CN113136438A

CN113136438A - Nucleic acid molecule, PCR primer pair and kit for detecting avian trichomonas beta-tubulin gene

Info

Publication number: CN113136438A
Application number: CN202110527892.0A
Authority: CN
Inventors: 蔡海明
Original assignee: Institute of Animal Health of Guangdong Academy of Agricultural Sciences
Current assignee: Institute of Animal Health of Guangdong Academy of Agricultural Sciences
Priority date: 2021-05-14
Filing date: 2021-05-14
Publication date: 2021-07-20
Anticipated expiration: 2041-05-14
Also published as: CN113136438B

Abstract

The invention discloses a nucleic acid molecule, a PCR primer pair and a kit for detecting a trichomonas avicularis beta-tubulin gene. The inventor clones the avian trichomonas beta-tubulin full-length gene coding sequence shown as SEQ ID NO. 1 in the avian trichomonas through extensive research, and further designs a PCR primer pair capable of detecting the avian trichomonas beta-tubulin gene and the transcription level thereof and corresponding PCR reaction conditions by utilizing the beta-tubulin full-length gene coding sequence to further assemble a kit for detecting the avian trichomonas beta-tubulin gene, thereby meeting the detection requirements of researchers in parasitic biology and life science, promoting the research progress of the gene level detection of the pathogen and the pathogenic mechanism thereof, and providing favorable technical support for research works such as gene function research, medicament development and the like.

Description

Nucleic acid molecule, PCR primer pair and kit for detecting avian trichomonas beta-tubulin gene

Technical Field

The invention relates to the field of molecular biological detection, in particular to a nucleic acid molecule, a PCR primer pair and a kit for detecting a trichomonas avicularis beta-tubulin gene.

Background

The pigeon breeding history in China is long, the pigeon production as a commodity has more than 100 years, the meat pigeon breeding industry is spread in various regions in China at the present stage, the scale is continuously increased, and the effective prevention and control of pigeon infectious diseases are more important. The trichomoniasis of pigeon (also called Pigeon Huang) is also a high-incidence consumption parasitic disease in pigeon breeding farms, and has important harmfulness to the production performance of meat pigeons and the survival rate of young pigeons. The most common characteristic changes are the formation of a coarse, button-like yellow deposit of the mucous membranes of the mouth and throat; wet, called wet ulcer; a dry ulcer is known as a cheese-like or scabby mass. When the umbilicus is infected, a lump is formed under the skin and presents cheese-like or ulcerative lesion; when internal organs are involved, cheese-like lesions with a sharp yellow rough boundary are caused, resulting in necrosis of parenchymal organ tissues. Sick pigeons are impaired in ingestion by oral ulcers and can cause a high mortality rate in young pigeons and growing pigeons. The etiological agent of trichomoniasis of pigeon is trichomonas avium, belonging to the sub phylum flagellata, class Acinetobacter, order Polyflagellates, family Trichomodidae, genus Trichomonas. The laboratory diagnosis generally refers to the discovery of polypide by microscopic examination of oral cavity, esophagus and crop secretion smear, or the preparation of smear by scraping the mucus at the lesion, and typical polypide is seen by staining microscopy. The bird caterpillar body is melon seed-shaped or pear-shaped, and 4 flagella extend out of the front end of the body through a feather matrix, so that the body can move rapidly. The one side of the worm body is provided with a wave film which starts from the front end of the worm body and ends at the rear side of the worm body, the front part of the worm body is provided with a 1-ellipse nucleus, the front part of the worm body is opposite to the wave film, one side of the worm body is provided with a cell opening, and the center of the worm body is provided with a slender shaft column which extends from front to back to the outside of the rear edge of the worm body. The worms spirally move in the body fluid and proliferate in a split manner, and can proliferate for one generation in about 4 hours. At present, the pathogenic mechanism of the pathogen is still rarely researched, and the reason is that an effective gene level detection means is lacked.

The Polymerase Chain Reaction (PCR) is a molecular biology technique for amplifying and amplifying specific DNA fragments, and can be regarded as special DNA replication in vitro, and the biggest characteristic of the PCR is that trace amount of DNA can be greatly increased. Among them, the fluorescence quantitative PCR is a common nucleic acid quantitative technique, and is a gene level detection means with low cost, high repeatability and convenient operation. According to the technology, nucleic acid fluorescent labeling substances such as SYBR and TB Green are introduced into a PCR reaction system, the amplification condition of PCR is monitored in real time by detecting the increase condition of a fluorescence value in the PCR reaction system after each thermal cycle, a fluorescence quantitative change curve of each sample is fitted, so that a Ct value (the cycle number when the fluorescence change reaches a threshold value) of each reaction tube is obtained, a standard curve is prepared by taking the logarithm of the initial template number as an abscissa and the Ct value as an ordinate, and the initial DNA template number of each reaction is quantitatively determined according to the standard curve and the Ct value of each sample.

The beta-tubulin protein is a major component of the cytoskeleton and plays an important role in parasite proliferation, morphological changes and adhesion to host epithelial cells. Therefore, the beta-tubulin gene and the detection of the transcription level thereof can provide favorable technical support for research works such as gene function research, drug development and the like.

However, there is no report on the study of the β -tubulin gene of avian trichomonas, which hinders the progress of the gene level detection of the pathogen and the mechanism of its pathogenesis.

Disclosure of Invention

In view of this, there is a need for a nucleic acid molecule displaying the coding sequence of the full-length gene for β -tubulin of avian trichomonas.

A nucleic acid molecule, the nucleotide sequence of which is shown as SEQ ID NO. 1.

The invention also provides a PCR primer pair which can detect the nucleic acid molecules through PCR amplification reaction.

In one embodiment, the primer set comprises an upstream primer with a nucleotide sequence shown as SEQ ID NO. 2 and a downstream primer with a nucleotide sequence shown as SEQ ID NO. 3, or comprises an upstream primer with a nucleotide sequence shown as SEQ ID NO. 4 and a downstream primer with a nucleotide sequence shown as SEQ ID NO. 5.

The invention also provides a recombinant vector containing the nucleic acid molecule as described above.

In one embodiment, the recombinant vector is constructed based on a pMD18T vector, a pUC18 vector, or a PBR322 vector.

The invention also provides application of the nucleic acid molecule, the PCR primer pair or the recombinant vector in preparing products for detecting, amplifying or expressing the trichomonas avicularis beta-tubulin gene.

The invention also provides a kit for detecting the avian trichomonas beta-tubulin gene, which comprises one or more of the nucleic acid molecule, the PCR primer pair and the recombinant vector.

In one embodiment, the kit further comprises one or more of fluorescent PCR dyes, DNA polymerases, dNTPs, and water.

In one embodiment, the kit further comprises a reverse transcriptase and a reverse transcription primer.

The invention also provides a detection method of the avian trichomonas beta-tubulin gene, which comprises the following steps:

extracting nucleic acid of a sample to be detected to obtain a nucleic acid sample;

adding the PCR primer pair to the extracted nucleic acid sample as a template to perform PCR amplification reaction;

and obtaining the result of the PCR amplification reaction.

The inventor clones the avian trichomonas beta-tubulin full-length gene coding sequence shown as SEQ ID NO. 1 in the avian trichomonas through extensive research, and further designs a PCR primer pair capable of detecting the avian trichomonas beta-tubulin gene and the transcription level thereof and corresponding PCR reaction conditions by utilizing the beta-tubulin full-length gene coding sequence to further assemble a kit for detecting the avian trichomonas beta-tubulin gene, thereby meeting the detection requirements of researchers in parasitic biology and life science, promoting the research progress of the gene level detection of the pathogen and the pathogenic mechanism thereof, and providing favorable technical support for research works such as gene function research, medicament development and the like. The PCR primer pair, the kit and the detection method can realize the fluorescent quantitative detection of the poultry trichomonas beta-tubulin gene and the transcription level thereof by using a real-time fluorescent quantitative PCR technology, and the PCR primer pair is used for the detection of the poultry trichomonas beta-tubulin gene and the transcription level thereof, and has the advantages of quick and simple use method, high repeatability, strong specificity, high sensitivity and no amplification signal to non-target genes.

Drawings

FIG. 1 is a graph showing the amplification curve of the β -tubulin gene of avian trichomonas in example 4;

FIG. 2 is a melting curve of the β -tubulin gene of avian trichomonas in example 4;

FIG. 3 is a standard curve of the avian trichomonas beta-tubulin gene in example 5.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Interpretation of terms

"Real-time fluorescent Quantitative PCR" (Quantitative Real-time PCR) is a method for measuring the total amount of products after each Polymerase Chain Reaction (PCR) cycle by using fluorescent chemical substances in DNA amplification reaction. Real-time PCR Real-time detection of PCR progress is performed by fluorescence signals during PCR amplification. In the exponential phase of PCR amplification, the Ct value of the template and the initial copy number of the template have a linear relationship, and therefore, the method becomes a basis for quantification. The detection principle includes a fluorescent dye method and a fluorescent probe method. The fluorescent dye method is characterized in that excessive fluorescent dye is added into a PCR reaction system, the dye is only combined with a minor groove of double-stranded DNA, is not combined with a single-stranded DNA chain, does not emit fluorescence in a free state, and can emit light only when being doped into a DNA double chain, so that in the PCR system, along with the exponential amplification of a specific PCR product, the dye is doped into the double-stranded DNA in each cycle of an extension stage, and the fluorescent signal intensity of the dye is in positive correlation with the quantity of the PCR product. When the fluorescence probe method is used for PCR amplification, a pair of primers is added, and a specific fluorescence probe is added at the same time, wherein the probe is a linear oligonucleotide, two ends of the linear oligonucleotide are respectively marked with a fluorescence reporter group and a fluorescence quenching group, when the probe is complete, a fluorescence signal emitted by the reporter group is absorbed by the quenching group, and the fluorescence signal cannot be detected by a PCR instrument; during PCR amplification (in an extension stage), the 5 '→ 3' exonuclease activity of Taq enzyme cuts and degrades the probe, so that the reporter fluorescent group and the quenching fluorescent group are separated, a fluorescence monitoring system can receive a fluorescence signal, namely, one fluorescent molecule is formed when one DNA chain is amplified, and the fluorescent signal accumulation and the PCR product formation are completely synchronous.

A "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma polyoma vacuolatum viruses (e.g., SV 40).

"reverse transcription", i.e., reverse transcription, is a process of synthesizing DNA using RNA as a template by reverse transcriptase, and is a specific mode of DNA biosynthesis. The reverse transcriptase functions by synthesizing a single DNA strand complementary to the RNA template in the 5 '→ 3' direction using dntps as substrates, RNA as a template, and tRNA (mainly tryptophan tRNA) as a primer, and this single DNA strand is called complementary DNA (cDNA), which forms an RNA-DNA hybrid with the RNA template. Then, under the action of reverse transcriptase, the RNA chain is hydrolyzed, and then the second DNA chain is synthesized by using cDNA as template. To this end, the RNA-directed DNA synthesis process is completed.

The nucleotide sequence of the nucleic acid molecule of one embodiment of the invention is shown in SEQ ID NO 1.

The inventor clones the full-length gene coding sequence of the poultry trichomonas beta-tubulin shown as SEQ ID NO. 1 in the poultry trichomonas through extensive research, can further design and obtain a PCR primer pair capable of detecting the poultry trichomonas beta-tubulin gene and the transcription level thereof by utilizing the full-length gene coding sequence of the beta-tubulin, and assembles various types of kits for detecting the poultry trichomonas beta-tubulin gene, thereby meeting the detection requirements of researchers in parasitic biology and life science, promoting the research progress of the gene level detection of the pathogen and the pathogenic mechanism thereof, and providing favorable technical support for the research works such as gene function research, medicine development and the like.

The PCR primer set of an embodiment of the present invention is capable of detecting the nucleic acid molecule as described above by a PCR amplification reaction.

The PCR primer pair is two artificially synthesized oligonucleotide sequences, one primer is complementary to one DNA template strand at one end of the target gene, and the other primer is complementary to the other DNA template strand at the other end of the target gene. In the PCR (polymerase chain reaction) technology, a nucleotide sequence of a target gene is known, a primer is synthesized according to the sequence, the target gene DNA is heated and denatured to melt into a single strand by utilizing the PCR amplification technology, the primer is combined with a corresponding complementary sequence of the single strand, then the extension is carried out under the action of high-temperature resistant DNA polymerase, the cycle is repeated, and a product obtained after the extension can be combined with the primer.

In a specific example, the PCR primer pair comprises an upstream primer with a nucleotide sequence shown as SEQ ID NO. 2 and a downstream primer with a nucleotide sequence shown as SEQ ID NO. 3, or an upstream primer with a nucleotide sequence shown as SEQ ID NO. 4 and a downstream primer with a nucleotide sequence shown as SEQ ID NO. 5. It is understood that the PCR primer set capable of amplifying the nucleic acid molecule is not limited thereto, and PCR primer sets having other sequences may be designed according to design rules.

The recombinant vector of one embodiment of the present invention contains the nucleic acid molecule as described above.

The vector functions to carry the gene of interest into the host cell, enabling it to be replicated and expressed. That is, the foreign DNA leaving the chromosome cannot be replicated, and the foreign DNA inserted into the replicon (replicon) DNA can be replicated in a recipient bacterium as a part of a replicon, which is a vector of a foreign gene. Therefore, the recombinant vector can be used for carrying the poultry trichomonas beta-tubulin gene to enter a host cell, so that the gene can be copied or expressed, and favorable technical support is provided for research works such as gene function research, drug development and the like.

It will be appreciated that the vector may also contain regulatory elements commonly used in genetic engineering, such as enhancers, promoters, etc., and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

In a specific example, the recombinant vector is constructed based on a pMD18T vector, a pUC18 vector, or a PBR322 vector. It will be appreciated that the particular type of carrier is not so limited and may be selected according to particular needs.

The host cell of an embodiment of the present invention has incorporated into its genome a nucleic acid molecule as described above. The host cell can be used for mass replication or protein expression of the poultry trichomonas beta-tubulin gene, thereby providing favorable technical support for research works such as gene function research, drug development and the like.

In one specific example, the host cell is E.coli. It is understood that the type of host cell is not limited thereto, and may be selected as desired.

The kit for detecting the avian trichomonas beta-tubulin gene of one embodiment of the invention comprises one or more of the nucleic acid molecule as described above, the PCR primer pair as described above and the recombinant vector as described above. For example, the nucleic acid molecule or recombinant vector can be used as a positive reference. The positive reference substance can provide a quantitative control basis for subsequent software analysis, for example, the positive reference substance after being subjected to value setting can be diluted into a plurality of concentrations by using a buffer solution, and the subsequent software can automatically obtain the value setting result of each sample through a standard curve drawn by a plurality of concentration gradient quantitative reference substances. It will be appreciated that the kit may also be used to detect the level of transcription of the β -tubulin gene of avian trichomonas.

In a specific example, the kit further comprises one or more of fluorescent PCR dyes, DNA polymerase, dNTPs and water, such that the avian trichomonas beta-tubulin gene can be detected by dye-based fluorescent quantitative PCR. It will be appreciated that the components of the kit may be varied as required depending on the mode of detection.

In a specific example, the kit further comprises a reverse transcriptase and a reverse transcription primer, so that RNA of a sample to be detected can be conveniently extracted and subjected to reverse transcription to obtain cDNA, and the cDNA is further used for detecting the transcription level of the poultry trichomonas beta-tubulin gene.

Further, the kit further comprises a negative control substance, and the negative control substance can adopt but is not limited to ultrapure water, physiological saline and the like.

In an alternative embodiment, the kit further comprises at least one of nucleic acid extraction reagents, PCR amplification buffers, DNA polymerase, dNTPs reagents.

In an alternative embodiment, the 10 XPCR amplification buffer comprises 200mmol/L Tris-HCl solution at pH 7.5, 30mmol/L magnesium chloride solution, 500mmol/L potassium chloride solution, 0.2% (v/v) Triton solution and 10% (v/v) formamide solution. The DNA polymerase may be, but is not limited to, hot start Taq enzyme, which is used at a concentration of 1U/. mu.L to 5U/. mu.L.

In an alternative embodiment, dNTPs include dATP, dGTP, dTTP, dCTP and dUTP, and further, UNG enzyme (uracil DNA glycosylase) is included in the kit at a concentration of 0.05U/. mu.L to 0.2U/. mu.L. UNG enzyme has the function of degrading PCR products containing dU, and the pollution of the PCR products can be prevented by using the UNG enzyme and dUTP in the PCR reaction solution. Further, in a specific embodiment, a DNA polymerase and UNG enzyme may be mixed to form an enzyme mixture.

In one specific example, the kit comprises TB Green Premix Ex Taq (2 ×) (Tli RNaseH Plus), an avian Trichomonas beta-tubulin gene template (i.e., the nucleic acid molecule or recombinant vector described above), a PCR primer pair, and ultrapure water. TB Green Premix Ex Taq (2 ×) (Tli RNaseH Plus) was purchased from TAKARA, PCR primer pairs were 200nmol/L, and ultrapure water was used for reverse osmosis having a purity of not less than 18.25 M.OMEGA.CM.

The detection method of the trichomonas avicularis beta-tubulin gene provided by the embodiment of the invention comprises the following steps of S1-S3:

s1, extracting nucleic acid of the sample to be detected to obtain a nucleic acid sample;

s2, adding the PCR primer pair to perform PCR amplification reaction by taking the extracted nucleic acid sample as a template;

s3, obtaining the result of the PCR amplification reaction.

Optionally, the result of the PCR amplification reaction is obtained by real-time detection of the PCR process by a fluorescent signal.

In one specific example, the PCR amplification reaction system is: TB Green Premix Ex Taq (2 ×) (Tli RNaseH Plus) 5. mu.L, 200nmol/L of each of the upstream and downstream primers 1. mu.L, cDNA of a sample to be detected or a gene template of the avian trichomonas beta-tubulin 0.5. mu.L, and 2.5. mu.L of ultrapure water.

The conditions of the real-time fluorescent PCR amplification reaction can be specifically determined and adjusted according to the salt ion concentration of the buffer solution and the length and nucleotide composition of the denatured nucleic acid, the reaction characteristics and the length of the nucleic acid, and the like, as in a preferred specific example, the following procedure can be followed: after instantaneous centrifugation, the mixture is loaded into a fluorescent quantitative PCR instrument, pre-denatured at 95 ℃ for 30s, then thermally cycled for 40 times according to denaturation at 95 ℃ for 3s and annealing at 60 ℃ for 30s, and fluorescence signals are detected and recorded at 60 ℃, and finally melting curve analysis is carried out at 65-95 ℃. In the real-time fluorescence PCR amplification process, the negative and positive of the poultry trichomonas beta-tubulin gene detection can be easily judged through the curve shape and the Ct value after the PCR amplification is finished through real-time fluorescence acquisition, so that a reliable experimental basis is provided for the detection and research of the poultry trichomonas.

In a specific example, the detection method of the poultry trichomonas beta-tubulin gene further comprises the step of performing the same treatment as that of a sample to be tested by using the nucleic acid molecule or the recombinant vector, so as to serve as a positive control or prepare a standard curve.

Further, in a specific example, the detection method further includes a step of performing the same treatment as the sample to be tested using a negative control such as ultrapure water or physiological saline as a negative control.

In one specific example, the detection method comprises the following steps: and extracting RNA of a sample to be detected, carrying out reverse transcription reaction to obtain a cDNA sample, adding the PCR primer pair to carry out PCR amplification reaction by taking the extracted cDNA sample as a template, and then obtaining the result of the PCR amplification reaction. Thus, the result of the detection is the transcription level of the avian trichomonas beta-tubulin gene.

The following are specific examples.

Example 1 preparation of Total cDNA of avian Trichomonas

Step (1): extraction of Trichomonas avicularis RNA

Taking (1-5) × 10⁶8000g of each bird caterpillar was centrifuged for 2 minutes, the medium was removed, 800. mu.L of PBS was added to rinse 3 times, and the supernatant was removed by centrifugation. Next, an avian trichomonas RNA sample was extracted by referring to the Omega E.Z.N.A. SE Total RNA plus kit I (R6836-01) kit operation.

Step (2): reverse transcription for preparing total cDNA of fowl trichomonas

Collecting 1.0 μ g of the above total RNA of the bird trichomonas, referring to TAKARA PrimeScript^TM1st Strand cDNA Synthesis Kit (6110A) reverse transcription Kit, prepare the avian trichomonas cDNA, and place it at-20 ℃ for gene coding sequence cloning or fluorescent quantitative detection.

EXAMPLE 2 cloning of the full-Length avian Trichomonas beta-tubulin-encoding Gene sequence

A cloning PCR reaction system of the full-length coding gene sequence of the trichomonas avian beta-tubulin is prepared in proportion according to the specification of TOYOBO KOD FX, and is specifically shown in Table 1. Shaking and mixing evenly, performing instant centrifugation, and then performing PCR reaction under the reaction condition of 94 ℃ for 2 min; 10s at 98 ℃, 30s at 59 ℃, 1min at 68 ℃ and 30 cycles; at 68 ℃ for 7 min. And (3) carrying out nucleic acid electrophoresis on the PCR product, cutting the gel, recovering a target band of about 1.3kb, connecting the target band with a pMD18T vector, and sending the vector to a tripartite company for sequencing to obtain the full-length coding gene of the trichomonas avian beta-tubulin, wherein the sequence is shown as SEQ ID NO. 1. The sequence of the upstream primer is as follows: 5'-ATGGTTCGTGAAATCGTTCACATCCAG-3', the sequence of the downstream primer is: 5'-TTATGCCTCGGCTTCCTCGTCTTCTT-3' are provided.

TABLE 1

Example 3 avian Trichomonas beta-tubulin Gene transcript level fluorescent quantitative PCR detection kit Assembly

The kit consists of TB Green Premix Ex Taq (2x) (Tli RNaseH Plus), a trichomonas avicularis beta-tubulin gene template, an upstream primer, a downstream primer and ultrapure water. The specific composition is shown in table 2.

TABLE 2

TB Green Premix Ex Taq II(2X)(Tli RNaseH Plus)	5mL
		Upstream primer	500μL
Downstream primer	500μL
		Poultry trichomonas beta-tubulin gene template	500μL
Ultrapure water	5mL

In the kit, TB Green Premix Ex Taq II (Tli RNaseH Plus) (2X) was purchased from TaKaRa, the mixture of fluorescent quantitative primers was 200nM upstream primer and 200nM downstream primer, the sequence of the upstream primer was 5'-AAGCTCGCTCCAACATGACA-3', the sequence of the downstream primer was 5'-TTTATGCCTCGGCTTCCTCG-3', and the ultrapure water was reverse osmosis water having a purity of not less than 18.25 M.OMEGA.CM.

Example 4 specific detection of the avian Trichomonas beta-tubulin transcript level fluorescent quantitative PCR detection kit

A fluorescence quantitative PCR reaction system for the gene transcription level of the trichomonas avian beta-tubulin is prepared in proportion according to the instruction of TB Green Premix Ex Taq (Tli RNaseH Plus) (2X), and is specifically shown in a table 3. After instantaneous centrifugation, the sample is loaded into a fluorescence quantitative PCR instrument (Bio-Rad CFX96), pre-denatured at 95 ℃ for 30s, then thermally cycled for 40 times according to denaturation at 95 ℃ for 3s and annealing at 60 ℃ for 30s, fluorescence signals are detected and recorded at 60 ℃, and finally melting curve analysis is carried out at 65-95 ℃. Each sample was replicated three times.

The instrument was read using Bio-Rad CFX Maestro software, where the gene fluorescence signal values fit the standard "S" curve, as shown in FIG. 1, and only a single peak was seen in the melting curve, indicating that the fluorescence quantification has high detection specificity, as shown in FIG. 2. The amplification curve and melting curve of all wells remained consistent, indicating that the kit was reproducible.

TABLE 3

Reaction solution Components	Volume/. mu.L
		TB Green Premix Ex Taq II(Tli RNaseH Plus)(2X)	10
200nM forward primer	2
		200nM downstream primer	2
Total cDNA of avian trichomonas	1
		Ultrapure water	5
Total volume	20

Example 5 sensitive detection of the avian Trichomonas beta-tubulin transcript level fluorescent quantitative PCR detection kit

The total cDNA of the avian trichomonas is detected10E-0, 10E-1, 10E-2, 10E-3, 10E-4 and 10E-5 serial dilutions were sequentially performed as nucleic acid templates, 10. mu.L per tube, for 6 tubes. A fluorescent quantitative Premix was prepared by mixing 10. mu.L of TB Green Premix Ex Taq II (Tli RNaseH Plus) (2X), 2. mu.L of 200nM forward primer, 2. mu.L of 200nM downstream primer and 5. mu.L of ultrapure water. Adding 1 mu L of the avian trichomonas total cDNA diluted in a gradient manner and 19 mu L of fluorescent quantitative premix liquid into each fluorescent quantitative reaction tube respectively, shaking, uniformly mixing, performing instant centrifugation, loading into a fluorescent quantitative PCR instrument (Bio-Rad CFX96), performing pre-denaturation at 95 ℃ for 30s, performing thermal cycle for 30s at the temperature of 95 ℃ for 3s and 60 ℃ for 30s, and detecting and recording a fluorescent signal at the temperature of 60 ℃. A standard curve of the avian trichomonas beta-tubulin gene was calculated by using Bio-Rad CFX Manager 3.1 software. As shown in FIG. 3, the standard curve R of the avian trichomonas beta-tubulin²Close to 1, is suitable for relative quantitative analysis of the gene.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

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Claims

1. A nucleic acid molecule, wherein the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 1.

2. A PCR primer pair capable of detecting the nucleic acid molecule of claim 1 by a PCR amplification reaction.

3. The PCR primer pair as claimed in claim 2, comprising an upstream primer having a nucleotide sequence shown in SEQ ID NO. 2 and a downstream primer having a nucleotide sequence shown in SEQ ID NO. 3, or an upstream primer having a nucleotide sequence shown in SEQ ID NO. 4 and a downstream primer having a nucleotide sequence shown in SEQ ID NO. 5.

4. A recombinant vector comprising the nucleic acid molecule of claim 1.

5. The recombinant vector according to claim 4, wherein the recombinant vector is constructed based on a pMD18T vector, a pUC18 vector or a PBR322 vector.

6. Use of the nucleic acid molecule of claim 1, the PCR primer pair of claim 2 or 3, or the recombinant vector of claim 4 or 5 for the preparation of a product for detecting, amplifying or expressing the β -tubulin gene of avian trichomonas.

7. A kit for detecting the β -tubulin gene of avian trichomonas, comprising one or more of the nucleic acid molecule of claim 1, the PCR primer pair of claim 2 or 3, and the recombinant vector of claim 4 or 5.

8. The kit of claim 7, further comprising one or more of fluorescent PCR dyes, DNA polymerase, dNTPs, and water.

9. The kit of claim 7, further comprising a reverse transcriptase and a reverse transcription primer.

10. A detection method of a poultry trichomonas beta-tubulin gene is characterized by comprising the following steps:

adding the PCR primer pair of claim 2 or 3 to the extracted nucleic acid sample as a template to perform a PCR amplification reaction;

and obtaining the result of the PCR amplification reaction.