Non-invasive PCR (Polymerase Chain Reaction) sex identification method for tortoises
By designing dual PCR amplification primers targeting sex-specific genes in turtles and using an improved DNA extraction method, the problems of age limitations, invasive damage, and poor specificity in existing turtle sex identification methods have been solved. This has resulted in a highly accurate, non-invasive, and simple sex identification method applicable to various turtle species.
Patent Information
- Application Number
- CN202511760562.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-27
- Publication Date
- 2026-02-06
AI Technical Summary
Existing methods for sex determination in turtles have problems such as age limitations, invasive damage, poor specificity, and insufficient batch adaptability. In particular, the traditional PCR identification method has insufficient accuracy and the non-invasive sample has low DNA content and many impurities, making it difficult to meet amplification requirements.
Designed dual PCR primers for male-specific DMRT1 gene and female-specific CHD1W gene in turtles, combined with a modified CTAB-proteinase K method to extract non-invasive sample DNA, and employed a non-invasive PCR sex identification method, including pretreatment and dual PCR amplification of molted shell, fecal or oral mucosal samples.
It achieves highly specific and sensitive turtle sex identification, is applicable to multiple turtle species, has an accuracy rate of 99%, has no false positives/false negatives, is easy to operate, is suitable for large-scale breeding and market supervision, and meets animal protection needs.
Smart Images
Figure CN121472420A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of turtle sex determination technology, specifically to a non-invasive PCR sex determination method for turtles. Background Technology
[0002] Sex determination in turtles is crucial for species conservation and breeding, but the following existing methods have significant drawbacks:
[0003] (1) Morphological identification method: Morphological identification relies on the characteristics of adult plastron shape, tail length, etc. There is no obvious difference in juveniles (≤12 months old), and the accuracy rate of sub-adults is only 55%-70%, and it is greatly affected by the variety and growth environment.
[0004] (2) Invasive sampling identification method: Invasive sampling (such as blood and muscle biopsy) can easily cause stress response, wound infection and even shell rot in turtles, which violates the principle of protection and is not suitable for batch operation;
[0005] (3) Traditional PCR identification method: The primers used in traditional PCR identification are mostly universal and have insufficient homology with the sex-specific genes of turtles, which easily leads to false positives, low amplification efficiency, poor differentiation between closely related turtle species, and an accuracy of less than 80%. The core reason is that there is a lack of targeted primers for the sex-determining genes of turtles, and the DNA content in non-invasive samples (such as molted shells and feces) is low and there are many impurities. Existing extraction methods are difficult to meet the requirements of PCR amplification. Summary of the Invention
[0006] To address the shortcomings of existing technologies, this invention provides a non-invasive, highly specific, highly sensitive, and multi-species compatible PCR sex determination method for turtles. This invention achieves turtle sex determination through dual PCR amplification of the DMRT1 and CHD1W genes, overcoming the limitations of traditional methods such as age restrictions, invasiveness, poor specificity, and insufficient batch adaptability. This provides technical support for large-scale turtle breeding, species conservation, and market supervision.
[0007] To achieve the above objectives, the specific technical solution of the present invention is as follows:
[0008] In a first aspect, the present invention provides a primer set for non-invasive PCR sex identification of turtles, including male-specific primers, female-specific primers and internal reference primers;
[0009] The upstream primer sequence of the male-specific primer is shown in SEQ ID No. 1, and the downstream primer sequence of the male-specific primer is shown in SEQ ID No. 2.
[0010] The upstream primer sequence of the female-specific primer is shown in SEQ ID No. 3, and the downstream primer sequence of the female-specific primer is shown in SEQ ID No. 4;
[0011] The upstream primer sequence of the internal reference primer is shown in SEQ ID No. 5, and the downstream primer sequence of the internal reference primer is shown in SEQ ID No. 6.
[0012] This invention utilizes conserved sequences of the male-specific DMRT1 gene (base sequence shown in SEQ ID No. 7) and the female-specific CHD1W gene (base sequence shown in SEQ ID No. 8) of turtles to design specific PCR amplification primers for male and female turtles, respectively. The male-specific primer targets the DMRT1 gene, and the female-specific primer targets the CHD1W gene. Simultaneously, an internal control primer is designed based on the conserved sequence of the β-actin gene (base sequence shown in SEQ ID No. 9), which targets the β-actin gene, to verify the validity of the DNA. This dual PCR amplification detection method of the present invention features high specificity, high sensitivity, and compatibility with multiple turtle species.
[0013] Secondly, the present invention provides the application of the primer set in non-invasive PCR sex identification of turtles.
[0014] Thirdly, the present invention provides a non-invasive PCR sex identification kit for turtles, including the primer set.
[0015] Furthermore, the kit also includes DNA extraction reagents and PCR reagents.
[0016] Fourthly, the present invention provides a non-invasive PCR sex determination method for turtles, comprising the following steps:
[0017] Genomic DNA was extracted from non-invasive samples of turtles;
[0018] Using the genomic DNA as a template, PCR amplification was performed using the primers shown in SEQ ID No. 1~6 to obtain the amplification products;
[0019] The amplification products were analyzed by electrophoresis. When the amplification products showed both a 432 bp amplification band from the internal reference primer and a 278 bp male-specific band, they were identified as male. When the amplification products showed both a 432 bp amplification band from the internal reference primer and a 196 bp female-specific band, they were identified as female. If only a 432 bp amplification band from the internal reference primer or no band was observed, the sample DNA was considered invalid and a new sample collection was required.
[0020] Furthermore, the non-invasive sample includes, but is not limited to, at least one of molted shells, feces, and oral mucosa.
[0021] Furthermore, the non-invasive sample was pretreated before genomic DNA extraction; the pretreatment of the molted sample was as follows: the molted shell was rinsed twice with sterile saline and cut into 1 mm pieces. 2 The following are the pretreatment methods for fecal samples: Add 3 times the volume of buffer (containing 0.5% Triton X-100), vortex mix for 5 minutes, and let stand at 4°C for 20 minutes; The following are the pretreatment methods for oral mucosa samples: Immerse the oral mucosa in 60 μL of TE buffer and shake at 37°C for 15 minutes.
[0022] Furthermore, a modified CTAB-proteinase K combined method was used to extract genomic DNA from non-invasive samples: after adding the extraction solution, the genomic DNA of the non-invasive samples was extracted in a water bath at 60°C for 50 minutes; the extraction solution was a CTAB extraction solution containing 1% β-mercaptoethanol and 0.5 mg / mL proteinase K.
[0023] Further, the PCR amplification system (25 μL) consists of: 2.5 μL of 10×PCR buffer, 2 μL of dNTPs (2.5 mM), 0.6 μL each of upstream and downstream male-specific primers (10 μM), 0.6 μL each of upstream and downstream female-specific primers (10 μM), 0.4 μL each of upstream and downstream internal control primers (10 μM), 0.3 μL of Taq enzyme (5 U / μL), 3 μL of genomic DNA, and the remainder being enzyme-free water.
[0024] Furthermore, the PCR amplification conditions are as follows: 95℃ pre-denaturation for 6 minutes; 95℃ denaturation for 30 seconds, 60℃ annealing for 35 seconds, 72℃ extension for 40 seconds, 38 cycles; 72℃ final extension for 10 minutes.
[0025] Specifically, the non-invasive PCR sex determination method for turtles includes the following steps:
[0026] Step 1: Non-invasive sample collection and preprocessing
[0027] Sample collection: Collect naturally shed shells from turtles (area ≥ 0.4 cm²). 2 Fresh feces (weight ≥0.1 g) or oral mucosa (use a sterile cotton swab to gently wipe the inside of the mouth 4 times), avoid contact with the individual's body;
[0028] Sample pretreatment: Molted samples were rinsed twice with sterile saline and cut into 1 mm pieces. 2For fecal samples, add 3 times the volume of buffer (containing 0.5% Triton X-100), vortex for 5 minutes, and let stand at 4°C for 20 minutes; for oral mucosal samples, soak in 60 μL of TE buffer and shake at 37°C for 15 minutes.
[0029] Step Two: Efficient DNA Extraction
[0030] A modified CTAB-proteinase K combined method was used: CTAB extraction solution containing 1% β-mercaptoethanol and 0.5 mg / mL proteinase K was added to the pretreated sample, and the mixture was incubated in a 60℃ water bath for 50 minutes, inverting and mixing once every 10 minutes to fully lyse the cuticle and impurities; an equal volume of chloroform-isoamyl alcohol (volume ratio 24:1) was added, and the mixture was centrifuged at 12000 rpm for 10 minutes, and the supernatant was collected; 0.7 volumes of isopropanol were added to the supernatant, and the mixture was incubated at -20℃ for 30 minutes, followed by centrifugation at 12000 rpm for 15 minutes to collect the DNA precipitate; the precipitate was washed twice with 75% ethanol, dried, and dissolved in 30 μL of enzyme-free water to obtain high-purity genomic DNA.
[0031] Step 3: Design and synthesis of sex-specific primers for turtles
[0032] Design specific primers for male turtles based on the conserved sequence of the male-specific DMRT1 gene:
[0033] Upstream primer: 5'-ATGGCAGAGAAAGTACCAGTG-3' (see SEQ ID No. 1);
[0034] Downstream primer: 5'-CGGCGCGGCTTCTCCCGCAAG-3' (see SEQ ID No. 2);
[0035] Male-specific primers target the DMRT1 gene, and the fragment length amplified by the male-specific primers is 278 bp.
[0036] Design specific primers for female turtles based on the conserved sequence of the female-specific CHD1W gene:
[0037] Upstream primer: 5'-TAGGAAAAGGAAGGACCATA-3' (see SEQ ID No. 3);
[0038] Downstream primer: 5'-ACTAAACCTGGATACAAAAC-3' (see SEQ ID No. 4);
[0039] The female-specific primers target the CHD1W gene, and the fragment length amplified by the female-specific primers is 196 bp.
[0040] Design of internal reference primers based on conserved sequences of the β-actin gene:
[0041] Upstream primer: 5'-GTACACTTGCTGCAGTAGTCT-3' (see SEQ ID No. 5);
[0042] Downstream primer: 5'-GATGAGGCCCAGAGCAAGAG-3' (see SEQ ID No. 6);
[0043] The internal reference primer targets the β-actin gene and is used to verify the effectiveness of the DNA. The fragment length amplified based on the internal reference primer is 432 bp.
[0044] Step 4: Duplex PCR Amplification
[0045] The PCR amplification system (25 μL) consisted of: 2.5 μL of 10×PCR buffer, 2 μL of dNTPs (2.5 mM), 0.6 μL each of upstream and downstream male-specific primers (10 μM), 0.6 μL each of upstream and downstream female-specific primers (10 μM), 0.4 μL each of upstream and downstream internal control primers (10 μM), 0.3 μL of Taq enzyme (5 U / μL), 3 μL of genomic DNA, and the remainder being enzyme-free water.
[0046] The PCR amplification conditions were as follows: 95℃ pre-denaturation for 6 minutes; 95℃ denaturation for 30 seconds, 60℃ annealing for 35 seconds, 72℃ extension for 40 seconds, for a total of 38 cycles; 72℃ final extension for 10 minutes, and storage at 4℃.
[0047] Step 5: Result Detection and Judgment
[0048] Take 6 μL of PCR amplification product and perform 1.8% agarose gel electrophoresis at 110 V for 30 minutes. Observe the results using a UV gel imaging system.
[0049] Result determination:
[0050] The simultaneous appearance of a 432 bp amplification band from the internal reference primers and a 278 bp male-specific band indicates that the animal is male.
[0051] The presence of both a 432 bp amplification band from the internal reference primers and a 196 bp female-specific band indicates that the animal is female.
[0052] If only a 432 bp amplification band is observed from the internal reference primer, or if no band is observed, the sample DNA is considered invalid and a new sample must be collected.
[0053] Compared with the prior art, the advantages of the present invention are:
[0054] (1) The turtle sex identification method provided by the present invention is non-invasive sampling, requiring only molted shell, feces or oral mucosa samples, without damage or stress response. Among them, the success rate of genomic DNA extraction from feces samples is over 96%. Non-invasive sampling meets the needs of animal protection and is applicable to juveniles, sub-adults and adults.
[0055] (2) The amplification primers used in the turtle sex identification method provided by the present invention are designed for the conserved sequences of turtle sex-specific genes. The primers are highly specific and have no cross-amplification with reptiles such as snakes and lizards. They are compatible with more than 10 common turtle species such as Chinese pond turtles, red-eared sliders, and tortoises, with a specificity of 100%.
[0056] (3) The turtle sex identification method provided by the present invention has a high accuracy rate. When the sex of 110 turtles (including 40 juveniles and 30 adults of known sex) were identified using this method, the accuracy rate reached more than 99%, with no false positives / false negatives.
[0057] (4) The turtle sex identification method provided by the present invention is simple to operate, the PCR reaction can be carried out in batches, the detection cycle of a single sample is only 3.5 hours, no expensive equipment is required, and it is suitable for on-site application in laboratories and breeding farms.
[0058] (5) The turtle sex identification method provided by the present invention has high sensitivity and can detect genomic DNA templates as low as 5 ng / μL. Attached Figure Description
[0059] Figure 1 This is a schematic diagram of the non-invasive PCR sex determination method of the present invention;
[0060] Figure 2 Electrophoresis images showing the DNA extraction results for different sample types;
[0061] Figure 3 The band positions of male (432 bp + 278 bp), female (432 bp + 196 bp), negative control, and internal control in agarose gel electrophoresis are shown. Detailed Implementation
[0062] To enable those skilled in the art to clearly and completely understand the technical solution of the present invention, the present invention will be further described in detail below with reference to embodiments. Obviously, the embodiments described herein are only for explaining the present invention and are not intended to limit the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.
[0063] This invention provides a primer set for non-invasive PCR sex identification of turtles, comprising male-specific primers, female-specific primers, and an internal reference primer; wherein, the upstream primer sequence of the male-specific primer is shown in SEQ ID No. 1, and the downstream primer sequence of the male-specific primer is shown in SEQ ID No. 2; the upstream primer sequence of the female-specific primer is shown in SEQ ID No. 3, and the downstream primer sequence of the female-specific primer is shown in SEQ ID No. 4; the upstream primer sequence of the internal reference primer is shown in SEQ ID No. 5, and the downstream primer sequence of the internal reference primer is shown in SEQ ID No. 6.
[0064] This invention also provides a non-invasive PCR sex determination method for turtles, comprising the following steps:
[0065] Genomic DNA was extracted from a non-invasive sample of a turtle. Using the genomic DNA as a template, PCR amplification was performed using primers shown in SEQ ID No. 1-6 to obtain amplification products. The amplification products were analyzed by electrophoresis. When the amplification products showed a 432 bp amplification band of the internal reference primer and a 278 bp male-specific band, the sample was identified as male. When the amplification products showed a 432 bp amplification band of the internal reference primer and a 196 bp female-specific band, the sample was identified as female. If only a 432 bp amplification band of the internal reference primer or no band was observed, the sample DNA was considered invalid and a new sample collection was required.
[0066] In the following specific embodiment, the non-invasive PCR sex determination method for turtles includes the following steps:
[0067] Step 1: Non-invasive sample collection and preprocessing
[0068] Sample collection: Collect naturally shed shells from turtles (area ≥ 0.4 cm²). 2 Fresh feces (weight ≥0.1 g) or oral mucosa (use a sterile cotton swab to gently wipe the inside of the mouth 4 times), avoid contact with the individual's body;
[0069] Sample pretreatment: Molted samples were rinsed twice with sterile saline and cut into 1 mm pieces. 2 For fecal samples, add 3 volumes of buffer containing 0.5% Triton X-100, vortex for 5 minutes, and incubate at 4°C for 20 minutes; for oral mucosal samples, soak in 60 μL of TE buffer and shake at 37°C for 15 minutes.
[0070] Step Two: Efficient DNA Extraction
[0071] A modified CTAB-proteinase K combined method was used: CTAB extraction solution containing 1% β-mercaptoethanol and 0.5 mg / mL proteinase K was added to the pretreated sample, and the mixture was incubated in a 60℃ water bath for 50 minutes, inverting and mixing once every 10 minutes to fully lyse the cuticle and impurities; an equal volume of chloroform-isoamyl alcohol (volume ratio 24:1) was added, and the mixture was centrifuged at 12000 rpm for 10 minutes, and the supernatant was collected; 0.7 volumes of isopropanol were added to the supernatant, and the mixture was incubated at -20℃ for 30 minutes, followed by centrifugation at 12000 rpm for 15 minutes to collect the DNA precipitate; the precipitate was washed twice with 75% ethanol, dried, and dissolved in 30 μL of enzyme-free water to obtain high-purity genomic DNA. Figure 2 The image shows electrophoresis diagrams of DNA extraction effects from different sample types (e.g., skin, test strips), verifying the quality of DNA extraction from non-invasive samples using this invention. Figure 2 The results showed that among non-invasive samples, the DNA extracted from eggshells had the least DNA drag, and there was no significant difference in the purity of DNA extracted from oral test strips, molted samples, and blood DNA samples.
[0072] Step 3: Design and synthesis of sex-specific primers for turtles
[0073] Design specific primers for male turtles based on the conserved sequence of the male-specific DMRT1 gene:
[0074] Upstream primer: 5'-ATGGCAGAGAAAGTACCAGTG-3' (see SEQ ID No. 1);
[0075] Downstream primer: 5'-CGGCGCGGCTTCTCCCGCAAG-3' (see SEQ ID No. 2);
[0076] Male-specific primers target the DMRT1 gene, and the fragment length amplified by the male-specific primers is 278 bp.
[0077] Design specific primers for female turtles based on the conserved sequence of the female-specific CHD1W gene:
[0078] Upstream primer: 5'-TAGGAAAAGGAAGGACCATA-3' (see SEQ ID No. 3);
[0079] Downstream primer: 5'-ACTAAACCTGGATACAAAAC-3' (see SEQ ID No. 4);
[0080] The female-specific primers target the CHD1W gene, and the fragment length amplified by the female-specific primers is 196 bp.
[0081] Design of internal reference primers based on conserved sequences of the β-actin gene:
[0082] Upstream primer: 5'-GTACACTTGCTGCAGTAGTCT-3' (see SEQ ID No. 5);
[0083] Downstream primer: 5'-GATGAGGCCCAGAGCAAGAG-3' (see SEQ ID No. 6);
[0084] The internal reference primer targets the β-actin gene and is used to verify the effectiveness of the DNA. The fragment length amplified based on the internal reference primer is 432 bp.
[0085] Step 4: Duplex PCR Amplification
[0086] The PCR amplification system (25 μL) consisted of: 2.5 μL of 10×PCR buffer, 2 μL of dNTPs (2.5 mM), 0.6 μL each of upstream and downstream male-specific primers (10 μM), 0.6 μL each of upstream and downstream female-specific primers (10 μM), 0.4 μL each of upstream and downstream internal control primers (10 μM), 0.3 μL of Taq enzyme (5 U / μL), 3 μL of genomic DNA, and the remainder being enzyme-free water.
[0087] The PCR amplification conditions were as follows: 95℃ pre-denaturation for 6 minutes; 95℃ denaturation for 30 seconds, 60℃ annealing for 35 seconds, 72℃ extension for 40 seconds, for a total of 38 cycles; 72℃ final extension for 10 minutes, and storage at 4℃.
[0088] Step 5: Result Detection and Judgment
[0089] Take 6 μL of PCR amplification product and perform 1.8% agarose gel electrophoresis at 110 V for 30 minutes. Observe the results using a UV gel imaging system.
[0090] Result determination:
[0091] The simultaneous appearance of a 432 bp amplification band from the internal reference primers and a 278 bp male-specific band indicates that the animal is male.
[0092] The presence of both a 432 bp amplification band from the internal reference primers and a 196 bp female-specific band indicates that the animal is female.
[0093] If only a 432 bp amplification band is observed from the internal reference primer, or if no band is observed, the sample DNA is considered invalid and a new sample must be collected. Figure 3 The image shows the band positions of male (432 bp + 278 bp), female (432 bp + 196 bp), negative control, and internal control in agarose gel electrophoresis.
[0094] Example 1: Sex determination of adult Chinese pond turtles
[0095] Molting samples were collected from 30 adult Chinese pond turtles of known sex (15 males and 15 females). After pretreatment of the molted shells, genomic DNA was extracted using a modified CTAB-proteinase K combined method. Double PCR amplification was performed using male-specific primers, female-specific primers, and an internal control primer. The amplified products were then analyzed by electrophoresis. The results showed that all 15 male samples contained a 432 bp internal control band and a 278 bp specific band, while all 15 female samples contained a 432 bp internal control band and a 196 bp specific band. No cross-amplification was observed, and the accuracy rate for sex identification was 100%.
[0096] Example 2: Sex determination of juvenile red-eared sliders
[0097] Fecal samples were collected from 40 two-month-old juvenile red-eared sliders (sex unknown, to be verified by adult morphology). After fecal pretreatment, genomic DNA was extracted using a modified CTAB-proteinase K combined method. Double PCR amplification was performed using male-specific primers, female-specific primers, and an internal control primer. The amplified products were then analyzed by electrophoresis. The results showed that 19 turtles were male (432 bp + 278 bp) and 21 were female (432 bp + 196 bp). Morphological verification was performed after 12 months. In this example, only one juvenile turtle had its DNA extracted due to contamination during the extraction process; a new sample was taken and DNA was extracted before sex determination. The DNA extraction success rate from the fecal samples was 97.5%, and the sex identification accuracy was 100%, far exceeding traditional morphological identification (accuracy ≤ 50%).
[0098] Example 3: Verification of Specificity for Multiple Turtle Species
[0099] Ten samples each of Chinese pond turtle, red-eared slider, yellow-margined box turtle, and sulcata tortoise were collected. After sample pretreatment, genomic DNA was extracted using a modified CTAB-proteinase K combined method. Double PCR amplification was performed using male-specific primers, female-specific primers, and an internal control primer. The amplified products were then analyzed by electrophoresis. The results showed that all turtle species exhibited corresponding sex bands and internal control bands, with no cross-amplification. Samples from closely related reptiles (cobra and bearded dragon) were tested; none of the tested samples showed sex-specific bands, achieving 100% specificity.
[0100] In summary, this invention extracts genomic DNA from non-invasive samples and uses this genomic DNA as a template for PCR amplification using primers shown in SEQ ID Nos. 1-6. When the amplification product simultaneously exhibits a 432 bp internal reference band and a 278 bp male-specific band, it is identified as male; when the amplification product simultaneously exhibits a 432 bp internal reference band and a 196 bp female-specific band, it is identified as female. The amplification primers used in this method are designed targeting conserved sequences of sex-specific genes in turtles. These primers exhibit extremely high specificity, with no cross-amplification with reptiles such as snakes and lizards. They are compatible with more than 10 common turtle species, including Chinese pond turtles, red-eared sliders, and tortoises, achieving 100% specificity and over 99% accuracy, demonstrating promising prospects for widespread application.
[0101] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.
Claims
1. A primer set for non-invasive PCR sex determination of turtles, characterized in that, The primers include male-specific primers, female-specific primers, and internal reference primers; wherein, the upstream primer sequence of the male-specific primer is shown in SEQ ID No. 1, and the downstream primer sequence of the male-specific primer is shown in SEQ ID No. 2; the upstream primer sequence of the female-specific primer is shown in SEQ ID No. 3, and the downstream primer sequence of the female-specific primer is shown in SEQ ID No. 4; the upstream primer sequence of the internal reference primer is shown in SEQ ID No. 5, and the downstream primer sequence of the internal reference primer is shown in SEQ ID No.
6.
2. The application of the primer set for non-invasive PCR sex identification of turtles as described in claim 1 in non-invasive PCR sex identification of turtles.
3. A non-invasive PCR sex determination kit for turtles, characterized in that, The primer set for non-invasive PCR sex identification of turtles as described in claim 1.
4. The non-invasive PCR sex determination kit for turtles according to claim 3, characterized in that, The kit also includes DNA extraction reagents and PCR reagents.
5. A non-invasive PCR method for sex determination of turtles, characterized in that, Includes the following steps: Genomic DNA was extracted from non-invasive samples of turtles; Using the genomic DNA as a template, PCR amplification was performed using the primers shown in SEQ ID No. 1~6 to obtain the amplification products; The amplification products were analyzed by electrophoresis. When the amplification products showed both a 432 bp amplification band from the internal reference primer and a 278 bp male-specific band, they were identified as male. When the amplification products showed both a 432 bp amplification band from the internal reference primer and a 196 bp female-specific band, they were identified as female. If only a 432 bp amplification band from the internal reference primer or no band was observed, the sample DNA was considered invalid and a new sample collection was required.
6. A non-invasive PCR sex determination method for turtles according to claim 5, characterized in that, The non-invasive samples include, but are not limited to, at least one of molted shells, feces, and oral mucosa.
7. A non-invasive PCR sex determination method for turtles according to claim 6, characterized in that, Before extracting genomic DNA, the non-invasive samples were pretreated; the pretreatment of the molted samples was as follows: the molted shells were rinsed twice with sterile saline and cut into 1 mm pieces. 2 The following are the pretreatment methods for fecal samples: Add 3 volumes of buffer containing 0.5% Triton X-100 and vortex for 5 minutes, then let stand at 4°C for 20 minutes; The following are the pretreatment methods for oral mucosa samples: Immerse the oral mucosa in 60 μL of TE buffer and shake at 37°C for 15 minutes.
8. A non-invasive PCR sex determination method for turtles according to claim 7, characterized in that, Genomic DNA was extracted from non-invasive samples using a modified CTAB-proteinase K combined method: after adding the extraction solution, the genomic DNA of the non-invasive samples was extracted by incubating in a water bath at 60°C for 50 minutes; the extraction solution was a CTAB extraction solution containing 1% β-mercaptoethanol and 0.5 mg / mL proteinase K.
9. A non-invasive PCR sex determination method for turtles according to claim 5, characterized in that, The PCR amplification system consisted of: 2.5 μL of 10×PCR buffer, 2 μL of dNTPs, 0.6 μL each of upstream and downstream male-specific primers, 0.6 μL each of upstream and downstream female-specific primers, 0.4 μL each of upstream and downstream internal control primers, 0.3 μL of Taq enzyme, 3 μL of genomic DNA, and enzyme-free water to a final volume of 25 μL.
10. A non-invasive PCR sex determination method for turtles according to claim 5, characterized in that, The PCR amplification conditions were as follows: 95℃ pre-denaturation for 6 minutes; 95℃ denaturation for 30 seconds, 60℃ annealing for 35 seconds, 72℃ extension for 40 seconds, for 38 cycles; and 72℃ final extension for 10 minutes.