Qpcr-based method for early sex identification of pigeon

Abstract

Provided is a qPCR-based method for early sex identification of a pigeon. The method specifically comprises the following steps: S1, extracting a genomic DNA from tissue or body fluid such as pigeon feathers and saliva; S2, adjusting primers, and carrying out a PCR reaction using the extracted DNA as a template; S3, preparing pure W and Z standard sequences by means of TA cloning; S4, identifying positive clones by bacterial colony PCR; S5, adjusting primer and probe sequences; and S6, detecting specificity by means of a fluorescent quantitative PCR test. The qPCR-based method for early sex identification of a pigeon achieves good specificity and high identification accuracy by using a fluorescent quantitative PCR probe method. In this method, on the basis of the conventional fluorescent quantitative PCR probe method, dual-probe multiplex detection is further used, the two probes respectively target Z gene and W gene fragments, and the two probes are respectively labeled with different fluorescence signals, enabling simultaneous detection in one tube. One advantage is that the results are intuitive: females exhibit signals from both Z and W probes, whereas males only exhibit a signal from the Z probe.

Description

A qPCR method for early sex identification of pigeons Technical Field

[0001] The present invention relates to the technical field of early sex identification of pigeons, and in particular to a method for early sex identification of pigeons using qPCR. Background Art

[0002] Pigeons lack visible external genitalia, and male and female birds are very similar in morphology. Furthermore, due to the large individual variability among pigeons, even birds of the same sex can vary in size and behavior. This makes sexing pigeons very difficult, especially for young birds.

[0003] While there are methods for identifying the sex of pigeons, they are not completely reliable. For example, identification based on appearance is easily affected by environmental and developmental factors, while identification based on sounds and behavior requires a thorough understanding of the pigeon's habits.

[0004] The CHD1 gene is a chromosome segregation protein gene that is widely present in many animals (including birds), and it plays an important role in determining the sex of males and females. The CHD1 gene exists in two subtypes: W and Z. The W type is only present in the chromosomes of female birds, while the Z type is present in the chromosomes of both male and female birds. Therefore, by detecting the subtype of the CHD1 gene, the sex of the bird can be determined. The CHD1 gene is widely present in birds and is highly conserved. The DNA sample required to detect the subtype of the CHD1 gene is very small, which is very convenient for field research and bird monitoring. Through technologies such as PCR amplification and gene sequencing, the subtype of the CHD1 gene can be quickly and accurately determined, and then the sex of the bird can be determined.

[0005] Summary of the Invention

[0006] (1) Technical problems solved

[0007] In view of the shortcomings of the existing technology, the present invention provides a method for identifying the early sex of pigeons by qPCR, which solves the problems mentioned in the above background technology.

[0008] (2) Technical solution

[0009] To achieve the above objectives, the present invention is implemented through the following technical solutions: a method for identifying the early sex of pigeons by qPCR, specifically comprising the following steps:

[0010] S1. Extract genomic DNA from pigeon feathers (medullary tissue, the tissue inside the cavity at the base of the feather), saliva, and other tissues or body fluids;

[0011] S2. Debug the primers and perform PCR reaction using the extracted DNA as a template;

[0012] S3, TA cloning to prepare standard sequences of pure W and Z;

[0013] S4, identification of positive clones by colony PCR;

[0014] S5, adjust the primer probe sequence;

[0015] S6. Fluorescence quantitative PCR test for detection specificity.

[0016] Preferably, in step S2, the PCR reaction procedure is: denaturation at 95°C for 3 min; denaturation at 94°C for 15 s, annealing at 55°C for 20 s, and extension at 72°C for 20 s, with the number of cycles being 35.

[0017] Preferably, in step S2, the reaction system of the PCR reaction is: qPCR Mix 15 μL, primer Mg at a concentration of 25 mM 2+ 2μL, 0.5μL of 10μM forward primer, 0.5μL of 10μM reverse primer, 2μL of DNA template, add ddH2O to 30μL.

[0018] Preferably, in step S4, the reaction system of the PCR reaction is: 2xMix 15 μL, primer Mg 2+ 3μL, Primer P5 0.5μL, Primer P3 0.5μL, ddH2O 9μL.

[0019] Preferably, in step S4, the PCR reaction procedure is: denaturation at 95°C for 3 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, with the number of cycles being 30.

[0020] Preferably, in step S5, an MGB probe is used.

[0021] (3) Beneficial effects

[0022] The present invention provides a method for identifying the sex of pigeons in their early stages by qPCR. Compared with existing technologies, the method has the following advantages: the method for identifying the sex of pigeons in their early stages by qPCR uses a fluorescent quantitative PCR probe method, which has good specificity and high identification accuracy;

[0023] In this technical solution, based on the conventional fluorescent quantitative PCR probe method, dual-probe multiplex detection is also adopted. The two probes target the Z gene and W gene fragments respectively. The two probes are labeled with different fluorescent signals and can be detected simultaneously in a single tube. The first advantage is that the results are clear at a glance. Females show detection of both Z and W probes, while males only have a signal from the Z probe. The second advantage is that the two probes confirm each other. Both females and males have signals, which is equivalent to having both a positive control and an internal control (Z probe).

[0024] By using MGB probes, the detection specificity is better and the sensitivity is high, reaching 10 copies / ul;

[0025] The CDH sequence information was cloned and sequenced to obtain gene sequence information that was different from that in the database. On this basis, unique primers and probes were designed that are more suitable for local pigeon breeds. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG1 shows the PCR amplification conditions provided in Example 2 of the present invention;

[0027] FIG2 is an electrophoresis analysis diagram after PCR amplification provided in Example 2 of the present invention;

[0028] FIG3 is a diagram of sequencing results provided in Example 3 of the present invention;

[0029] FIG4 is a diagram showing the specificity of fluorescent quantitative PCR testing using a simple Z sequence plasmid as a template in Example 4 of the present invention;

[0030] FIG5 is a diagram showing the specificity of fluorescent quantitative PCR testing using a simple W sequence plasmid as a template in Example 4 of the present invention;

[0031] FIG6 is a graph showing the specificity of fluorescent quantitative PCR testing using four gradients of Z template diluted to 4.18, 41.8, 418, and 4180 copies / ul in Example 4 of the present invention;

[0032] FIG7 is a graph showing the specificity of fluorescent quantitative PCR testing using four gradients of W template diluted to 4.3, 43, 430, and 4300 copies / ul in Example 4 of the present invention;

[0033] FIG8 is a diagram showing the results of a fluorescent quantitative PCR test on four female pigeons of known sex in Example 5 of the present invention;

[0034] FIG9 is a diagram showing the results of a fluorescent quantitative PCR test on four male pigeons of known sex in Example 5 of the present invention;

[0035] FIG10 is a diagram showing the results of a fluorescent quantitative PCR test on four pigeons of known sex in Example 5 of the present invention;

[0036] FIG11 is a graph showing the stability results of the fluorescence quantitative PCR test method after the samples were placed at room temperature for one week. DETAILED DESCRIPTION

[0037] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0038] In this technical solution, the sample nucleic acid lysis and extraction method listed here is the classic and universal "phenol-chloroform method", which is applicable to various tissue and body fluid samples such as pigeon muscle, feathers, and saliva. In actual application, corresponding modifications and adjustments can be made, and different extraction methods can be used according to the sample type. For example, the centrifugal column extraction method and high-salt precipitation method for tissue DNA can be used. For body fluid samples such as saliva, a one-step method can be used for rapid lysis, eliminating the phenol-chloroform extraction and improving efficiency. For large sample quantities, the magnetic bead method can be used for automated extraction, which is fast and efficient. These are not listed here one by one.

[0039] Example 1

[0040] S1. Pluck several pigeon feathers (cut about 1-2 mm from the root of the feather medullary tissue) or collect pigeon saliva by oral swab (use a clean cotton swab to stir the pigeon's mouth several times to obtain the saliva). Place the saliva in 500 μl of digestion solution, add proteinase K, and place in a 55°C water bath for overnight digestion. The next day, remove the centrifuge tube from the water bath after overnight storage, add 550 μl of DNA extraction phenol reagent (lower layer), mix well, and centrifuge at 12,000 rpm for 10 minutes.

[0041] S2. Take a 1.5 ml centrifuge tube and transfer the supernatant obtained after centrifugation in the previous step to a new tube as much as possible. Add 1 / 2 of the supernatant volume of DNA extraction phenol reagent and 1 / 2 of chloroform, mix well, and centrifuge at 12000 rpm for 10 minutes.

[0042] S3. Take another 1.5 ml centrifuge tube and transfer the supernatant obtained from the previous step to a new centrifuge tube as much as possible. Add chloroform equal to the volume of the supernatant, mix well, and centrifuge at 12000 rpm for 10 minutes.

[0043] S4. Take another 1.5 ml centrifuge tube and transfer as much of the supernatant obtained from the previous step to a new centrifuge tube. Add isopropanol equal to the volume of the supernatant, mix well, and place in a -20 degree refrigerator for 15 minutes. Then take out and centrifuge at 12000 rpm for 10 minutes.

[0044] S5. Discard the liquid after the previous centrifugation step and keep the pellet. Add 1 ml of 70% ethanol, flick the pellet to wash it, and centrifuge it again at 12000 rpm for 5 minutes.

[0045] S6, same as above;

[0046] S7. Discard the liquid after centrifugation in the previous step and use a small-scale gun to remove as much of the remaining liquid as possible. Place the sample in a 60°C metal dry bath for drying. Add 30 μl of ddH2O, wait until the precipitate is completely dissolved in water, mix thoroughly, and measure the concentration. The results are shown in Table 1:

[0047] Table 1

[0048] Example 2

[0049] PCR amplification conditions

[0050] PCR uses a 30 μL reaction system:

[0051] qPCR Mix 15μL; Mg 2+ (25mM) 2μL; Forward Primer (10μM) 0.5μL; Reverse Primer (10μM) 0.5μL; Template DNA 2μL; add ddH2O to 30μL.

[0052] PCR reaction parameters: denaturation at 95°C for 3 min; denaturation at 94°C for 15 s, annealing at 55°C for 20 s, and extension at 72°C for 20 s, with 35 cycles.

[0053] The statistics of annealing temperature gradient PCR amplification results are shown in Table 2:

[0054] Table 2

[0055] Example 3

[0056] The target bands of the PCR products from the genomes of pigeon tissues of different individuals were recovered by electrophoresis and placed in a tip containing a filter. The tip was then placed in a 1.5ml EP tube and centrifuged at 8000r / m for 2 minutes. The liquid flowing out of the tip filter contained the PCR product nucleic acid solution.

[0057] TA cloning

[0058] (1) Ligation: Prepare the ligation system according to the TA cloning ligation system preparation requirements, as shown in Table 3, and ligate in a PCR instrument at 16°C for 30 minutes to 1 hour (do not heat the lid).

[0059] The molecular ratio of b:c or c:b is maintained between 1:3-10 times, and the volume of a = the volume of b + c.

[0060] (2) Take out the competent cells from the -80℃ freezer and immediately place them on ice. After thawing on ice, add 10ul of the ligation product to the competent E. coli solution (just add 10ul of the ligation product, do not pipette to mix), and place on ice for 15-30 minutes;

[0061] (3) Heat shock: Heat shock in a 42°C water bath for 45 seconds, then ice bath for 2 minutes;

[0062] (4) Cultivation: Add 1 ml of LB culture medium without antibiotics and shake slowly at 37°C for 45 minutes (recovery).

[0063] (5) Collect bacteria: centrifuge at 8000 rpm for 2 minutes.

[0064] (6) Plate coating: Pour off the supernatant, leaving about 100 μl of LB culture medium. Mix the precipitate by pipetting (let the precipitate disappear to form a single-cell suspension). Pipette all the bacterial solution and evenly drop it onto the LB agar plate containing ampicillin resistance. Use an alcohol burner to burn a cooled glass rod and spread it evenly on the LB agar plate until it dries. Mark and date it. Place it upside down in a 37°C constant temperature incubator and culture overnight.

[0065] (7) Amplification: Take out the LB agar plate that has been cultured overnight at 37°C, and mark 5-10 single clone colonies as templates. Prepare the PCR reaction system in the negative area, and dispense 28ul into each tube after preparation. Keep one tube as a negative control and cover it tightly. Take the prepared and dispensed reaction system to the sample processing area, burn the inoculation loop with an alcohol lamp, let it cool slightly, and then dip some single colonies into the PCR reaction system to rinse; cover the tube cap, write a mark, centrifuge it instantly, and put it into the PCR instrument for amplification (the number of cycles should not exceed 30 times);

[0066] (8) Identification of positive clones by colony PCR

[0067] PCR reaction system preparation:

[0068] PCR reaction procedure:

[0069] (9) Gel electrophoresis: Based on the electrophoresis results, positive monoclonal colonies with the correct amplification size and sufficient abundance were selected and inoculated at 4-5 pm.

[0070] (10) Prepare LB culture medium containing AMP resistance: In a clean bench, take a 50ml centrifuge tube after autoclaving and pour 15ml of LB culture medium into it. Add 15ul of 1000X Amp mother liquor and mix well. Then divide the mixture into four 10ml centrifuge tubes (3-4ml each).

[0071] (11) Inoculation: Burn the tweezers with an alcohol burner. After cooling slightly, use the tweezers to pick up a 10 μl pipette tip after high pressure and dip it into a positive monoclonal colony. Place the colony in four 10 ml centrifuge tubes. Shake the tubes: Place the four 10 ml centrifuge tubes in a shaker at 37°C and 160 rpm overnight.

[0072] (12) Plasmid extraction: The next day, bacterial culture preservation and plasmid extraction

[0073] (13) Sequencing: first-generation sequencing.

[0074] Sequencing verification revealed that some sites were different from the database. The primer and probe sequences were adjusted according to the sequencing results:

[0075] Example 4

[0076] Fluorescence quantitative PCR test detection specificity

[0077] (1) The Z sequence plasmid was used as a template for verification, as shown in Figure 4.

[0078] Channel 1 is the FAM signal, also known as the W signal; channel 3 is the ROX signal, also known as the Z signal.

[0079] (2) The pure W sequence plasmid was used as a template for verification, as shown in Figure 5.

[0080] Channel 1 is the FAM signal, also known as the W signal; channel 3 is the ROX signal, also known as the Z signal.

[0081] (3) The Z template was diluted gradiently to 4.18, 41.8, 418, and 4180 copies / ul, and the four gradients were used as templates for detection, as shown in Figure 6.

[0082] 4) The W template was diluted in a gradient manner to 4.3, 43, 430, and 4300 copies / ul, and the four gradients were used as templates for detection, as shown in Figure 7.

[0083] Example 5

[0084] Fluorescence quantitative PCR test to detect test samples (feathers) sent by pigeon farms

[0085] 5) The test results of the four female pigeons whose genders are known are shown in FIG8 .

[0086] 6) The test results of the four male pigeons whose genders are known are shown in Figure 9:

[0087] 7) The test results of the four pigeons whose genders are known are shown in Figure 10:

[0088] Identification results:

[0089] 8) After the samples were placed at room temperature for one week, the stability of the detection method was tested by fluorescence quantitative PCR, as shown in FIG11 .

[0090] In this technical solution, MGB probes are used to achieve highly sensitive and specific detection. The sensitivity of the Z template can reach 4.18 copies / ul, and the sensitivity of the W template can reach 4.3 copies / ul. The samples preserved in the lysate are stored at room temperature of about 25-30 degrees for one week. Testing shows that this method does not affect the test results.

[0091] Meanwhile, the contents not described in detail in this specification belong to the prior art known to those skilled in the art.

[0092] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus.

[0093] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for identifying the early sex of pigeons by qPCR, characterized in that: The specific steps include: S1, genomic DNA extraction from pigeon feathers, saliva and other tissues or body fluids; S2. Debug the primers and perform PCR reaction using the extracted DNA as a template; S3, TA cloning to prepare standard sequences of pure W and Z; S4, identification of positive clones by colony PCR; S5, adjust the primer probe sequence; S6. Fluorescence quantitative PCR test for detection specificity.

2. The method for early sex identification of pigeons by qPCR according to claim 1, characterized in that: In step S2, the PCR reaction procedure is: denaturation at 95°C for 3 minutes; denaturation at 94°C for 15 seconds, annealing at 55°C for 20 seconds, and extension at 72°C for 20 seconds, with the number of cycles being 35.

3. The method for identifying the early sex of pigeons by qPCR according to claim 1, characterized in that: In step S2, the reaction system of the PCR reaction is: 15 μL of qPCR Mix, 25 mM primers Mg 2+ 2μL, 0.5μL of 10μM forward primer, 0.5μL of 10μM reverse primer, 2μL of DNA template, add ddH2O to 30μL.

4. The method for identifying the early sex of pigeons by qPCR according to claim 1, characterized in that: In step S4, the reaction system of PCR reaction is: 2xMix 15 μL, primer Mg 2+ 3μL, Primer P5 0.5μL, Primer P3 0.5μL, ddH2O 9μL.

5. The method for identifying the early sex of pigeons by qPCR according to claim 1, characterized in that: In step S4, the PCR reaction procedure is: denaturation at 95° C. for 3 min; denaturation at 94° C. for 30 s, annealing at 55° C. for 30 s, and extension at 72° C. for 30 s, with the number of cycles being 30.

6. The method for identifying the early sex of pigeons by qPCR according to claim 1, characterized in that: In step S5, an MGB probe is used.

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