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Primers and probes for detecting dengue virus

A dengue virus and probe technology, which is applied in the field of primers and probes for detecting dengue virus, can solve the problems of untimely detection, insufficient sensitivity, time-consuming and cumbersome, etc.

Pending Publication Date: 2021-06-04
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Traditional isolation culture and serological detection methods are time-consuming and cumbersome, resulting in untimely detection and insufficient sensitivity. Therefore, it is urgent to develop new technologies for rapid and accurate detection to screen suspicious cases, control the spread of the epidemic, and give timely symptomatic treatment

Method used

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  • Primers and probes for detecting dengue virus
  • Primers and probes for detecting dengue virus
  • Primers and probes for detecting dengue virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] The design of embodiment 1 primer and probe

[0081] 1. Experimental method

[0082] By comparing and analyzing the gene sequences of all known dengue viruses, select highly conserved segments, and design multiple pairs of primers and probes. The length of the primers is generally about 20 bases.

[0083] Analyze the information of the above-mentioned virus-specific genes, and use the sequence analysis software DNASTAR to exclude inter-primer / inner dimers, and verify the specificity of the primers and the homology of similar pathogens by BLAST, and design primers and primers for the detection of the above-mentioned pathogens. probe.

[0084] 2. Experimental results

[0085] The optimal primer-probe sequence combination is as follows:

[0086] Set 1 Primers and Probes for Detection and Characterization of Dengue Virus Type 1 (DENV1),

[0087] The nucleotide sequence of the upstream primer is shown in SEQ ID NO: 1: CAAAAGGAAGTCGYGCAATA,

[0088] The nucleotide sequen...

Embodiment 2

[0102] Embodiment 2 A kind of detection kit that detects dengue virus

[0103] 1. Composition

[0104] Nucleotide sequence such as the primer shown in SEQ ID NO: 1 to 8, the nucleotide sequence such as shown in SEQ ID NO: 9 to 12, reverse transcription reaction reagent ( II Q Select RT SuperMix for qPCR:), fluorescence quantitative PCR reagent (Vazyme 2×AceQ Mix)

[0105] Specifically, primers and probes include:

[0106] The nucleotide sequence of the upstream primer for detecting and identifying dengue virus type 1 (DENV1) is shown in SEQ ID NO: 1: CAAAAGGAAGTCGYGCAATA,

[0107] The nucleotide sequence of the downstream primer for detecting and identifying dengue virus type 1 (DENV1) is shown in SEQ ID NO: 2: CTGAGTGAATTCTCTCTGCTRAAC,

[0108] The nucleotide sequence of the probe for detecting and identifying dengue virus type 1 (DENV1) is shown in SEQ ID NO: 9, its 5' end is marked with a fluorescent marker group FAM, and its 3' end is marked with a fluorescent marker g...

Embodiment 3

[0145] Embodiment 3 is to the detection of positive sample

[0146] 1. Experimental method

[0147] The kit in Example 2 was used to detect Aedes albopictus infected with dengue virus type 1-4 strains, all of which were provided by our laboratory. At the same time, virus titer titration (TCID50 / ml) was carried out. TCID50 is the half infectious dose of tissue cell culture, and each group of virus strains infects Aedes albopictus with a virus amount of 1TCID50 / ml.

[0148] Use one Aedes albopictus infected with dengue virus types 1-4 as the sample to be tested, and extract the total RNA. According to the measured concentration, all the RNA is diluted with RNase-free water to a final concentration of 1 μg / μl.

[0149] Using the kit of Example 2, the detection and result determination were performed according to the method used in Example 2.

[0150] 2. Experimental results

[0151] see results Figure 1 to Figure 4, suggesting that the above primers and probes have good sp...

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Abstract

The invention discloses a group of primers and probes for detecting a dengue virus. Single-tube fluorescent PCR is established to detect four types of dengue viruses at the same time, and can be used for clinical specimen detection. According to the invention, multiple PCR is combined with a Tagman fluorescent probe technology, multiple pathogens which are possibly caused are simultaneously detected in one detection process, and the purposes of rapid diagnosis and treatment guidance are achieved, so that the limitation of conventional PCR is overcome, the bottleneck problem of clinical detection flux is solved, the working time is shortened, the raw material loss is reduced, and the primers and probes have the characteristics of higher sensitivity and specificity, capability of effectively solving the problems of PCR pollution and the like, higher automation degree and the like. A fluorescent probe quantitative PCR detection technology ingeniously utilizes the advantages of efficient DNA amplification of the PCR technology, high specificity of the probe technology and sensitivity and quantitative analysis of the spectrum technology. Besides, the defects that the traditional PCR technology is easy to pollute, high in false positive rate and incapable of quantifying are overcome.

Description

technical field [0001] The invention relates to the technical field of virus detection, in particular to a set of primers and probes for detecting dengue virus. Background technique [0002] Dengue virus (dengue virus, DENV) is a single-stranded positive-sense RNA virus of the genus Flavivirus, about 11kb in length, and is divided into four serotypes: type I, type II, type III and type IV according to the antigenicity of the E protein . Dengue virus is transmitted by Aedes aegypti and Aedes albopictus. Aedes is an insect of the genus Aedes in the Culex family. Aedes is a small to medium-sized black mosquito species with silvery white markings. There is a central white longitudinal stripe on the mid-thorax scutellum, which extends from the front end to the front of the small scutellum at the level of the wing base and forks. The 1st to 4th segments of the posterior tarsus have base white rings, and the distal segments are all white. There are basal leucorrhea in 2-6 segme...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6851C12Q2600/16C12Q2531/113C12Q2537/143C12Q2563/107C12Q2561/101
Inventor 黎孟枫李隽朱勋何振健吴珏珩谭姹辉陈德林林翠姬谭泳谣曹开源袁洁蔡俊超
Owner SUN YAT SEN UNIV
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