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Nucleic acid molecule, PCR primer pair and kit for detecting beta-tubulin gene of trichomonad

A technology of nucleic acid molecules and primer pairs, which is applied in the field of kits for detecting the β-tubulin gene of Trichomonas fowl, can solve the problems that there is no research report on the β-tubulin gene, hindering the research progress of pathogenic gene level detection of pathogenic mechanism, etc.

Active Publication Date: 2021-07-20
INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, there is no research report on the β-tubulin gene of Trichomonas avium, which hinders the research progress of the gene-level detection of the pathogen and its pathogenic mechanism

Method used

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  • Nucleic acid molecule, PCR primer pair and kit for detecting beta-tubulin gene of trichomonad
  • Nucleic acid molecule, PCR primer pair and kit for detecting beta-tubulin gene of trichomonad
  • Nucleic acid molecule, PCR primer pair and kit for detecting beta-tubulin gene of trichomonad

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Preparation of Total CDNA of Poultry Michter

[0062] Step (1): Extract the poultry drops RNA

[0063] Take (1 ~ 5) × 10 6 A poultry dripper was centrifuged for 2 minutes, removed from the medium, and added 800 μl of PBS and washed 3 times, and centrifugally removed. Then, refer to Omega E.Z.N.A SE TOTAL RNA PLUS KIT I (R6836-01) kit operation, extracting the poultry mesentery RNA sample.

[0064] Step (2): Reversal Recording Preparation of Pouglet Total CDNA

[0065]Take the total RNA of the poultry droppers, refer to Takara Primescript TM 1st Strand CDNASYNThesis Kit (6110a) Anti-transcription kit specification, preparing a poultry pymoni cDNA, and is stored at -20 ° C for gene encoding sequence clone or fluorescent quantitative detection.

Embodiment 2

[0066] Example 2 A full length coding gene sequence clone in a poultry mesentery β-Tubulin

[0067] Referring to the Toyobo Kod FX manual, a cloning PCR reaction system of the full-length coding gene sequence of poultry mese-Tubulin is prepared, as shown in Table 1. After the shock is mixed, after instantaneous centrifugation, the PCR reaction was carried out, and the reaction conditions were 94 ° C for 2 min; 98 ° C 10s, 59 ° C 30s, 68 ° C for 1 min, 30 cycles; 68 ° C for 7 min. The PCR product was electrophoresed, and the rubber was recovered from about 1.3 kb, and the PMD18T carrier was connected, and the three-party company sequenced, and the full length coding gene of the poultry mesenter β-Tubulin was obtained. Sequence, such as SEQ ID NO: 1. Upstream primer sequences are: 5'-AtggttcgtgaAatcgttcacatccag-3 ', downstream primer sequences are: 5'-TtatgcctcggctTcctcgtctTCTTTT-3'.

[0068] Table 1

[0069]

[0070]

Embodiment 3

[0071] Example 3 Poultry Michi β-Tubulin Gene Transcription Horizontal Fluorescent Quantitative PCR Detection Kit

[0072] This kit is composed of TB Green Premix Ex TAQ (2x) (TLI RNaseh Plus), a poultry mesenamulin β-Tubulin gene template, upstream primer, downstream primer and ultrapure water. The specific composition is shown in Table 2.

[0073] Table 2

[0074] TB Green Premix EX TAQ II (2X) (TLI RNaseh Plus) 5ml Upstream primer 500μL Downstream primer 500μL Poultry mesentery β-Tubulin gene template 500μL Ultra-pure water 5ml

[0075] In the kit, TB Green Premix EX TAQ II (TLI RNaseh Plus) (2X) was purchased from Takara, the fluorescent quantitative primer mixture was 200 nm of upstream primers 200 nm and downstream primers, and the upstream primer sequence is 5'-aagctcgctccaacatgaca-3 ', downstream primers. The sequence is 5'-tttatgcctcggcttcctcctcg-3 ', ultrapure water is a reverse osmosis with a purity of no lower than 18.25mΩ · cm.

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Abstract

The invention discloses a nucleic acid molecule, a PCR primer pair and a kit for detecting a beta-tubulin gene of trichomonad. Through extensive research, the inventor clones in trichomonad to obtain a trichomonad beta-tubulin full-length gene coding sequence as shown in SEQ ID NO: 1, and further designs, by utilizing the beta-tubulin full-length gene coding sequence, a PCR primer pair capable of detecting the trichomonad beta-tubulin gene and the transcriptional level of the trichomonad beta-tubulin gene and corresponding PCR reaction conditions; and further, the kit for detecting the beta-tubulin gene of the trichomonad is assembled. According to the invention, the detection requirements of parasitic biology and life science researchers can be met, the gene level detection of the pathogen and the research progress of the pathogenic mechanism of the pathogen can be promoted, and favorable technical support is provided for the research work of gene function research, drug development and the like.

Description

Technical field [0001] The present invention relates to the field of molecular biological detection, and more particularly to a nucleic acid molecule, a PCR primer pair, and a kit for detecting a poultry aphid β-Tubulin gene. Background technique [0002] my country's raising pigeons have a long history, and the pigeons are also more than 100 years. At present, the pigeon pigeon breeding industry has spread all over my country, and the scale is constantly growing, and the effective prevention and control of pigeon infectious diseases is more important. Among them, the pigeon hairpin (also known as "pigeon") is also a high-risk disabled disease in the pigeon field, which has an important harmful to the production performance of meat pigeons and the survival rate of young birds. The most common change is the oral and throamer mucosa form a rough buttonned yellow chronicle; the moisture, referred to as a wet ulcer; a cheese or sputum shape is called dry ulcers. When the umbilical is...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/6888C12Q1/686C12Q2561/113C12Q2563/107C12Q2545/114Y02A50/30
Inventor 蔡海明
Owner INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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