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Primer and probe for direct fluorescence RT-PCR detection on typing of foot-and-mouth disease viruses and kit

A technology of RT-PCR and foot-and-mouth disease virus, applied in the field of primers, probes and kits for direct fluorescent RT-PCR detection of foot-and-mouth disease virus typing, can solve the problems of not finding primers and probes, shorten the detection time, improve The effect of improving detection efficiency and detection speed

Inactive Publication Date: 2016-07-13
深圳澳东检验检测科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the fact that primers and probes are easily affected by complex factors in samples, no primers and probes that can be used in this way have been found yet.

Method used

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  • Primer and probe for direct fluorescence RT-PCR detection on typing of foot-and-mouth disease viruses and kit
  • Primer and probe for direct fluorescence RT-PCR detection on typing of foot-and-mouth disease viruses and kit
  • Primer and probe for direct fluorescence RT-PCR detection on typing of foot-and-mouth disease viruses and kit

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0045] Experimental Example 1 is used for the detection test of clinical samples

[0046] 1. Sample processing:

[0047] For serum samples, take a certain amount of sample and add 3 times the volume of methanol, mix it, let it stand for five minutes, and centrifuge at 3000r / min for 15min, and take the supernatant for later use; Centrifuge for 10 min, and take the supernatant for later use.

[0048] 2. Reagent preparation (Table 2):

[0049] (1) The direct RT-PCR reaction solution includes: PCR buffer and PCR enhancer. Wherein the PCR buffer includes: Tris-HCl (pH8.3), ammonium sulfate, potassium chloride, TritonX-100, magnesium chloride and deoxyribonucleotide triphosphates (dNTPs), wherein the concentration of Tris-HCl in the PCR buffer is 35.6mmol / L, the concentration of ammonium sulfate in PCR buffer is 15mmol / L, the concentration of potassium chloride in PCR buffer is 47.22mmol / L, the volume ratio of TritonX-100 and PCR buffer is 0.43, magnesium chloride The concentrat...

Embodiment 2

[0060] The analysis sensitivity comparison of embodiment 2 direct fluorescent RT-PCR and single conventional PCR

[0061] Type A, O type, Asian type 1 inactivated standard product strain cell culture positive sample of foot-and-mouth disease virus is diluted to the nucleic acid concentration of 0.001LD50; Carry out typing diagnosis according to step 2, step 4 in embodiment 1 direct fluorescence RT -PCR and single-fold conventional PCR amplification detection, each concentration was repeated 40 times, and the results are shown in Table 4.

[0062] Table 4 Analytical Sensitivity Results

[0063]

[0064] As can be seen from the results shown in table 4, the analytical sensitivity of direct fluorescent RT-PCR of the present invention detects foot-and-mouth disease virus is compared with the detection sensitivity of conventional reagent single-fold RT-PCR with purified RNA as template, and this result illustrates direct fluorescent RT-PCR The analytical sensitivity of the meth...

Embodiment 3

[0065] The specificity of embodiment 3 direct fluorescence RT-PCR detection kit

[0066] The samples of type A, type O, and type 1 of foot-and-mouth disease virus added to the direct fluorescent RT-PCR kit of foot-and-mouth disease typing were amplified, and the specificity of the kit was tested according to the amplification curves of different fluorescent labels. from figure 1 , 2 , 3 It can be seen that there is no cross-reaction in the direct PT-PCR specific detection of type A, O and Asian type I FMD. The above results show that this kit has good specificity to the direct fluorescent RT-PCR detection kit for FMDV typing.

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Abstract

The invention discloses a primer and probe for direct fluorescence RT-PCR detection of typing on foot-and-mouth disease viruses and a kit. The primer and the probe comprise universal primers and probes, wherein the universal primer are used for detecting the foot-and-mouth disease viruses, and the base sequences of the universal primers are represented by SEQ ID NO:1 and SEQ ID NO:2; and the probes are used for detecting the foot-and-mouth disease viruses, the base sequences of the probes are represented by SEQ ID NO:3-5, 5' terminals of the probe sequences are marked with fluorescence report groups, and 3' terminals of the probe sequences are marked with fluorescence quenching groups. By adopting the primer and the probe or the kit and combining other conventional reagent components, the release, the inverse transcription and the fluorescence quantitative PCR detection of RNA of the viruses can be continuously and automatically carried out in one PCR tube, a tedious step of purifying RNA is omitted, the operation steps are reduced, the foot-and-mouth disease viruses can be rapidly and accurately detected, the detection time is shortened, the detection efficiency is improved, and the cost is saved.

Description

technical field [0001] The invention relates to the technical field of virus detection, in particular to a primer, a probe and a kit for direct fluorescent RT-PCR detection of foot-and-mouth disease virus typing. Background technique [0002] Foot-and-mouth disease virus (Foot-and-MouthDiseaseVirus, FMDV) is the pathogen of foot-and-mouth disease (Foot-and-MouthDisease, FMD), this virus can infect pigs, cattle, sheep and other artiodactyls and cause acute, severe, contact infectious diseases. Livestock infected with foot-and-mouth disease virus will cause a decrease in food intake, lower body resistance and immunity, and other viral and bacterial diseases will easily occur, resulting in a decline in the production performance of infected animals and endangering the normal production and development of animal husbandry. The World Organization for Animal Health (OIE) lists foot-and-mouth disease as an animal infectious disease that must be reported, and my country classifies i...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/701C12Q1/686C12Q2600/172C12Q2563/107C12Q2547/101C12Q2521/101
Inventor 翟建新潘艳萍吴晓卫钟文杨张利
Owner 深圳澳东检验检测科技有限公司
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