A degenerate primer RT-PCR detection method for Eimeria virus

A technology of RT-PCR and degenerate primers, which is applied in the direction of biochemical equipment and methods, and the determination/inspection of microorganisms. Effect

Inactive Publication Date: 2019-08-06
JILIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to disclose a kind of degenerate primer RT-PCR detection method that is used for Eimeria coccidia virus, is degenerate primer RT-PCR detection method, solves the problem that there is still no detection of Eimeria genus virus at present. The difficulty of the method, the method uses the low variation characteristics of the highly conserved region, uses degenerate primers to detect Eimeria virus, achieves the combination of specificity and versatility, and provides a new method for the detection of Eimeria virus new viable approach

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  • A degenerate primer RT-PCR detection method for Eimeria virus
  • A degenerate primer RT-PCR detection method for Eimeria virus
  • A degenerate primer RT-PCR detection method for Eimeria virus

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Primer design and synthesis

[0027] 1. Materials: Molecular biology software MEGA4, DNAMAN and NCBI network resources.

[0028] 2. Methods and results:

[0029] Sequence obtained: through Eimeria brucei virus ( E. brunetti RNA virus 1) Genome analysis, select the coat gene as the target sequence, and obtain the gene sequence from the GenBank public database, the accession number is: NC_002701. Use the coat protein sequence as the query sequence, and perform BlustP alignment on NCBI to obtain two highly conserved protein regions from 330 to 346 (IALGTAAAVAHCDPLVP) and 435 to 446 (PFFWIEPTTVLP), and compare the nucleic acid sequences of the corresponding positions of the rest of the reference sequence Separate downloads yielded Gremmeniella abietina RNA virus L2, Gremmeniella abietina RNA virus L1, Helicobasidium mompa totivirus 1-17, Helminthosporium victoriae virus, Beauveria bassiana RNA virus 1, Tolypocladium cylindrosporum virus 1, Aspergillus foetidus slowman vi...

Embodiment 2

[0035] Nucleic acid sample preparation

[0036] 1. Materials: Eimeria tenella, 2×STE buffer (Ph8.0), water-saturated phenol, chloroform, β-mercaptoethanol, 10% SDS, 70% ethanol, isopropanol, high-speed centrifuge .

[0037] 2. Methods and results:

[0038] 1) Broken Eimeria tenella oocysts: about 10 7 The coccidia to be tested were ground with liquid nitrogen, and dissolved in 1ml of 2×STE (pH8.0) buffer after grinding.

[0039] 2) Protein extraction: Add 30 μl of β-mercaptoethanol, 0.6 ml of water-saturated phenol, 0.4 ml of chloroform, and 140 μl of 10% SDS to the above coccidia solution. Thoroughly homogenize for 5 minutes. Centrifuge at 12000r / min for 5 minutes at 4°C, and take the supernatant.

[0040] 3) Acquisition of total nucleic acid: mix the supernatant with an equal volume of cold isopropanol, let stand at -80°C for 10 minutes, centrifuge at 15,000r / min for 15 minutes at 4°C, wash the pellet with 70% cold ethanol solution, and store at 4°C Under conditions, c...

Embodiment 3

[0044] Detection of Eimeria tenella virus by RT-PCR with degenerate primers

[0045] 1. Materials: Eimeria tenella RNA crude extract, PrimeScript™ Reverse Transcriptase (2680Q) reverse transcriptase kit, TaKaRa LA Taq (RRO2MQ) nucleic acid polymerase kit, agarose, ethidium bromide solution, Biorad PCR instrument.

[0046] 2. Methods and results:

[0047] 1) 1st-Stand cDNA synthesis: Prepare the following mixture in Microtube:

[0048]

[0049] Denature at 99°C for 1 minute, and immediately transfer to -40°C for quick freezing.

[0050] The amount of template RNA was used for gradient experiments, which were divided into six gradients, 50ng / μl, 5ng / μl, 0.5ng / μl, 0.05ng / μl, 0.005ng / μl, 0.0005ng / μl. When the template RNA concentration is 0.05ng / μl, PCR results can still be clearly interpreted. (see attached figure 2 )

[0051] Add the following components in the above reaction solution:

[0052]

[0053] Reaction at 30°C for 10 minutes, reaction at 42°C for 45 minut...

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Abstract

The invention discloses a degenerate primer RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection method for eimeria viruses. A pair of brand-new degenerate primers is provided, and specific degenerate primer RT-PCR amplification is performed on the eimeria viruses by using reverse transcription and touchdown PCR techniques. The degenerate primer RT-PCR detection method is capable of effectively detecting the existence of an eimeria tenella virus and an eimeria stiedae virus, and good in specificity (an electrophoretic band is single). The degenerate primer RT-PCR detection method is used for detecting the eimeria viruses for the first time, and is capable of quickly and accurately identifying whether a coccidium sample to be detected contains a virus-carrying strain or not.

Description

technical field [0001] The invention discloses a degenerate primer RT-PCR detection method for Eimeria coccidia virus, provides a pair of brand-new degenerate primers, and uses reverse transcription and landing PCR technology to detect Eimeria coccidia virus Perform RT-PCR amplification with specific degenerate primers. The applied technologies are degenerate primer design, reverse transcription, and touch-down PCR (TD-PCR), belonging to the field of molecular biology detection technology. Background technique [0002] Chicken coccidiosis (Coccidiosis) is caused by one or more Eimeria coccidia, and the damage is very serious parasitic disease. As an intracellular parasite, Eimeria causes enormous losses to the global poultry industry, estimated at $8 billion annually. Double-stranded RNA viruses were first discovered in fungi in 1948, and dsRNA viruses were later discovered in protozoa. Protozoa virus is a kind of virus particles existing in protozoa, most of which are do...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/686
CPCC12Q1/686C12Q1/70C12Q2531/113C12Q2521/107C12Q2521/101
Inventor 李建华武斌张西臣宫鹏涛信彩岩张国才杨举李赫杨正涛
Owner JILIN UNIV
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