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Nucleic acid molecule, PCR primer pair and kit for detecting poultry trichomonad TBP gene

A technology of nucleic acid molecules and primer pairs, which can be applied in the fields of plant gene improvement, genetic engineering, and microbial detection/inspection. It can solve the problems of lack of research reports on transcriptional regulatory signals and hinder the progress of research on transcriptional regulatory signals at the level of pathogenic genes.

Pending Publication Date: 2021-07-23
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, Trichomonas fowl is an important pathogenic parasite in meat pigeons, and the research reports on its transcriptional regulatory signals are extremely scarce, and the TBP gene has not been published in nucleic acid sequence databases such as NCBI and Uniprot, which hinders the gene level of the pathogen. Advances in detection and transcriptional regulatory signals

Method used

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  • Nucleic acid molecule, PCR primer pair and kit for detecting poultry trichomonad TBP gene
  • Nucleic acid molecule, PCR primer pair and kit for detecting poultry trichomonad TBP gene
  • Nucleic acid molecule, PCR primer pair and kit for detecting poultry trichomonad TBP gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Trichomonas fowl total cDNA preparation

[0061] Step (1): Extract Trichomonas fowl RNA

[0062] Take (1~5)×10 6 Trichomonas fowl were centrifuged at 8000g for 2 minutes, the culture medium was removed, 800 μL PBS was added to rinse and washed 3 times, and the supernatant was removed by centrifugation. Next, according to the operation of the Omega E.Z.N.A SE Total RNA plus kit I (R6836-01) kit, the RNA sample of Trichomonas avium was extracted.

[0063] Step (2): Preparation of Trichomonas fowl total cDNA by reverse transcription

[0064] Take 1.0 μg of the total RNA of Trichomonas fowl above, refer to TAKARA PrimeScript TM 1st Strand cDNASynthesis Kit (6110A) Reverse Transcription Kit Manual, prepare Trichomonas fowl cDNA and store it at -20°C for gene coding sequence cloning or fluorescence quantitative detection.

Embodiment 2

[0065]Example 2 Cloning of Trichomonas fowl TBP full-length coding gene sequence

[0066] Referring to the instruction manual of TOYOBO KOD FX, a PCR reaction system for cloning the full-length coding gene sequence of TBP of Trichomonas fowl was prepared in proportion, as shown in Table 1. Shake and mix well, and perform PCR reaction after brief centrifugation. The reaction conditions are 94°C for 2min; 98°C for 10s, 51.5°C for 30s, 68°C for 40s, 30 cycles; 68°C for 7min. The PCR product was subjected to nucleic acid electrophoresis, the gel was cut to recover a target band of about 729 bp, connected to the pMD18T vector, and sent to Sanfang Company for sequencing to obtain the full-length coding gene of Trichomonas fowl TBP, the sequence of which is shown in SEQ ID NO:1. The upstream primer sequence is: 5'-ATGGAAGCATCATCATTCTTTG-3', and the downstream primer sequence is: 5'-TTATGAGTTATATTTGCTGTCCTC-3'.

[0067] Table 1

[0068]

[0069]

Embodiment 3

[0070] Example 3 Trichomonas fowl TBP gene transcription level fluorescent quantitative PCR detection kit assembly

[0071] This kit consists of TB Green Premix Ex Taq (2×) (Tli RNaseH Plus), Trichomonas fowl TBP gene template, fluorescent quantitative primer mix and ultrapure water. The specific composition is shown in Table 2.

[0072] Table 2

[0073] TB Green Premix Ex Taq II(2X)(Tli RNaseH Plus) 5mL Fluorescence Quantitative Primer Mix 500μL Trichomonas fowl TBP gene template 500μL Ultra-pure water 5mL

[0074] In the kit, TB Green Premix Ex Taq II (Tli RNaseH Plus) (2X) was purchased from TaKaRa Company, the fluorescent quantitative primer mixture was upstream primer 8 μM and downstream primer 8 μM, the sequence of the upstream primer was 5’-GGGAGCCAAAAGCTACAGGT-3’, and the downstream primer The sequence is 5'-TCCGAGGATTTGAACGAGCA-3', and the ultrapure water is reverse osmosis water with a purity not 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 poultry trichomonad TBP gene. Through extensive research, the inventor clones in poultry trichomonad to obtain a poultry trichomonad TBP full-length gene coding sequence as shown in SEQ ID NO: 1, and further designs by utilizing the TBP full-length gene coding sequence to obtain a PCR primer pair capable of detecting the poultry trichomonad TBP gene and the transcriptional level of the poultry trichomonad TBP gene and corresponding PCR reaction conditions. Further, the kit for detecting the poultry trichomonad TBP gene is assembled, so that 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 transcriptional regulation signals of the pathogen are promoted, and favorable technical support is provided for the research work of gene function research, drug development and the like. The PCR primer pair is high in repeatability, strong in specificity and high in sensitivity, and does not have amplification signals for non-target genes.

Description

technical field [0001] The invention relates to the field of molecular biology detection, in particular to a nucleic acid molecule, a pair of PCR primers and a kit for detecting the TBP gene of Trichomonas fowl. Background technique [0002] my country has a long history of raising pigeons, and the production of pigeons as a commodity has a history of more than 100 years. At this stage, the breeding industry of meat pigeons has spread all over my country, and the scale continues to grow. The effective prevention and control of pigeon infectious diseases is becoming more and more important. Wherein, pigeon trichomoniasis (also known as "pigeon yellow") is also a consumptive parasitic disease with high incidence in pigeon farms, which is very harmful to the production performance of meat pigeons and the survival rate of young birds. The most common characteristic change is the formation of rough button-shaped yellow deposits on the mucous membranes of the mouth and throat; wet...

Claims

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Application Information

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IPC IPC(8): C12N15/30C12Q1/6893C12Q1/6851C12N15/11
CPCC07K14/44C12Q1/6893C12Q1/6851C12Q2600/158C12Q2531/113C12Q2521/107C12Q2563/107
Inventor 孙铭飞
Owner INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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