阅读说明：本技术 一种基于荧光pcr鉴定鲟鱼性别的引物、荧光探针及试剂盒 (Primer, fluorescent probe and kit for identifying gender of sturgeon based on fluorescent PCR ) 是由 黄薇 廖茜 宋永康 黄镇 陈贝 于 2021-02-01 设计创作，主要内容包括：本发明公开一种基于荧光PCR技术鉴定鲟鱼性别的引物、荧光探针及试剂盒,所述引物和荧光探针包括基于鲟鱼W染色体特异DNA序列设计的引物WF、引物WR和荧光探针W以及基于鲟鱼Z染色体特异DNA序列设计的引物ZF、引物ZR和荧光探针Z,所述试剂盒包括荧光PCR反应液、Taq DNA聚合酶、阴性对照和阳性对照,所述荧光PCR反应液包括上述引物和荧光探针；鲟鱼基因组样品在进行荧光PCR扩增后,进行Ct值分析；若两组引物的荧光PCR结果均为阳性,则待测样品为雌性；若W染色体特异DNA序列引物的荧光PCR结果为阴性而Z染色体特异DNA序列引物的荧光PCR结果为阳性时待测样品为雄性；本发明的鉴定方法实现在鲟鱼生命早期阶段进行性别鉴定,操作简便、结果准确、适用于生产实践。(The invention discloses a primer, a fluorescent probe and a kit for identifying the sex of sturgeons based on a fluorescent PCR technology, wherein the primer and the fluorescent probe comprise a primer WF, a primer WR and a fluorescent probe W which are designed based on a specific DNA sequence of a sturgeon W chromosome, and a primer ZF, a primer ZR and a fluorescent probe Z which are designed based on a specific DNA sequence of a sturgeon Z chromosome, the kit comprises a fluorescent PCR reaction solution, a Taq DNA polymerase, a negative control and a positive control, and the fluorescent PCR reaction solution comprises the primer and the fluorescent probe; carrying out fluorescence PCR amplification on the sturgeon genome sample, and then carrying out Ct value analysis; if the fluorescence PCR results of the two groups of primers are positive, the sample to be detected is female; if the fluorescence PCR result of the W chromosome specific DNA sequence primer is negative and the fluorescence PCR result of the Z chromosome specific DNA sequence primer is positive, the sample to be detected is male; the identification method provided by the invention realizes sex identification in the early life stage of the sturgeon, is simple and convenient to operate, has accurate results, and is suitable for production practice.)

1. A primer and a fluorescent probe for identifying the sex of sturgeons based on fluorescent PCR are characterized in that: the primer and the fluorescent probe comprise a primer and a fluorescent probe W designed based on a sturgeon W chromosome specific DNA sequence, and a primer and a fluorescent probe Z designed based on a sturgeon Z chromosome specific DNA sequence;

the primers designed based on the sturgeon W chromosome specific DNA sequence are respectively a primer WF and a primer WR;

the primer WF is shown as SEQ ID NO 1 in the sequence table;

the primer WR is shown as SEQ ID NO. 2 in the sequence table;

the fluorescent probe W designed based on the sturgeon W chromosome specific DNA sequence is shown as SEQ ID NO. 3 in the sequence table; the 5' end and the 3' end of the fluorescent probe W are respectively connected with a fluorescent reporter group and a quenching group for modification, and the fluorescent reporter group for modifying the 5' end is FAM; modifying the quenching group at the 3' end to be MGB;

the primers designed based on the sturgeon Z chromosome specific DNA sequence are an upstream primer ZF and a downstream primer ZR respectively;

the upstream primer ZF is shown as SEQ ID NO. 4 in the sequence table;

the downstream primer ZR is shown as SEQ ID NO. 5 in the sequence table;

the fluorescent probe Z designed based on the sturgeon Z chromosome specific DNA sequence is shown as SEQ ID NO. 6 in the sequence table; the 5' end and the 3' end of the fluorescent probe Z are respectively connected with a fluorescent reporter group and a quenching group for modification, and the fluorescent reporter group for modifying the 5' end is VIC; and modifying the quenching group at the 3' end into MGB.

2. A kit for identifying the sex of sturgeons based on fluorescence PCR is characterized in that: comprises fluorescent PCR reaction solution, Taq DNA polymerase, positive control and negative control; the fluorescent PCR reaction solution comprises the primer WF and the primer WR as set forth in claim 1, a fluorescent probe W, an upstream primer ZF, a downstream primer ZR and a fluorescent probe Z.

3. The kit for identifying the gender of the sturgeon based on the fluorescence PCR according to claim 2, which comprises: the fluorescent PCR reaction solution also comprises dNTP and Mg²⁺Taq buffer and RNase-Free water.

4. The kit for identifying the gender of the sturgeon based on the fluorescence PCR according to claim 2, which comprises: the positive control is StuW plasmid containing sturgeon W chromosome specific DNA sequence and stuZ plasmid containing sturgeon Z chromosome specific DNA sequence.

5. The kit for identifying the gender of the sturgeon based on the fluorescence PCR according to claim 2, which comprises: the sturgeon W chromosome specific DNA sequence is a West miscellaneous W chromosome specific DNA sequence SEQ ID NO. 7, and the sturgeon Z chromosome specific DNA sequence is a West miscellaneous Z chromosome specific DNA sequence SEQ ID NO. 8; the west hybrids are hybrid varieties of Siberian sturgeons in females and sturgeons in males.

6. The kit for identifying the gender of the sturgeon based on the fluorescence PCR according to claim 2, which comprises: the negative control included 0.1 × TE solution.

Technical Field

The invention belongs to the technical field of molecular biology, relates to biological molecular detection, and particularly relates to a primer, a fluorescent probe and a kit for identifying the sex of sturgeons based on fluorescent PCR.

Background

Sturgeons including sturgeons of Acipenser and Huso of Huso are the only existing subjects in the class of chondral scleroderma, are called "activating stones", and sturgeon seed sauce is known as "black gold". China is the largest sturgeon breeding country and the main sturgeon caviar export country, the export quantity of the Chinese sturgeon caviar in 2019 is 139.8 tons, and the export amount is 3253.5 ten thousand dollars. However, sturgeons have long growth period, slow sexual maturation and no obvious side-effect, are difficult to distinguish males and females from appearance in the early development period, can be distinguished after being cultured for 3-4 years, and then male fishes are eliminated as commercial fishes to be put on the market; female fish breeding needs to be continuously bred for 7-8 years and used for caviar production, in the current sturgeon breeding market, intensive breeding or breeding according to a certain sex proportion cannot be performed according to the sex of the female and male sturgeons, resource waste of breeding too many male individuals can be caused, breeding cost is increased, rapid development of sturgeon breeding industrialization is hindered, therefore, the sex of the sturgeons can be judged as early as possible, feed investment on male fish can be reduced, breeding cost is saved, and the method is very important in production.

In production practice, the main methods for the sex identification of sturgeons are: (1) the method comprises the following steps of (1) an ultrasonic method, (2) an abdominal cavity lateral operation method and (3) an abdominal cavity puncture method, wherein the identification can be carried out only when the gonad of the sturgeon grows to a certain stage (3-4 years old), and the problems of long dehydration time, complex operation, long identification time and the like of a fish body exist in the identification process, so that the fish body is easily damaged. In recent years, with the rapid development of molecular biology, a technology for sex determination by using molecules is becoming popular, and compared with the conventional determination method, the molecular determination method has the advantages of convenient sampling, small damage to organisms, simple operation and more accurate result. The existing research shows that sturgeons are male and female variant sturgeons, the genetic sex determination system is ZZ/ZW type, male sturgeons are ZZ, and female sturgeons are ZW, so that the sex identification of the male and female sturgeons in the early life stage of the sturgeons can be realized by utilizing a molecular detection method. In the prior art, chinese patent No. CN201910066586.4 discloses a female specific DNA fragment SSM2 and application for sturgeon gender identification, chinese patent No. CN201910066600.0 discloses a female specific DNA fragment SSM1 and application for sturgeon gender identification, chinese patent No. CN201910066534.7 discloses a specific DNA fragment for female sturgeon and application, the above patents provide some specific molecular marker fragments and primers related to sturgeon, which can be used for identifying sturgeon gender, but the fragments disclosed in the above patents are only specific DNA fragments on female W chromosome, the designed primers can only amplify one band, once the extracted DNA quality is not good, PCR or gel electrophoresis operation is wrong, the electrophoresis diagram will not appear, and the sample is misjudged as male, and false negative condition appears; in addition, the existing sturgeon sex molecular identification method is based on a common PCR amplification mode and a gel electrophoresis mode is carried out on products, the sample amount detected by the method is limited by a gel sample lattice (generally twenty-thirty comb holes), and the method is not suitable for large-scale sturgeon sex identification work in production in cost and efficiency.

In the sturgeon sex molecular identification method, a sex-specific DNA marker is an important tool for judging the sex; if two pairs of specific primers can be designed according to the specific DNA fragment on the W chromosome and the specific DNA fragment on the Z chromosome to carry out PCR amplification on the sturgeon genome, two positive results can appear in female (ZW), and one positive result appears in male (ZZ) sturgeon, so that the possibility of false negative can be eliminated by judging the male and female through the double positive results, and the result is more reliable and accurate.

In addition, the fluorescence PCR technology is advanced relative to the common PCR technology, and the characteristics thereof comprise: 1. the accuracy is high, the fluorescent PCR technology uses specificity specially designed for a target sequence to identify quantitative molecules, the accuracy is very high, the fluorescent PCR technology is controlled by the target sequence and the probe, the specificity of the common PCR technology is greatly improved, and the false positive rate of detection is reduced; 2. the method is simple and convenient to operate and high in environment-friendly performance, detection by using a real-time fluorescence PCR technology is completed in one tube, gel electrophoresis is not needed, and the emission of related pollutants can be reduced; 3. the method has the advantages that the speed is high, the efficiency is high, electrophoresis is not needed by using a fluorescent PCR technology, quantitative analysis of 96 sample wells or 384 sample wells can be completed within 2-3 hours, and the labor amount of workers is effectively reduced; therefore, the fluorescence PCR technology is more suitable for identifying the male and female sturgeons in practical production.

Disclosure of Invention

In order to solve the technical problems of misjudgment of sturgeon gender samples, false negative, low efficiency, complex operation and the like in the prior art, the invention provides the primer, the fluorescent probe and the kit for identifying the sturgeon gender based on the fluorescent PCR.

The technical scheme of the invention is as follows:

the invention discloses a primer and a fluorescent probe for identifying the sex of sturgeons based on fluorescent PCR (polymerase chain reaction); the primer and the fluorescent probe comprise a primer and a fluorescent probe W designed based on a sturgeon W chromosome specific DNA sequence, and a primer and a fluorescent probe Z designed based on a sturgeon Z chromosome specific DNA sequence;

the primers designed based on the sturgeon W chromosome specific DNA sequence are respectively a primer WF and a primer WR;

the primer WF is shown as SEQ ID NO 1 in the sequence table;

the primer WR is shown as SEQ ID NO. 2 in the sequence table;

the fluorescent probe W designed based on the sturgeon W chromosome specific DNA sequence is shown as SEQ ID NO. 3 in the sequence table; the 5' end and the 3' end of the fluorescent probe W are respectively connected with a fluorescent reporter group and a quenching group for modification, and the fluorescent reporter group for modifying the 5' end is FAM; modifying the quenching group at the 3' end to be MGB;

the primers designed based on the sturgeon Z chromosome specific DNA sequence are an upstream primer ZF and a downstream primer ZR respectively;

the upstream primer ZF is shown as SEQ ID NO. 4 in the sequence table;

the downstream primer ZR is shown as SEQ ID NO. 5 in the sequence table;

the fluorescent probe Z designed based on the sturgeon Z chromosome specific DNA sequence is shown as SEQ ID NO. 6 in the sequence table; the 5' end and the 3' end of the fluorescent probe Z are respectively connected with a fluorescent reporter group and a quenching group for modification, and the fluorescent reporter group for modifying the 5' end is VIC; and modifying the quenching group at the 3' end into MGB.

The invention also discloses a kit for identifying the sex of the sturgeon based on the fluorescence PCR technology, which comprises fluorescence PCR reaction liquid, Taq DNA polymerase, positive control and negative control; the fluorescent PCR reaction solution comprises the primer WF, the primer WR, a fluorescent probe W, an upstream primer ZF, a downstream primer ZR and a fluorescent probe Z.

Furthermore, the fluorescent PCR reaction solution also comprises dNTP, Mg2+, Taq buffer and RNase-Free water.

Furthermore, the positive control is StuW plasmid containing sturgeon W chromosome specific DNA sequence and stuZ plasmid containing sturgeon Z chromosome specific DNA sequence.

Further, the sturgeon W chromosome specific DNA sequence is a West hybrid W chromosome specific DNA sequence SEQ ID NO. 7, and the sturgeon Z chromosome specific DNA sequence is a West hybrid Z chromosome specific DNA sequence SEQ ID NO. 8; the west hybrids are hybrid varieties of Siberian sturgeons in females and sturgeons in males.

Further, the negative control included 0.1 × TE solution.

The invention has the beneficial effects that:

1. the invention provides the fluorescent PCR primer, the probe and the kit for identifying the sex of the sturgeon for the first time, the provided identification method avoids an electrophoresis process required by common PCR identification, the operation process is greatly simplified, the time consumption is short, the flux is higher, the identification efficiency is greatly improved, and the method is more suitable for production practice.

2. The invention provides a method for rapidly and accurately identifying the sex of sturgeons, wherein gene sequences with the same exon sequence and different intron sequences are found in W chromosome and Z chromosome of sturgeons, specific primers are designed aiming at the specific gene sequences, PCR amplification is carried out, if the gene sequences are female (ZW), two positive results can appear, and if the gene sequences are male (ZZ), a positive result appears, and the judgment of the male and female is carried out through the double positive results, so that the false negative errors can be eliminated, and the accuracy of the identification result is improved; positive control and negative control are arranged in the kit, and the PCR accuracy is controlled by the target sequence and the probe, so that the false positive result is obviously reduced; the detection result is more reliable.

3. The sturgeon sample required by the sturgeon gender identification method provided by the invention can be swabs of tail fins, dorsal fins, blood, puncture fluid, oral secretion, intestinal secretion and the like of sturgeons, and basically has no harm to living sturgeons.

4. The sturgeon gender identification method provided by the invention is simple to operate, short in time and high in universality, greatly advances the sturgeon gender identification time, can be used for gender identification even for small sturgeons which are just hatched, is beneficial to separate breeding and culture of male and female sturgeons, sells male sturgeons in advance, reduces the culture cost of the sturgeons, and greatly promotes the development of sturgeon culture industry.

Drawings

FIG. 1 is a schematic anatomical view of female and male sturgeons according to example 3 of the present invention;

FIG. 2 is a fluorescence PCR map of a W chromosome specific DNA sequence of a sturgeon sample in example 1 of the present invention;

FIG. 3 is a fluorescence PCR map of a Z chromosome specific DNA sequence of a sturgeon sample in example 1 of the present invention;

Detailed Description

The invention will be further described with reference to the accompanying drawings and specific examples, which are given by way of illustration only and are not to be construed as limiting the invention; unless otherwise specified, the reagent raw materials used in the following examples are biochemical reagent raw materials which are conventionally commercially available or commercially available, and the laboratory instruments used are laboratory conventional instruments, and unless otherwise specified, the methods and apparatuses used in the following examples are those conventionally used in the art.

Example 1 design and Synthesis of primers

The public sturgeon genome sequence is collected, a sturgeon W chromosome specific DNA sequence and a sturgeon Z chromosome specific DNA sequence are screened out through multiple comparison and analysis, and two groups of original primers and fluorescent probes based on a fluorescent PCR technology are designed according to the sequences.

Wherein, a primer WF (nucleotide sequence is SEQ ID NO:1), a primer WR (nucleotide sequence is SEQ ID NO:2) and a fluorescent probe W (SEQ ID NO:3) are designed based on the sturgeon W chromosome specific DNA sequence; the 5' end and the 3' end of the fluorescent probe W are respectively connected with a fluorescent reporter group and a quenching group for modification, and the fluorescent reporter group for modifying the 5' end is FAM; modifying the quenching group at the 3' end to be MGB;

the primer WF: 5'-CTGACATTGCTGAAGAGGTTGA-3', respectively;

the primer WR: 5'-TGGAATCTCAGAGCCAAGTTAA-3', respectively;

the fluorescent probe W: FAM-5'-TCCGTCACATATTC-3' -MGB;

wherein, an upstream primer ZF (nucleotide sequence is SEQ ID NO:4), a downstream primer ZR (SEQ ID NO:5) and a fluorescent probe Z (nucleotide sequence is SEQ ID NO:6) are designed based on the sturgeon Z chromosome specific DNA sequence; the 5' end and the 3' end of the fluorescent probe Z are respectively connected with a fluorescent reporter group and a quenching group for modification, and the fluorescent reporter group for modifying the 5' end is VIC; modifying the quenching group at the 3' end to be MGB;

the upstream primer ZF: 5'-TGTGTCATAGCAAAGGGGGT-3', respectively;

the downstream primer ZR: 5'-AAGTGGGGGAAAAAATCTACATA-3', respectively;

the fluorescent probe Z: VIC-5'-ACTTTTCAGTTTTTTATTTG-3' -MGB;

and the two groups of primers are completely different from the reported primers for identifying the sex of the sturgeon through retrieval and comparison.

Example 2 kit and preparation method

The invention provides a kit for identifying the sex of sturgeons based on a fluorescence PCR technology, which comprises a fluorescence PCR reaction solution, Taq DNA polymerase, a positive control and a negative control;

the fluorescent PCR reaction solution comprises: 10 μ M primer WF, 10 μ M primer WR, 10 μ M fluorescent probe W, 10 μ M primer ZF, 10 μ M primer ZR, 10 μ M fluorescent probe Z, 25mM Mg²⁺100mM dNTP, 10 XTaq buffer solution and RNase-Free water;

the concentration of the Taq DNA polymerase is 5U/mu L;

the positive control is 20ng/mL stuW plasmid containing sturgeon W chromosome specific DNA sequence and 20ng/mL stuZ plasmid containing sturgeon Z chromosome specific DNA sequence; specifically, the sturgeon W chromosome specific DNA sequence is a West miscellaneous W chromosome specific DNA sequence SEQ ID NO. 7, and the sturgeon Z chromosome specific DNA sequence is a West miscellaneous Z chromosome specific DNA sequence SEQ ID NO. 8; wherein the Xiza is a hybrid variety of Siberian sturgeon as female and Acipenser schrenki as male;

the positive control was prepared as follows:

extracting the genomic DNA of the West hybrid, taking the genomic DNA as a template, firstly, carrying out PCR amplification by adopting an upstream primer SEQ ID NO. 9 and a downstream primer SEQ ID NO. 10 to obtain a West hybrid W chromosome specific DNA sequence SEQ ID NO. 7, recovering and purifying a PCR product, connecting the PCR product into a plasmid PUC-T vector, transforming escherichia coli DH5 alpha, carrying out sequencing verification on a recombinant transformant, extracting a plasmid after culturing the recombinant transformant to obtain a StuW plasmid containing the West hybrid W chromosome specific DNA sequence SEQ ID NO. 7; then, carrying out PCR amplification by adopting an upstream primer SEQ ID NO. 11 and a downstream primer SEQ ID NO. 12 to obtain a specific DNA sequence SEQ ID NO. 8 of the Xiza Z chromosome, recovering and purifying a PCR product, connecting the PCR product into a plasmid PUC-T vector, transforming escherichia coli DH5 alpha, carrying out sequencing verification on a recombinant transformant, extracting a plasmid after culturing the recombinant transformant, and obtaining a StuZ plasmid containing the specific DNA sequence SEQ ID NO. 8 of the Xiza W chromosome;

upstream primer SEQ ID NO 9: 5'-TTATTTTCTGTTTTATATTTG-3', respectively;

downstream primer SEQ ID NO: 10: 5'-TTAATTTGCTTTGTTGGAGATAG-3', respectively;

upstream primer SEQ ID NO: 11: 5'-GGATCGCCCTTGTGTCAT-3', respectively;

downstream primer SEQ ID NO: 12: 5'-GGATCGCCCTTTCACAGGGCC-3', respectively;

the negative control included 0.1 × TE solution.

Example 3 method for identifying the sex of sturgeon Using the kit of the present invention

(1) Extracting DNA of sturgeon sample to be detected

30 3-year-old sturgeons of 4 varieties are provided by Fujian Longhuso sturgeon Limited, and the specific conditions of sample information are shown in Table 1; respectively taking 1cm multiplied by 1cm samples of the tail fin tissue of the sturgeon to be detected, storing the samples in absolute ethyl alcohol, and identifying the sex of the sturgeon by adopting a dissection method, wherein the sex-gland diagram of an example of female sturgeon and an example of male sturgeon is shown in the attached figure 1; then, extracting the DNA of the sturgeon tail fin genome by using a DNA extraction kit, diluting to 50 ng/mu L, and storing at-20 ℃ for later use;

(2) sample application

Adding 2 mul of sample to be detected or negative control sample or positive control sample, 17.8 mul of fluorescent PCR reaction solution and 0.2 mul of Taq DNA into the PCR reaction tube, covering the tube cover tightly, and centrifuging instantly for amplification;

(3) PCR amplification and fluorescence detection

Placing the prepared PCR reaction tube in a fluorescent PCR instrument, and carrying out amplification reaction and detection according to the edited sample information under the following conditions: 94-3 min; 94-5 s, 57-15 s, 72-30 s; the cycle number is 40; the setting of the reaction conditions has great influence on the interpretation of the detection result such as amplification efficiency, fluorescence value and the like, the optimal reaction conditions of the kit are as described above, the cycle can be carried out for 40 times, and different cycle times can be set according to practical requirements;

(4) analysis of detection results

When the kit is used for detecting a sample, the negative and positive controls simultaneously meet the following conditions, otherwise, the experiment is regarded as invalid and needs to be redone;

positive quality control: the positive control Ct value is less than or equal to 36;

negative quality control: negative control Ct values >36, or "underdetermined";

then, Ct value of each sample amplification curve is observed, and positive result and negative result are judged

Ct value determination criteria:

the Ct value of the sample is less than or equal to 36, and a positive result is obtained; sample Ct value >36 or "undeterified", negative result.

(5) Male and female sex determination

When the fluorescence PCR reaction result of the specific DNA sequence of the chromosome W is positive and the fluorescence PCR reaction result of the specific DNA sequence of the chromosome Z is positive, the sample to be detected is female;

when the fluorescence PCR reaction result of the W chromosome specific DNA sequence is negative and the fluorescence PCR reaction result of the Z chromosome specific DNA sequence is positive, the sample to be detected is male;

if the fluorescence PCR reaction result of the Z chromosome specific DNA sequence is negative, the test fails;

in summary, the detection results of 30 4 sturgeons of 3-year old using the kit provided by the invention are shown in table 1:

TABLE 1 determination of identity

As can be seen from the results of sex determination of the samples in Table 1, the positive control detected by the kit provided by the present invention shows an amplification curve, and the negative control does not show an amplification curve; the sex of the sturgeon judged according to the Ct value result obtained by the fluorescence PCR of the sturgeon individual samples of the 4 sturgeon varieties is basically consistent with the result of the identification by the dissection method (the fluorescence PCR result of the sample 23 is undetermined, and the test fails), thereby further showing that the kit provided by the invention has very good specificity.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.