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Detecting method of blue tongue virus and reagent box

A detection method, bluetongue virus technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as undetectable BTV, inapplicable bluetongue disease, and abnormal proliferation

Inactive Publication Date: 2004-12-22
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, BTV that is present at low levels in samples or that does not proliferate normally in tissue culture or in animals cannot be detected by this method.
Therefore, nucleic acid probe technology is not suitable for the quarantine of bluetongue virus, and can only be used as a laboratory identification method for bluetongue virus

Method used

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  • Detecting method of blue tongue virus and reagent box
  • Detecting method of blue tongue virus and reagent box
  • Detecting method of blue tongue virus and reagent box

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0111] The preparation of embodiment 1 positive control

[0112] A. Ligation reaction

[0113] The RT-PCR dsDNA product low melting point agarose of BTV-10S75'-NCR fragment was recovered,

[0114] For ligation reaction with pUCm-T carrier, add to the reaction system with a total volume of 10μl

[0115] T4 DNA ligase 1.0μl

[0116] S7PCR recovery product 1.0μl

[0117] PEG4000 1.0μl

[0118] pUCm-T vector 1.0μl

[0119] Sterile water 6.0μl

[0120] Mix well, put in 16℃ water bath for 2hr

[0121] B. Recombinant pUCm-T-BTV-S7 plasmid transforms E. coli TG1 strain and amplifies it

[0122] (A) Preparation of Competent Cells:

[0123] (1) Inoculate 20 μl of Escherichia coli TG1 strain preserved in 25% glycerol into 20 ml (1:1000) of LB liquid medium containing Amp (50 μg / ml), culture overnight at 37° C. on a shaker at 200 rpm.

[0124] (2) On the second day, absorb 1 ml of the bacterial culture from the cultured bacterial liquid into 20 ml of LB liquid medium, shak...

Embodiment 2

[0159] The preparation of various solutions of embodiment 2

[0160] tool enzyme

[0161] M-MLV reverse transcriptase, BamHI and HindIII are products of Promega Company.

[0162] Taq DNA polymerase is a product of Sangon Bioengineering Company.

[0163] RNAsin is a product of Huamei Bioengineering Company.

[0164] main chemical

[0165] DEPC is a product of GIBCO.

[0166] Agarose was a product of Huamei Bioengineering Company.

[0167] Low-melting point agarose was a product of Promega.

[0168] The pUCm-T vector is a product of Promega.

[0169] TG1 engineered Escherichia coli is a product of Sigma.

[0170] Reagents and solutions for RNA extraction

[0171] Lysis solution formula: 6mol / L guanidine isothiocyanate, 7.5mol / L sodium citrate (pH7.0), 0.75% (W / V) sodium dodecylsulfonate, 0.15mol / L β-mercaptoethanol (2 -ME)

[0172] Water-saturated phenol: saturated with DEPC-treated double-distilled water (pH4.0)

[0173] 3mol / LNaAc(pH4.0)

[0174] Chloroform: isoamy...

Embodiment 3

[0180] The composition of embodiment 3 detection reagents

[0181] Sample treatment solution I: PBS solution

[0182] Sample treatment solution II: 6mol / l guanidine isothiocyanate, 7.5mol / l sodium citrate (pH7.0), 0.75% (W / V) sodium dodecylsulfonate, 0.15mol / L β-mercaptoethanol ( 2-ME)

[0183] Sample treatment solution III: phenol: chloroform: isoamyl alcohol (25:24:1)

[0184] Sample Treatment Solution IV: Isopropanol

[0185] Sample treatment solution V: 3mol / L NaAc

[0186] Sample treatment solution VI: 75% ethanol

[0187] Sample treatment solution VII: DEPC treated double distilled water

[0188] Reverse transcription reaction solution I: 25 μmol / L sense primer P1 (5′-AGC CAT ATG TTG AGT ATT-3′) and 25 μmol / L antisense primer P2 (5′-TAG AGA TGG ACA CTATGG C-3′), coke Double distilled water treated with diethyl carbonate.

[0189] Reverse transcription reaction solution II: 10× reverse transcription buffer, 10mmol / L dNTP, 200u / μl M-MLV reverse transcriptase.

[01...

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Abstract

A method and reagent kit for detecting blue tongue virus are disclosed. Said method includes culturing cells while reproducing viruses, adding specimen-treating liquid in test tubes to prepare the PCR templates of specimen to be detected and negative and positive references, respectively adding the specimen template of the virus to be detected and reverse transcription reaction solution to reaction test tubes for synthesizing the cDNA of specimen dsRNA, adding PCR reaction system, and PCR amplification to synthesize target sequence.

Description

technical field [0001] The invention relates to a detection method for bluetongue virus, and meanwhile, the invention also relates to a detection kit for bluetongue virus. Background technique [0002] my country is a big country for raising sheep and cattle. Cattle and sheep are extremely important economic animals, and cattle are also the agricultural productivity of our country. Bluetongue virus seriously harms and restricts the development of sheep breeding and the raising of cattle for agricultural production in my country. my country has always believed that there is no epidemic of this virus, and it was only around 1980 that bluetongue disease caused by this virus was discovered. Afterwards, the disease spread rapidly, forming a nationwide animal epidemic and causing significant economic losses. Therefore, since the early 1980s, the Chinese government has attached great importance to the prevention and treatment of bluetongue. "China Import and Ex...

Claims

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Application Information

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IPC IPC(8): C12Q1/04C12Q1/68
Inventor 董长垣桂亦瑞张芄玮陈冬峨刘军
Owner WUHAN UNIV