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Nucleotide sequence and detection kit for detecting bovine enterovirus and foot-and-mouth disease virus

A technology of foot-and-mouth disease virus and nucleotide sequence, applied in the field of nucleotide sequence and detection kits for detecting bovine enterovirus and foot-and-mouth disease virus

Inactive Publication Date: 2011-09-14
PEOPLES REPUBLIC OF CHINA BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, after each outbreak, only the slaughter and collective burning of infected livestock is necessary to prevent future infections.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Embodiment 1, preparation and use of kit

[0096] 1. The composition of kit A is shown in Table 3. Wherein, the formula of the RT-PCR reaction solution is shown in Table 1.

[0097] Table 3 Kit preparation composition

[0098] Composition (48 tests / box)

quantity

lysate

5.0mL×6 tubes

RT-PCR reaction solution

900μL×1 tube

Taq DNA polymerase (5U / μL)

240μL×1 tube

Reverse transcriptase M-MLV (200U / μl)

250μL×1 tube

DEPC water

1mL×3 tubes

negative control

1mL×5 tubes

[0099] Bovine enterovirus positive control

1mL×5 tubes

Foot and Mouth Disease Virus Positive Control

1mL×5 tubes

[0100] 2. How to use kit A

[0101] 2.1 RNA extraction:

[0102] Take n 1.5ml sterilized centrifuge tubes (n = number of samples + 1 tube of negative control + 1 tube of positive control) and mark them. First add 600 μL of lysate (the lysate is highly corrosive, do not get it on th...

Embodiment 2

[0133] Embodiment 2, the sensitivity test of kit

[0134] 1. Materials:

[0135] Methods The virus strains used in the research process were bovine enterovirus and foot-and-mouth disease virus isolated and preserved in our laboratory.

[0136] 2. Method

[0137] Bovine enterovirus and foot-and-mouth disease virus cell cultures were used as 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 、10 -9 、10 -10 The multiple dilutions were carried out for multiple RT-PCR detection, and at the same time, the virus of each dilution was inoculated into BK cells, and the sensitivity of the two methods was compared.

[0138] 3. Results

[0139] The cell cultures of bovine enterovirus and foot-and-mouth disease virus were serially diluted by 10 times, and the assembled kits A and B were used for detection respectively. The test results showed that the detection limit could reach 10 -6 , which is equivalent to cell separation and culture identification, but the detection tim...

Embodiment 3

[0140] Embodiment 3, the specificity test of kit

[0141] 1. Materials

[0142] Table 5 Virus strains used in method research

[0143] Virus

source

bovine enterovirus

The lab saves

bovine infectious rhinotracheitis virus

The lab saves

bovine viral diarrhea virus

The lab saves

foot and mouth disease virus

The lab saves

Vesicular stomatitis virus

The lab saves

[0144] 2. Method

[0145] Use the established RT-PCR method to detect a variety of common cattle viruses (including bovine enterovirus, bovine infectious rhinotracheitis virus, bovine viral diarrhea virus, foot-and-mouth disease virus, vesicular stomatitis virus) to verify the method specificity.

[0146] 3. Results

[0147] The results showed that no matter whether kit A or kit B was used, the established method had no cross-reaction with the above-mentioned viruses, and the specificity was good.

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PUM

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Abstract

The invention discloses a nucleotide sequence for detecting bovine enterovirus and foot-and-mouth disease virus, and the nucleotide sequence for detecting the bovine enterovirus and the foot-and-mouth disease virus is as shown in SEQ (sequence) ID (identity) NO: 1 to SEQ ID NO: 4 in a sequence table, wherein the SEQ ID NO: 1 and the SEQ ID NO: 2 are a sense primer and an antisense primer for detecting the bovine enterovirus respectively; and the SEQ ID NO: 3 and the SEQ ID NO: 4 are the sense primer and the antisense primer for detecting the foot-and-mouth disease virus respectively. The invention further discloses a kit containing the primers for detecting the bovine enterovirus and the foot-and-mouth disease virus. The kit has the characteristics of quickness, sensitiveness, specificity, stability and good repeatability.

Description

technical field [0001] The invention relates to a nucleotide sequence and a detection kit for detecting bovine enterovirus and foot-and-mouth disease virus, especially a detection nucleotide sequence and a kit for multiplex RT-PCR detection of bovine enterovirus and foot-and-mouth disease virus, not only applicable to animals The accurate detection of tissue samples can also be applied to the detection of living animal samples (throat swabs, anal swabs, feces, serum, herpes skin, herpes fluid, conjunctival secretions, etc.), and can be used for domestic screening of bovine enteroviruses. Inspection and entry-exit quarantine, as well as simultaneous detection and differential diagnosis of bovine enterovirus and foot-and-mouth disease virus, etc., belong to the field of animal disease detection, inspection and quarantine. Background technique [0002] Both bovine enterovirus and foot-and-mouth disease virus are members of the Picornaviridae family. Picornavirus is an extremel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 吴丹张鹤晓吴涛高志强周琦刘来福蒲静张伟汪琳谷强张向东乔彩霞柏亚铎段向英齐玮郭铮蕾
Owner PEOPLES REPUBLIC OF CHINA BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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