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Real-time fluorescence PCR primer and probe for Asia-Europe type foot-and-mouth disease virus detection

A foot-and-mouth disease virus and real-time fluorescence technology, which is applied in fluorescence/phosphorescence, biochemical equipment and methods, and microbial measurement/inspection. Good specificity, strong versatility, and high-efficiency amplification

Inactive Publication Date: 2010-06-09
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, studies have shown that the seven serotypes of FMDV, namely O, A, C, Asia 1 (Asia1), South Africa 1, 2, and 3 (SAT1, SAT2, SAT3) can be divided into two groups by nucleic acid hybridization, O, A, and C Type 1 and Asia are Eurasian FMDV, and South Africa 1, 2, and 3 are South African FMDV. The nucleic acid homology of each type in the group is 60%-70%, but only 25%-40% between the two groups ( Lu Zengjun, 2003), so it is still difficult to design fluorescent RT-PCR primers and probes with high specificity for Eurasian and South African types

Method used

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  • Real-time fluorescence PCR primer and probe for Asia-Europe type foot-and-mouth disease virus detection
  • Real-time fluorescence PCR primer and probe for Asia-Europe type foot-and-mouth disease virus detection
  • Real-time fluorescence PCR primer and probe for Asia-Europe type foot-and-mouth disease virus detection

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Experimental program
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Effect test

Embodiment 1

[0023] The preparation of embodiment 1 primer and probe

[0024] 1 Design and synthesis of primers and probes

[0025]The genome sequence of foot-and-mouth disease virus (FMDV) was obtained from GenBank, and MegAlign software was used to compare and analyze the above-mentioned sequences, select a relatively conservative 5'-UTR (untranslated region) sequence, and according to the design principles of primers and probes, use The software BeaconDesigner 7.0 screened a pair of Eurasian foot-and-mouth disease virus universal primers with common base sequences in the conserved regions of these sequences. The primer pair was determined to be Eurasian FMDV (including A, O, C and Asia Type 1) is different from the specific primer pair of South African type FMDV (including SAT-1, SAT-2, SAT-3 type). And set a universal fluorescent TaqMan probe in the amplification region of the primer pair.

[0026] The primer sequences are:

[0027] FMDVU6 (forward): 5'-TACCACYTTCCGCCTACTTG-3'

[0...

Embodiment 2

[0034] The establishment of embodiment 2 Asia-Europe type foot-and-mouth disease virus fluorescent PCR detection method

[0035] 1 Extraction of viral RNA

[0036] Take 100 μL of inactivated cytotoxicity, add 1 mL of Trizol solution, extract total RNA according to the operation manual of Trizol, dissolve it with 20 μL of DEPC-treated sterile double-distilled water, and store it in a -86°C refrigerator for later use.

[0037] 2 One-step amplification of the target gene

[0038] The primers FMDVU6 and FMDVL6 prepared in Example 1 were used to perform RT-PCR amplification on the extracted RNA. The RT-PCR reaction system was carried out according to the instructions of One Step RNA PCRKit. Amplified on a PTC-200 gradient PCR instrument (MJ), the reaction program was: reverse transcription at 50°C for 30 minutes, pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 30 seconds, and amplification of 35 ...

Embodiment 3

[0051] Application of embodiment 3 Eurasian foot-and-mouth disease virus fluorescent PCR detection method

[0052] 1 Extraction of RNA from tissue disease materials

[0053] In a sterile environment, put the collected animal body tissues (such as tongue, nose, and hoof blister skin) into a mortar, cut them into pieces, and grind them with sterilized quartz sand. For other body tissues (such as lymph nodes, tonsils, etc.), remove the capsule and other connective tissues, select the inner substantial part, put it in a mortar, cut it into pieces, and add sterilized quartz sand to grind. Add 0.01mol / L PBS (pH7.6-7.8) or MEM (pH7.6-7.8) to make a 1:5 suspension. Freeze and thaw twice at -20°C to -30°C, centrifuge at 3000r / min for 10min, and take the supernatant to extract total RNA. Liquid samples, such as vesicle fluid and OP fluid, are directly used to extract total RNA. While extracting RNA from the combined disease material, cell-cultured Eurasian FMD virus should be set up ...

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Abstract

The invention provides a real-time fluorescence PCR primer and probe for Asia-Europe type foot-and-mouth disease virus detection, wherein the primer pair can specifically amplify the conservative region of foot-and-mouth disease virus gene group 5'-UTR sequence, the specificity of the probe directs at the GC high content region in the amplification region of the primer pair, the 5' end of the probe is provided with a report fluorescence dye mark, and the 3' end is provided with a quenching dye mark. The invention makes the best of high efficiency amplification of the fluorescence PCR technology, favourable specificity of the nucleotide hybridization and rapid sensitivity of the fluorescence detection technology, and whether the tissue sample to be detected contains Asia-Europe type foot-and-mouth disease virus is judged according to amplification curve after reaction is finished. The gene group region directed by PCR amplification is Asia-Europe type foot-and-mouth disease virus specified sequence and has no cross reaction with South Africa type foot-and-mouth disease virus, and universality is stronger while favourable specificity is obtained.

Description

technical field [0001] The invention belongs to the field of epidemiology and food sanitation detection, specifically, the present invention relates to real-time fluorescent PCR primers and probes for the detection of Eurasian foot-and-mouth disease virus, and a method for detection thereof, more specifically, General real-time fluorescent PCR primers and probes for detecting whether A, O, C and Asia 1 foot-and-mouth disease viruses are carried in artiodactyl animals such as pigs, cattle, sheep and related animal products, and a method for using them for detection. Background technique [0002] Foot-and-mouth disease (FMD) is an acute, febrile, high-contact infectious disease common to cloven-hoofed animals caused by foot-and-mouth disease virus (FMDV) infection (Jiang Pengfei et al., 1999) , is also a serious "political and economic disease", listed as the first class A infectious disease stipulated by the OIE, and is also an important quarantine object in the international...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11G01N21/64
Inventor 林祥梅吴绍强韩雪清梅琳
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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