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Primer probe group and reagent box for detecting rift valley fever virus with RAA (Recombinase Aided Amplification) fluorescence method

A Rift Valley fever virus, primer probe technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, microbial measurement / inspection, etc., can solve the problems of high false positive rate and high requirements for target genes, and achieve good specificity, The effect of high specificity and easy operation

Inactive Publication Date: 2018-10-09
浙江国际旅行卫生保健中心 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, RT-LAMP technology requires multiple pairs of primers and has high requirements for target genes, and the false positive rate is relatively high

Method used

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  • Primer probe group and reagent box for detecting rift valley fever virus with RAA (Recombinase Aided Amplification) fluorescence method
  • Primer probe group and reagent box for detecting rift valley fever virus with RAA (Recombinase Aided Amplification) fluorescence method
  • Primer probe group and reagent box for detecting rift valley fever virus with RAA (Recombinase Aided Amplification) fluorescence method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] The present invention extracts Rift Valley fever virus sample RNA for verification, and according to the gene name Rift Valley fever virus (Rift Valley fever virus, RVFV), finds the corresponding full gene sequence (www.ncbi.nlm.nih.gov) in genebank, uses DNASTAR The software performed homology analysis and b1ast sequence analysis, and screened out the highly conserved sequence of Rift Valley fever virus, as shown in SEQ ID NO.4: GTTGATGAGAGCCTCCACAGTTGCTTTGCCTTCTTTCGACATTTTCATCATCATCCTCCGGGGCTTGTTGCCACGAGTCAGAGCCAGAACAATCATTTTCTTGGCATCCTTCTCCCAGTCAGCCCACCATACTGCTTTAAGAGTTCGATAGACCCTACGAGCATCAACCACCTTGATAACCCTACGAGCATCAACCCTT.

[0045] According to the highly conserved sequence as the target gene for detection, the invention synthesizes the recombinant DNA plasmid of Rift Valley fever virus and designs primer probes.

[0046] According to the above sequence, Sangon Bioengineering (Shanghai) Co., Ltd. was entrusted to synthesize the recombinant DNA plasmid of Rift Valley ...

Embodiment 2

[0069] Sensitivity experiment

[0070] Upstream primers:

[0071]5'-CATTTTCATCATCATCCTCCKGGGCTTRTTG-3';

[0072] Downstream primers:

[0073] 5'-GARCTCYTAAAGCAGTATGGTGGGGCTGACT-3'.

[0074] The probe sequence is:

[0075] GGGAGAAGGATGCCAAGAAAATGATTGTTCTGGCTCTRACTCGTG

[0076] The fluorescent reporter group uses FAM, and the fluorescent quencher group uses BHQ1;

[0077] The modified probe is:

[0078] GGGAGAAGGATGCCAAGAAAATGATTGT(BHQ1-dT)(THF)(FAM-dT)GGCTCTRACTCGTG;

[0079] The composition of the kit is shown in Table 1:

[0080] Table 1 Kit composition list

[0081]

[0082] Rift Valley fever virus recombinant DNA plasmid positive standard prepared different concentrations of working standards, respectively:

[0083] Working standard (positive quality control) 1, containing 1.0×10 6 Copies / μL Rift Valley fever virus recombinant DNA plasmid.

[0084] Working standard (positive quality control) 2, containing 1.0×10 5 Copies / μL Rift Valley fever virus recombinant...

Embodiment approach

[0090] 1 Preparation of reaction buffer

[0091] Draw 301 μL of reaction buffer solution from the reaction buffer tube in the kit and add it to the pre-prepared 1.5mL PE tube, then add 16 μL of the mixture of probe and primer (the concentration of the probe is 0.02mmol / L, and the concentration of the primer is 0.05mmol / L ), and mix thoroughly to obtain the mixed reaction buffer.

[0092] 2 RAA fluorescent basic reaction reagent redissolution

[0093] Prepare 7 RAA fluorescent basic reaction reagents, draw 45 μL of the reaction buffer mixed in step 1 and add them to the prepared 7 RAA fluorescent basic reaction reagent tubes, so that the lyophilized powder is fully dissolved and mixed to form an RAA reaction system .

[0094] 3. Sample addition reaction

[0095] Add 5 μL of negative quality control, 5 μL of working standard 6, 5 μL of working standard 5, 5 μL of working standard 4, 5 μL of working standard 3, 5 μL of working standard into the above 7 prepared RAA fluorescent...

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Abstract

The invention relates to a primer probe group and a reagent box for detecting a rift valley fever virus with an RAA (Recombinase Aided Amplification) fluorescence method, and belongs to the technicalfield of biological detection. The primer probe group for detecting the rift valley fever virus with the RAA fluorescence method comprises an upstream primer, a downstream primer and a probe, whereinthe nucleotide sequence of the upstream primer is shown in SEQ ID NO. 1; the nucleotide sequence of the downstream primer is shown in SEQ ID NO. 2; the probe is a substance with the nucleotide sequence shown in SEQ ID NO. 3, wherein the 28th basic group is a modified fluorescence reporter group, the 29th basic group is replaced with a tetrahydrofuran residue, and the 30th basic group is a modifiedquenching group. The primer probe group provided by the invention is quick, accurate and sensitive in detection and is convenient in operation, specificity is 100%, and the detection of the rift valley fever virus can be finished within 15 min.

Description

technical field [0001] The invention relates to the technical field of molecular biology detection, in particular to a primer probe group and a kit for detecting Rift Valley fever virus by RAA fluorescence method. Background technique [0002] Rift Valley fever (Rift Valley fever, RVF) is a zoonotic disease caused by Rift Valley fever virus (RVFV). It is a febrile and acute infectious disease transmitted by arthropods, mainly through mosquitoes. The main hosts are sheep, goats and cattle. The epidemic is characterized by rapid abortion in pregnant animals and high mortality in young animals, accompanied by morbidity and death in the population. The disease is endemic in Africa and the Arabian Peninsula. The epidemic is often accompanied by cycles of heavy rainfall. It occurs less frequently in semi-arid areas or more frequently in grassland areas with relatively high rainfall. The lesion is characterized by liver damage. WHO has listed it as one of the biological warfare ag...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2531/119C12Q2563/107C12Q2545/113
Inventor 郑伟吴忠华麻慧君黄雷杨永耀郭利川汤赛君王智宏应清界
Owner 浙江国际旅行卫生保健中心
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