CN113416743B - Nucleic acid molecule, PCR primer pair and kit for detecting trichomonas fowl rpoB gene - Google Patents

Abstract

The invention discloses a nucleic acid molecule, a PCR primer pair and a kit for detecting an avian trichomonas rpoB gene. Through long-term research, the inventor clones and obtains the full-length gene coding sequence of the trichomonas avionica rpoB shown as SEQ ID NO. 1 in trichomonas avionica, and further designs and obtains a PCR primer pair and corresponding PCR reaction conditions capable of detecting the trichomonas avionica rpoB gene and the transcription level thereof by utilizing the full-length gene coding sequence of the rpoB, and further assembles the kit for detecting the trichomonas avionica rpoB gene, thereby meeting the detection needs of researchers in parasitic biology and life science, promoting the progress of gene level detection and pathogenic mechanism research of the pathogen, and providing favorable technical support for research works such as gene function research, drug development and the like. The PCR primer pair has high repeatability, strong specificity and high sensitivity, and does not amplify non-target genes.

Description

Nucleic acid molecule, PCR primer pair and kit for detecting trichomonas fowl rpoB gene

Technical Field

The invention relates to the field of molecular biology detection, in particular to a nucleic acid molecule, a PCR primer pair and a kit for detecting an avian trichomonas rpoB gene.

Background

The pigeon raising history in China is long, the pigeon is taken as commodity production for over 100 years, the meat pigeon raising industry in the present stage is spread over various areas in China, the scale is continuously strong, and the effective prevention and control of the pigeon infectious diseases are also of great importance. Wherein, trichomonas pigeon (also called as Pitaer Huang) is also a highly-developed consumption parasitic disease in pigeon farms, and has important harm to the production performance of meat pigeons and the survival rate of young pigeons. The most common characteristic change is the formation of coarse button-like yellow deposits on the mucous membranes of the mouth and throat; wet persons, called wet ulcers; cheese-like or crusted forms are called dry ulcers. When the umbilical region is infected, a tumor is formed subcutaneously and is in the shape of cheese or ulcerative lesions; when the viscera are affected, a cheese-like focus with a clear rough yellow border is caused, and the tissue necrosis of the parenchyma organs is caused. The ill pigeons are hampered by oral ulcers, which can cause high mortality in young and growing pigeons. The pathogen of trichomonas pigeon is trichomonas avionica, belonging to the subgenera, the animal flagellidae, the order of the multiple flagellidae, the genus trichomonas. Laboratory diagnosis usually detects the insects by microscopic examination of the oral cavity, esophagus, crop secretions smears, or scrapes mucus from lesions to make smears, staining microscopy to see typical insects. The trichomonas fowl body is melon seed-shaped or pear-shaped, and the front end of the trichomonas fowl body is provided with a hair matrix which extends out of 4 flagella, so that the trichomonas fowl body can move rapidly. One side of the insect body is provided with a fluctuation film, the fluctuation film starts from the front end of the insect body, the front part of the insect body is provided with a1 oval nucleus at the rear part of the insect body, the front part of the insect body is opposite to the fluctuation film, one side of the insect body is provided with a cell opening, and the center of the insect body is provided with an elongated shaft column extending from front to back to the outside of the rear edge of the insect body. The insects proliferate in a split manner by virtue of spiral movement in body fluid, and proliferate for about 4 hours for one generation. At present, the pathogenic mechanism of the pathogen is rarely studied, and the reason is that an effective gene level detection means is lacking.

Polymerase Chain Reaction (PCR) is a molecular biological technique for amplifying specific DNA fragments, which can be regarded as specific DNA replication in vitro, and the greatest feature of PCR is the ability to greatly increase minute amounts of DNA. The fluorescent quantitative PCR is a common nucleic acid quantitative technology, and is also a gene level detection means with low cost, high repeatability and convenient operation. According to the technology, nucleic acid fluorescent marker substances such as SYBR, TB Green and the like are introduced into a PCR reaction system, the increase condition of a fluorescent value in the PCR reaction system after each thermal cycle is detected, the PCR amplification condition is monitored in real time, a fluorescent quantitative change curve of each sample is fitted, so that the Ct value (cycle number when the fluorescent change reaches a threshold) 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 measured according to the standard curve and the Ct value of each sample.

The rpoB protein (DNA-directed RNA polymerase, beta subset) is the β subunit of RNA polymerase, involved in RNA synthesis, and is an important drug target, such as rifampicin target, to inhibit mycobacterium tuberculosis rpoB gene, and thus inhibit bacterial growth. In addition, the gene is widely existed in eukaryotic organisms and prokaryotes, but in different organisms, the sequence of the coding gene is different, so that the gene is also an effective genetic typing tag. Moreover, detection of rpoB gene and its transcription level can provide a favorable technical support for research works such as gene function research and drug development.

However, no report has been published on the rpoB gene of trichomonas fowl, which hinders the progress of gene level detection of the pathogen and research on its pathogenic mechanism.

Disclosure of Invention

Based on this, it would be desirable to provide a nucleic acid molecule that displays the full-length gene coding sequence of rpoB of trichomonas avians.

A nucleic acid molecule with a nucleotide sequence shown in SEQ ID NO. 1.

The invention also provides a PCR primer pair capable of detecting a nucleic acid molecule as described above by a PCR amplification reaction.

In one embodiment, the primer 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.

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

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

The invention also provides application of the nucleic acid molecule, the PCR primer pair or the recombinant vector in preparation of products for detecting, amplifying or expressing the trichomonas fowl rpoB gene.

The invention also provides a kit for detecting an avian trichomonas rpoB gene, comprising one or more of the nucleic acid molecules described above, the PCR primer pairs described above, and the recombinant vectors described above.

In one embodiment, the kit further comprises one or more of fluorescent PCR dyes, DNA polymerase, 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 trichomonas fowl rpoB 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;

obtaining the result of PCR amplification reaction.

Through long-term research, the inventor clones and obtains the full-length gene coding sequence of the trichomonas avionica rpoB shown as SEQ ID NO. 1 in trichomonas avionica, and further designs and obtains a PCR primer pair and corresponding PCR reaction conditions capable of detecting the trichomonas avionica rpoB gene and the transcription level thereof by utilizing the full-length gene coding sequence of the rpoB, and further assembles the kit for detecting the trichomonas avionica rpoB gene, thereby meeting the detection needs of researchers in parasitic biology and life science, promoting the progress of gene level detection and pathogenic mechanism research of the pathogen, and providing favorable technical support for research works such as gene function research, drug development and the like. The PCR primer pair, the kit and the detection method can realize the fluorescent quantitative detection of the trichomonas avionica rpoB gene and the transcription level thereof by using a real-time fluorescent quantitative PCR technology. The PCR primer pair is used for detecting the trichomonas avid rpoB gene and the transcription level thereof, and the use method is rapid, simple and convenient, high in repeatability, strong in specificity and high in sensitivity, and does not amplify signals for non-target genes.

Drawings

FIG. 1 is an amplification curve of the trichomonas avia rpoB gene in example 4;

FIG. 2 is a melting curve of the rpoB gene of avian trichomonas in example 4.

Detailed Description

In order that the invention may be readily understood, a more complete description of the invention will be rendered by reference to the appended 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 herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

Interpretation of the terms

"real-time fluorescent quantitative PCR" (Quantitative Real-time PCR) is a method of measuring the total amount of product after each Polymerase Chain Reaction (PCR) cycle in a DNA amplification reaction using fluorescent chemicals. Real-time PCR detects the PCR progress in Real time by fluorescent signals during the PCR amplification process. Since the Ct value of a template and the initial copy number of the template have a linear relationship in the exponential phase of PCR amplification, the quantitative basis is established. The detection principle comprises a fluorescent dye method and a fluorescent probe method. In the fluorescent dye method, excessive fluorescent dye is added into a PCR reaction system, the dye is only combined with a small 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 by being doped into the DNA double-stranded DNA, so that the fluorescent dye is doped into the double-stranded DNA in each cycle extension stage along with exponential amplification of a specific PCR product in the PCR system, and the fluorescent signal intensity is positively correlated with the quantity of the PCR product. When the fluorescent probe method is used for amplifying by PCR, a pair of primers is added, and a specific fluorescent probe is added, wherein the probe is a linear oligonucleotide, a fluorescent reporter group and a fluorescent quenching group are respectively marked at two ends, and when the probe is complete, a fluorescent signal emitted by the reporter group is absorbed by the quenching group, and the fluorescent signal cannot be detected by a PCR instrument; during PCR amplification (in the extension stage), the 5 '. Fwdarw.3' exonuclease activity of Taq enzyme is used for enzyme digestion degradation of the probe to separate the reporting fluorescent group from the quenching fluorescent group, so that a fluorescent signal can be received by a fluorescent monitoring system, namely, one fluorescent molecule is formed every time one DNA chain is amplified, and the accumulation of the fluorescent signal and the formation of PCR products are completely synchronous.

"vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of 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 such that 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 Chromosome (YAC), bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuolation virus (e.g., SV 40).

Reverse transcription, i.e., reverse transcription, is a process of synthesizing DNA by reverse transcriptase using RNA as a template, and is a special mode of DNA biosynthesis. Reverse transcriptase acts to synthesize a single strand of DNA complementary to the RNA template, called complementary DNA (cDNA), in the 5 'to 3' direction, using dntps as substrate, RNA as template, trnas (mainly tryptophan trnas) as primer, which forms an RNA-DNA hybrid with the RNA template. Then, under the action of reverse transcriptase, RNA strand is hydrolyzed, and cDNA is used as template to synthesize the second DNA strand. Thus, the RNA-guided DNA synthesis process is completed.

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

Through extensive research, the inventor clones and obtains the full-length gene coding sequence of the trichomonas fowl rpoB shown as SEQ ID NO. 1 in trichomonas fowl, can further design and obtain PCR primer pairs capable of detecting the trichomonas fowl rpoB gene and the transcription level thereof by utilizing the full-length gene coding sequence of the rpoB, and assembles various types of kits for detecting the trichomonas fowl rpoB gene, thereby meeting the detection needs of researchers of parasitic biology and life science, promoting the gene level detection of the pathogen and the progress of the research on pathogenic mechanism thereof, and providing favorable technical support for research works such as gene function research, drug development and the like.

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

The PCR primer pair is two oligonucleotide sequences synthesized artificially, 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 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 melted into a single chain after being denatured by heating by using a PCR amplification technology, the primer is combined with a corresponding complementary sequence of the single chain, then the primer is extended under the action of high-temperature-resistant DNA polymerase, the repeated cycle is performed, and a product obtained after the extension can be combined with the primer.

In a specific example, the PCR primer pair includes 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. It will be appreciated that the PCR primer pairs capable of amplifying the above nucleic acid molecules are not limited thereto, and PCR primer pairs of 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 function of the vector is to carry the target gene into the host cell, so that the target gene can be replicated and expressed. That is, the foreign DNA leaving the chromosome cannot replicate, and the foreign DNA inserted into a replicon (replicon) DNA, which is a vector of the foreign gene, can replicate as a part of a replicon in a recipient bacterium. Therefore, the recombinant vector can be used for carrying the trichomonas avionica rpoB gene into host cells, so that the trichomonas avionica rpoB gene can be replicated or expressed, and a 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, and the like, as well as 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, pUC18 vector or PBR322 vector. It will be appreciated that the particular type of carrier is not limited thereto and may be selected according to particular needs.

In one embodiment of the invention, the host cell has a genome into which the nucleic acid molecule as described above is incorporated. The host cell can be used for carrying out mass replication or protein expression of the trichomonas avionica rpoB gene, thereby providing a favorable technical support for research works such as gene function research, drug development and the like.

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

The kit for detecting the trichomonas avia rpoB gene according to an embodiment of the present invention comprises one or more of the nucleic acid molecules described above, the PCR primer pairs described above, and the recombinant vectors described above. For example, the nucleic acid molecules or recombinant vectors described above can be used as positive references. The positive reference can provide quantitative control basis for subsequent software analysis, for example, the positive reference after fixed value can be diluted into a plurality of concentrations by buffer solution, and the subsequent software can automatically obtain the fixed value result of each sample through standard curves drawn by a plurality of concentration gradient quantitative references. It will be appreciated that the kit may also be used to detect the transcript level of the trichomonas fowl rpoB gene.

In a specific example, the above-described kit further comprises one or more of fluorescent PCR dyes, DNA polymerase, dNTPs, and water, such that the trichomonas avia rpoB gene can be detected by dye-based fluorescent quantitative PCR. It will be appreciated that the composition of the different kits may be adapted as desired depending on the assay format.

In a specific example, the kit further comprises reverse transcriptase and reverse transcription primers, so that RNA of a sample to be tested is conveniently extracted and subjected to reverse transcription to obtain cDNA, and the kit is further used for detecting the transcription level of the trichomonas fowl rpoB gene.

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

In an alternative specific example, the kit further comprises at least one of a nucleic acid extraction reagent, a PCR amplification buffer, a DNA polymerase, dNTPs reagent.

In an alternative specific example, the 10 XPCR amplification buffer includes 200mmol/L tris hydrochloride solution, 30mmol/L magnesium chloride solution, 500mmol/L potassium chloride solution, 0.2% (v/v) triton solution, and 10% (v/v) formamide solution at pH 7.5. The DNA polymerase may be, but is not limited to, a hot start Taq enzyme, which is used at a concentration of 1U/. Mu.L to 5U/. Mu.L.

In an alternative specific example, 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. The UNG enzyme has the function of degrading the PCR product containing dU, and the UNG enzyme and dUTP in the PCR reaction liquid can play a role in preventing pollution of the PCR product. Further, in a specific embodiment, the DNA polymerase may be mixed with the UNG enzyme to form an enzyme mixture.

In one specific example, the kit includes TB Green Premix Ex Taq (2×) (Tli RNaseH Plus), an avian trichomonas rpoB gene template (i.e., the nucleic acid molecule or recombinant vector described above), a PCR primer pair, and ultrapure water. Wherein TB Green Premix Ex Taq (2×) (Tli RNaseH Plus) was purchased from TAKARA, and the PCR primer pair was 8. Mu. Mol/L, respectively, and the ultrapure water was reverse osmosis water having a purity of not less than 18.25 M.OMEGA.CM.

The invention relates to a detection method of an avian trichomonas rpoB gene, which comprises the following steps of S1 to S3:

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

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

s3, obtaining a result of the PCR amplification reaction.

Alternatively, the result of the PCR amplification reaction is obtained by detecting the progress of PCR in real time by a fluorescent signal.

In one specific example, the system of the PCR amplification reaction is: TB Green Premix Ex Taq (2X) (Tli RNaseH Plus) 5. Mu. L, PCR primer set 1. Mu.L, cDNA of the sample to be tested or avian trichomonas rpoB gene template 1. Mu.L, ultrapure water 8. Mu.L.

The conditions of the real-time fluorescent PCR amplification reaction may be specifically determined and adjusted according to the buffer salt ion concentration and the length and nucleotide composition of the denatured nucleic acid, the reaction characteristics, the nucleic acid length, and the like, and as a preferred specific example, may be performed according to the following procedure: after instantaneous centrifugation, loading the sample into a fluorescent quantitative PCR instrument, pre-denaturing for 30s at 95 ℃, then carrying out thermal cycle for 40 times according to denaturation for 3s at 95 ℃ and annealing for 30s at 60 ℃, detecting and recording fluorescent signals at 60 ℃, and finally carrying out melting curve analysis at 65-95 ℃. In the real-time fluorescence PCR amplification process, after the PCR amplification is finished, the negative and positive of the detection of the trichomonas fowl rpoB gene can be easily judged through the curve shape and the Ct value, so that a reliable experimental basis is provided for the detection and research of the trichomonas fowl.

In a specific example, the method for detecting the trichomonas fowl rpoB gene further comprises the step of performing the same treatment as the sample to be tested using the above nucleic acid molecule or recombinant vector, as a positive control or preparing 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 includes the steps of: extracting RNA of a sample to be detected, carrying out reverse transcription reaction to obtain a cDNA sample, taking the extracted cDNA sample as a template, adding the PCR primer pair to carry out PCR amplification reaction, and then obtaining the result of the PCR amplification reaction. Thus, the result of the detection is the transcription level of the trichomonas fowl rpoB gene.

Through extensive research, the inventor clones and obtains the full-length gene coding sequence of the trichomonas avionica rpoB shown as SEQ ID NO. 1 in trichomonas avionica, and further designs and obtains a PCR primer pair and corresponding PCR reaction conditions capable of detecting the trichomonas avionica rpoB gene and the transcription level thereof by utilizing the full-length gene coding sequence of the rpoB, and further assembles the kit for detecting the trichomonas avionica rpoB gene, thereby meeting the detection needs of researchers in parasitic biology and life science, promoting the progress of gene level detection and pathogenic mechanism research of the pathogen, and providing favorable technical support for research works such as gene function research, drug development and the like. The PCR primer pair, the kit and the detection method can realize the fluorescent quantitative detection of the trichomonas avionica rpoB gene and the transcription level thereof by using a real-time fluorescent quantitative PCR technology. The PCR primer pair is used for detecting the trichomonas avid rpoB gene and the transcription level thereof, and the use method is rapid, simple and convenient, high in repeatability, strong in specificity and high in sensitivity, and does not amplify signals for non-target genes.

The present invention will be described in further detail below mainly with reference to the detailed description and the accompanying drawings.

EXAMPLE 1 preparation of total cDNA of avian trichomonas

Step (1): extraction of trichomonas fowl RNA

Taking (1-5) x 10 ⁶ Each trichomonas was centrifuged at 8000g for 2min, the medium was removed, washed 3 times with 800. Mu.L PBS, and the supernatant was removed by centrifugation. Next, an avian trichomonas RNA sample was extracted by reference to the Omega E.Z.N.A SE Total RNA plus kit I (R6836-01) kit procedure.

Step (2): preparation of total cDNA of trichomonas fowl by reverse transcription

The total RNA of the trichomonas was taken at 1.0 mug, and the trichomonas cDNA was prepared and stored at-20 ℃ for cloning of the gene coding sequence or fluorescent quantitative detection by referring to the instruction of the reverse transcription kit of TAKARAPRIMECTRITTM 1st Strand cDNA Synthesis Kit (6110A).

EXAMPLE 2 cloning of the coding Gene sequence of trichomonas avionica rpoB

Referring to TOYOBO KOD FX specification, a cloning PCR reaction system of an avian trichomonas rpoB coding gene sequence is prepared proportionally, and is shown in table 1. Shaking and mixing uniformly, carrying out PCR reaction after instantaneous centrifugation, wherein the reaction condition is 94 ℃ for 2min;98℃for 10s,55℃for 30s,68℃for 3.5min,30 cycles; and at 68℃for 7min. And (3) carrying out nucleic acid electrophoresis on the PCR product, cutting gel, recovering a target band of about 3.7kb, connecting a pMD18T vector, and sending to a three-party company for sequencing to obtain the trichomonas fowl rpoB coding gene, wherein the sequence is shown as SEQ ID NO. 1 (coding sequence CDS).

The upstream primer sequence is as follows: 5'-TGTTAAGCCAGTCTTTTTCG-3', the downstream primer sequences are: 5'-TTCTTTCCTGTGCATCAAATATCTT-3', primers are designed based on orf regions.

TABLE 1

Example 3 assembly of fluorescent quantitative PCR detection kit for the rpoB Gene transcript level of avian trichomonas

The kit consists of TB Green Premix Ex Taq (2×) (Tli RNaseH Plus), an avian trichomonas rpoB gene template, a fluorescent quantitative primer mixture and ultrapure water. The specific composition is shown in Table 2.

TABLE 2

TB Green Premix Ex Taq II(2X)(Tli RNaseH Plus)	5mL
		Fluorescent quantitative primer mixture	500μL
Avian trichomonas rpoB gene template	500μL
		Ultrapure water	5mL

In the kit, TB Green Premix Ex Taq II (Tli RNaseH Plus) (2X) was purchased from TaKaRa, and the fluorescent quantitative primer mixture was 8. Mu.M for the upstream primer and 8. Mu.M for the downstream primer, 5'-CCGATCGCTACTTCGTTCCA-3' for the upstream primer sequence, 5'-ATCCTGGCATGATGGGCAAA-3' for the downstream primer sequence, and water for reverse osmosis having a purity of not less than 18.25 M.OMEGA.CM.

Example 4 specific detection of avian trichomonas rpoB transcriptional level fluorescent quantitative PCR detection kit

The fluorescent quantitative PCR reaction system of the transcription level of the trichomonas fowl rpoB gene is prepared according to the proportion, and is specifically shown in Table 3. After transient centrifugation, the sample is put into a fluorescence quantitative PCR instrument (Bio-Rad CFX 96) and is pre-denatured for 30s at 95 ℃, then is denatured for 3s at 95 ℃ and annealed for 30s at 60 ℃ for 40 times, and fluorescence signals are detected and recorded at 60 ℃, and finally melting curve analysis is carried out at 65-95 ℃. Each sample was repeated three times.

The results were read on the instrument using Bio-Rad CFX Maestro software, wherein the gene fluorescence signal values met the standard "S" profile, as shown in FIG. 1, and the melting profile was only seen as a single peak, indicating that the fluorescence quantification had a high detection specificity, as shown in FIG. 2. The amplification curves and melting curves were kept consistent for all wells, indicating that the kit was reproducible.

TABLE 3 Table 3

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Sequence listing

<110> animal health institute of academy of agricultural sciences in Guangdong province

<120> nucleic acid molecule, PCR primer pair and kit for detecting trichomonas fowl rpoB gene

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tacactcact gcgaaattca tccatcactt attctcggtg tctgcggcac cctcattccg 2160

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ccagccggta tcaacgtctg cgtcgccatt tgctgctaca caggttataa ccaggaagac 2400

tctctcatgc tcaactcttc tggcatcgac cgcggtctct tccgttcatt cttcttcaga 2460

atgtacaagg aaacagccga aggtgatgat gcaaatggtg catcagaagt cttcacagtg 2520

ccagacccag ataacgtcaa aggcatcaag taccacgata catcaaagat cgacaaagat 2580

ggcctcgcta ttccaggtac tgttgtctca gcacaagatg tcatcatcgg caagatttct 2640

ccagataaag ccaccgatgt cgataagcgt acatacagag atacatcatg catggtaaga 2700

ccaatggaaa caggtatcgt cgacttggtt ctcaggacag tcacaaagaa gaacattcag 2760

ctggctaagg tcagaacacg ccaggctaga cgtccagaaa tcggtgataa gttctgctct 2820

cgccacggcc agaaaggtgt ctgtggcatg acataccgcc aggaagatat gccattcaca 2880

cgcgacggca tggtcccaga catcatcatg aacccgcacg ctattccgtc acgtatgaca 2940

atcggtcacc ttatcgagtg cctcctcgga aaagtcacag ccatcgcgcc agaagtttct 3000

ccagatggtg atgccacacc attcactggt gtcacaatgg aagaaatcgc cgacaagctc 3060

caggcttgcg gcttcgagcg ctacggtaac gaaacactct acaacggtcg tagcggcaag 3120

cgcatgaaag ctaagatcta cttcggccca acatactacc aacgtctgaa gcatatggtc 3180

ggtgataagg ctcacgcacg tgcacacggc aagaagtcac ttcttctccg tcagccagtc 3240

gaaggtcgtg caagaaacgg tggtcttcga ttcggagaaa tggaacgtga ctgcctcatc 3300

gcccacggtg tttcagctat gatcagagat cgtctccttg agaactccga tcgctacttc 3360

gttccagtct gcaagaactg cggactcatc gctatcgagc aaacagatgg tacaatggtt 3420

tgcccatcat gccaggatga gggcagactc gtccgcgtcg aaatgccata cgccttcaag 3480

ctcgttattc aagagcttat gtccatgtgc atcgctcctc gtctcaatgc aatcgaggac 3540

taa 3543

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

tgttaagcca gtctttttcg 20

<210> 3

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

ttctttcctg tgcatcaaat atctt 25

<210> 4

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

ccgatcgcta cttcgttcca 20

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

atcctggcat gatgggcaaa 20

Claims (9)

1. The trichomonas fowl rpoB gene is characterized in that the nucleotide sequence of the trichomonas fowl rpoB gene is shown as SEQ ID NO. 1.

2. A PCR primer pair, wherein the PCR primer pair is capable of detecting the avian trichomonas rpoB gene of claim 1 by a PCR amplification reaction, the PCR primer pair 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.

3. A recombinant vector comprising the trichomonas fowl rpoB gene of claim 1.

4. The recombinant vector according to claim 3, wherein the recombinant vector is constructed based on a pMD18T vector, pUC18 vector or PBR322 vector.

5. Use of the trichomonas avionica rpoB gene of claim 1, the PCR primer pair of claim 2, or the recombinant vector of claim 3 or 4 in the preparation of a product for detecting, amplifying, or expressing the trichomonas avionica rpoB gene.

6. A kit for detecting an avian trichomonas rpoB gene comprising one or more of the avian trichomonas rpoB gene of claim 1, the PCR primer pair of claim 2, and the recombinant vector of claim 3 or 4.

7. The kit of claim 6, further comprising one or more of a fluorescent PCR dye, a DNA polymerase, dNTPs, and water.

8. The kit of claim 6, further comprising a reverse transcriptase and a reverse transcription primer.

9. A method for detecting an avian trichomonas rpoB gene for non-disease diagnosis and treatment purposes, comprising the steps of:

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

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

obtaining the result of PCR amplification reaction.

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