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Primers, probes and kit for on-site detection of a variety of serotype foot and mouth disease viruses

A foot-and-mouth disease virus, primer probe technology, applied in biochemical equipment and methods, microbe determination/testing, DNA/RNA fragments, etc., to achieve good specificity, high sensitivity, and fast detection speed

Active Publication Date: 2015-09-30
DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the current research on RPA technology is still in its infancy, and there is no report on the application of RPA technology to FMDV detection at home and abroad.

Method used

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  • Primers, probes and kit for on-site detection of a variety of serotype foot and mouth disease viruses
  • Primers, probes and kit for on-site detection of a variety of serotype foot and mouth disease viruses
  • Primers, probes and kit for on-site detection of a variety of serotype foot and mouth disease viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1: the preparation of positive standard plasmid

[0047] 1. Design and synthesis of primers

[0048] By comparing the sequences of the foot-and-mouth disease virus 2B gene in GeneBank, the conserved region was determined, and a pair of universal primers were designed, which are SEQ ID NO.4 and SEQ ID NO.5 in the sequence table, which can detect 7 serotypes of foot-and-mouth disease virus. PCR fragments were amplified for the construction of positive standard plasmids; all primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0049] 2. Preparation of foot-and-mouth disease virus cDNA

[0050] According to the instructions of the virus DNA / RNA extraction kit, extract the RNA of type O, type A, and Asia 1 foot-and-mouth disease virus respectively, and perform reverse transcription according to the instructions of the reverse transcription kit to obtain cDNA.

[0051] 3. Preparation of Positive Plasmids

[0052] Use the cDNA prepared by the abo...

Embodiment 2

[0054] Example 2: Optimization and Establishment of Foot-and-Mouth Disease Virus RPA Lateral Flow Chromatography Test Strip Detection Method

[0055] 1. Design of RPA primers and probes

[0056] The key to RPA amplification is the design of amplification primers and probes. There is little data on RPA primer and probe design, and there is currently no software or established principles for design. PCR primers are not suitable for RPA, and RPA primers are longer than general PCR primers, about 30-35 bases. Too short primers will reduce the recombination rate, affect the amplification speed and detection sensitivity; and the increase in the length of the primers will easily form secondary structures within and between the primers, and the increase in the length will also increase the difficulty of primer design and selection. Therefore, the primers used for RPA amplification need to be screened and optimized through a large number of experiments.

[0057] In general, the desi...

Embodiment 3

[0078] Embodiment 3: Sensitivity, repeatability, specificity detection of foot-and-mouth disease virus RPA lateral flow chromatography test strip detection method

[0079] 1. Sensitivity analysis and repeatability detection of RPA lateral flow chromatography test strip detection method

[0080] Set the titer to 10 7 TCID 50 After 10-fold serial dilution with PBS per 100 μL of foot-and-mouth disease virus liquid, the virus RNA was extracted using the kit, and the cDNA was reverse-transcribed; under optimized RPA conditions, RPA amplification was performed using the 2B primer-probe set: upstream and downstream primers each 2.0 μL (10μmol / L), probe 0.6μL (10μmol / L), buffer 29.5μL, template cDNA 1μL, use ddH 2 O 12.4 μL, add the above mixture into a 0.2mL TwistAmp nfo eight-way reaction tube containing lyophilized enzyme powder, and pipette repeatedly until it is completely dissolved. Finally, 2.5 μL of magnesium acetate (280 mmol / L) was added to start the reaction. 38°C for 4...

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Abstract

The invention discloses a universal primer and probe combination for detecting foot and mouth disease viruses through RPA (recombinase polymerase amplification)-lateral flow assay technology. The forward primer sequence is shown as SEQ ID No.1, the reverse primer sequence is shown as SEQ ID No.2, and the probe sequence is shown as SEQ ID No.3. The invention also discloses a kit for detecting foot and mouth disease viruses. Detection with the primers and probes involved in the invention only needs virus RNA extraction on clinical samples and isothermal amplification after one-step process reverse transcription into cDNA, thermal cycling reaction is not needed, amplification in a PCR instrument is unnecessary, and the result can be clearly displayed on a lateral flow chromatography test strip. The primer and probe combination and the kit provided by the invention have the advantages of high sensitivity, strong specificity, simple reaction procedure, short detection time and the like, and are suitable for rapid, accurate and simple detection of foot and mouth disease viruses on a cattle farm.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a primer, a probe for on-site detection of multiple serotypes of foot-and-mouth disease virus by using a recombinase polymerase amplification technique (Recombinase Polymerase Amplification, RPA) combined with a lateral flow chromatography technique (Lateral Flow Assay). Needles and their kits. Background technique [0002] Foot-and-mouth disease is an acute, febrile, and highly contagious infectious disease caused by foot-and-mouth disease virus (FMDV) that occurs on cloven-hoofed animals such as pigs, cattle, and sheep. It is listed as "Class A severe infectious disease" by the World Organization for Animal Health. In case of an outbreak, the infected animals must be culled and contacted with the infected animals, causing huge direct economic losses, and seriously endangering the healthy development of animal husbandry and the foreign trade of related products, which ha...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6844C12Q1/70C12Q2521/507C12Q2522/101C12Q2531/119C12Q2565/625
Inventor 王洪梅何洪彬
Owner DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI
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