shanghai) Co., Ltd.), and the sequencing results were imported into SeqMan software for splicing, and then uploaded to the NCBI gene library for comparison. The redundant sequences were discarded, and the sequences were saved with a unified name. The reference sequence with the highest homology in the gene library was used as the molecular identification result. The species corresponding to the reference sequence was the species of the rodent to be tested.
[0057] Comparative Example 1 A kit for detecting species of rodents, comprising the following reagents: (1) Premix Taq Buffer (provided by RR902A kit of TaKaRa Company); (2) 20 μmol / L forward primer IF1 (sequence see Table 1 above); (3) 20 μmol / L reverse primer IR1 (sequence see Table 1 above).
[0058] The kit of this embodiment is used to perform species detection of murine animals, and the steps are as follows: S1: Adoption The Blood&Tissus kit is used to extract DNA from the liver tissue of the mouse-shaped animal to be tested. The specific steps are the same as those in Example 1 to obtain the DNA sample to be tested.
[0059] S2: PCR reaction: S2.1: Prepare the PCR reaction system according to the formula in Table 6.
[0060] Table 6 PCR reaction system formula of comparative example 1 S2.2: Using the PCR reaction system prepared in step S2.1, perform PCR reaction according to the PCR reaction program in Table 7 to obtain PCR products, which are stored at 4°C.
[0061] Table 7 PCR reaction program of comparative example 1 S3: Sequencing to determine species: The PCR product was sequenced (performed by Sangon Biotech (Shanghai) Co., Ltd.), and the sequencing results were imported into SeqMan software for splicing, and then uploaded to the NCBI gene library for comparison. The redundant sequences were discarded, and the sequences were saved with a unified name. The reference sequence with the highest homology in the gene library was used as the molecular identification result. The species corresponding to the reference sequence was the species of the rodent to be tested.
[0062] Comparative Example 2 A kit for detecting species of rodents, comprising the following reagents: (1) 2× Multiplex PCR Buffer (provided by TaKaRa's RR062A and RR902A kits, containing Mg 2+ and dNTPs); (2) Premix Taq Buffer (provided by TaKaRa RR902A kit) (3) Taq enzyme (provided by RR062A kit of TaKaRa Company); (4) 20 μmol / L forward primer WF1 (sequence see Table 8); (5) 20 μmol / L reverse primer WR1 (sequence see Table 8); (6) 20 μmol / L forward primer WF2 (sequence see Table 1); (7) 20 μmol / L reverse primer WR2 (sequence see Table 1); (8) 20 μmol / L forward primer IF1 (sequence see Table 1); (9) 20 μmol / L reverse primer IR1 (sequence see Table 1).
[0063] Table 8 Primer sequences in the kit of Comparative Example 2 Primer name sequence Sequence number WF3 5'-GGR TAC TCC TCAGTR GCY ATAG-3' SEQ ID NO.7 WR3 5'-TGG GCG GGT TGT TGR TT-3' SEQ ID NO.8 The kit of this embodiment is used to perform species detection of murine animals, and the steps are as follows: S1: Adoption The Blood&Tissus kit is used to extract DNA from the liver tissue of the mouse-shaped animal to be tested. The specific steps are the same as those in Example 1 to obtain the DNA sample to be tested.
[0064] S2: First PCR reaction: S2.1: Prepare a PCR reaction system according to the formula in Table 9.
[0065] Table 9: Formula of the single PCR reaction system of Comparative Example 2 Reagents Dosage 2×Multiplex PCR Buffer 25μL Taq enzyme 0.25μL 20 μmol / L forward primer WF3 0.5μL 20 μmol / L reverse primer WR3 0.5μL 20 μmol / L forward primer WF2 0.5μL 20 μmol / L reverse primer WR2 0.5μL DNA sample to be tested 5.0μL Sterile water 17.75μL S2.2: Using the primary PCR reaction system prepared in step S2.1, perform the first PCR reaction according to the PCR reaction program in Table 10 to obtain a primary PCR product, which is stored at 4°C.
[0066] Table 10: Primary PCR reaction program of Comparative Example 2 S2.3: The primary PCR product is subjected to agarose gel electrophoresis. When the electrophoresis result shows a negative result (no band appears in the electrophoresis pattern of the primary PCR product), the primary PCR product is directly used to prepare the secondary PCR reaction system in step S3; when the electrophoresis result shows a positive result (a band appears in the electrophoresis pattern of the primary PCR product), the primary PCR product is diluted 20 times and then used to prepare the secondary PCR system in step S3.
[0067] S3: Second PCR reaction: S3.1: Prepare the secondary PCR reaction system according to the formula in Table 11.
[0068] Table 11 Secondary PCR reaction system formula of comparative example 2 Reagents Dosage Premix Taq Buffer 25μL 20 μmol / L forward primer IF1 1μL 20 μmol / L reverse primer IR1 1μL One PCR product 5.0μL Sterile water 18μL S2.2: Using the secondary PCR reaction system prepared in step S2.1, perform a second PCR reaction according to the PCR reaction program in Table 12 to obtain a secondary PCR product, which is stored at 4°C.
[0069] Table 12 Secondary PCR reaction program of comparative example 2 S4: Sequencing to confirm species: The secondary PCR product was sequenced (performed by Sangon Biotech (Shanghai) Co., Ltd.), and the sequencing results were imported into SeqMan software for splicing, and then uploaded to the NCBI gene library for comparison. The redundant sequences were discarded, and the sequences were saved with a unified name. The reference sequence with the highest homology in the gene library was used as the molecular identification result. The species corresponding to the reference sequence was the species of the rodent to be tested.
[0070] Test Example 1: Effect of Detection on Multiple Species of Mouse-like Animals Using the kit and steps in Example 1, 138 rodents were tested by species. The test results are shown in Table 12.
[0071] Table 12 Results of detection of rodent-like animals by species Species name Quantity (pcs) Brown rat (Rattus norvegicus) 45 Yellow-haired rat (Rattus losea) 27 Rattus flavipectus 10 Apodemus agrarius 5 Niviventer niviventer 3 House mouse (Mus musculus) 1 Black House Rat (Rattus rattus) 2 Apodemus draco 2 Burmese Rat (Rattus exulans) 2 Stinky shrew (Suncus murinus) 37 Giant Musk Shrew (Crocidura lasiura) 2 Small Musk Shrew (Crocidura suaveolens) 2 It can be seen from Table 13 that the kit of the present invention has good universality for rodents, and can be used for the species detection of Rattus norvegicus, Rattus norvegicus, Rattus flavipectus, Apodemus agrarius, Rattus musculus, Rattus house mouse, Rattus musculus, Apodemus chinensis and Burmese mouse in the family Muridae of the order Rodentia, and the species detection of Odorea odorifera, Rattus dasyphylla and Rattus scrofa in the family Shrews of the order Insectivora.
[0072] Test Example 2: Effect of increasing PCR product concentration One each of Rattus norvegicus, Rattus norvegicus, Rattus flavipectus, Apodemus agrarius, Rattus edulis, Rattus musculus, Rattus musculus, Rattus chinensis, Apodemus chinensis, Rattus burmese, Rattus odoratus, Rattus sibiricus and Rattus sibiricus was randomly selected, and the kits and steps in Example 1 and Comparative Example 1 were used for species detection. The secondary PCR product obtained in step S2.2 of Example 1 and the PCR product obtained in step S2.2 of Comparative Example 1 were taken for agarose gel electrophoresis, 5 μL of sample was added to each well, and a DNA molecular weight reference (DL2000) was added to another well. After electrophoresis at 100 V for 60 min, the specific bands and their brightness were observed under a gel imager, and the obtained electrophoresis pattern was shown in FIG. Figure 1 and Figure 2 .
[0073] Compare Figure 1 and Figure 2It can be seen that: before using primers IF1 and IR1 for PCR amplification, the present invention first uses two primer pairs (primer pair WF1 and WR1, and primer pair WF2 and WR2) for PCR amplification, which can effectively increase the concentration of the target gene fragment in the PCR amplification product, and can play the above-mentioned role in a variety of rodent-shaped animals (including the common brown rat, yellow-haired rat, yellow-breasted rat, black-lined field mouse, community mouse, house mouse, black house mouse, Chinese field mouse and Burmese mouse in the family of rodents, and the common stinky shrew, big musk shrew and small musk shrew in the family of Insectivora).
[0074] Test Example 3: The influence of one PCR primer selection 50 rodent-like animals (including 25 rodents and 25 shrews) were randomly selected, and the kits and steps in Example 1 and Comparative Example 2 were used for species detection. The secondary PCR products obtained in step S2.2 of Example 1 and Comparative Example 2 were taken for agarose gel electrophoresis, 5 μL was added to each well, and a DNA molecular weight reference (DL2000) was added to another well. After electrophoresis at 100V for 60 minutes, the specific bands and their brightness were observed under a gel imager. The results showed that 100% of the secondary PCR products of Example 1 could meet the sequencing concentration requirements (all 50 samples met the sequencing concentration requirements); only 86% of the secondary PCR products of Comparative Example 2 could meet the sequencing concentration requirements (43 of the 50 samples met the sequencing concentration requirements). The aforementioned judgment criteria of "being able to meet the sequencing concentration requirements" are as follows: There are 6 reference standard bands in the DNA molecular weight reference standard (DL2000), the brightest of which is the 750bp band (150ng / 5μL). If the brightness of the electrophoresis band of the PCR product is greater than the brightness of the 750bp band in the DNA molecular weight reference standard (that is, the concentration of the PCR product is higher than 150ng / 5μL), then it is judged that the PCR product can meet the sequencing standards.
[0075] The above results show that the selection of a PCR primer will affect the concentration of the target gene fragment in the PCR amplification product. The present invention can achieve a better amplification effect by designing the PCR primer sequence, so that the concentration of the target gene fragment in the product reaches a higher level, thereby improving the sensitivity of rodent species detection.
[0076] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.
Claims
1. A PCR primer set for detecting species of murine animals, characterized in that: It includes a primary PCR primer set and a secondary PCR primer set; the primary PCR primer set includes the upstream and downstream primers shown as SEQ ID NO.1 and SEQ ID NO.2, respectively, and also includes the upstream and downstream primers shown as SEQ ID NO.3 and SEQ ID NO.4, respectively; the secondary PCR primer set includes the upstream and downstream primers shown as SEQ ID NO.5 and SEQ ID NO.6, respectively.
2. A kit for detecting species of murine animals, characterized in that: The kit comprises the PCR primer set according to claim 1.
3. The kit according to claim 2, characterized in that The kit also includes a DNA polymerase and a Mg 2+ and dNTP buffer.
4. Use of the PCR primer set according to claim 1 or the kit according to claim 2 or 3 in detecting species of murine animals.
5. The use according to claim 4, characterized in that: The mouse-shaped animal is a rodent of the family Muridae or an Insectivora of the family Shrew.
6. The use according to claim 4, characterized in that: The following steps are involved: S1: extracting DNA from the mouse-shaped animal to be tested to obtain a DNA sample to be tested; S2: preparing a primary PCR system containing the primary PCR primer set and the DNA sample to be tested, performing a first PCR reaction, and obtaining a primary PCR product; S3: preparing a secondary PCR system containing the secondary PCR primer set and the primary PCR product, and performing a second PCR reaction to obtain a secondary PCR product; S4: Sequence the secondary PCR product, and determine the species of the mouse-like animal to be tested according to the sequencing results.
7. The use according to claim 6, characterized in that: After step S2, the primary PCR product is subjected to electrophoresis detection. When the result is negative, the primary PCR product is directly used to prepare the secondary PCR system in step S3; when the result is positive, the primary PCR product is diluted 10 to 100 times and then used to prepare the secondary PCR system in step S3.
8. The use according to claim 5, characterized in that: In step S2, the first PCR reaction is carried out according to the following steps: 1) React at 94-95°C for 4-6 min; 2) Perform 35-40 cycles as follows: 94-95°C 60-90 s → 55-57°C 60-90 s → 70-73°C 90-100 s; 3) React at 71-73°C for 10-15 min.
9. The use according to claim 5, characterized in that: In step S3, the second PCR reaction is carried out according to the following steps: 1) React at 94-95°C for 4-6 min; 2) Perform 35-40 cycles as follows: 94-95°C 60-90 s → 60-62°C 60-90 s → 70-73°C 90-100 s; 3) React at 71-73°C for 10-15 min.
10. The use according to claim 6, characterized in that: The primary PCR system and the secondary PCR system both contain DNA polymerase, Mg 2+ and dNTPs.