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Primers, probes and methods for detecting dengue virus and its typing

A technology of dengue virus and dengue, which is applied in the field of biochemistry and biological detection, can solve the problems of inability to realize early diagnosis of cases, complex detection condition setting, low specificity and sensitivity, and achieve fast detection speed, stable results, The effect of rapid typing test

Inactive Publication Date: 2016-04-27
河北国际旅行卫生保健中心
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

However, these methods have more or less defects: virus isolation not only has strict requirements on samples, but also has high technical requirements, and it takes at least one week to obtain the results of etiological diagnosis, which cannot achieve the purpose of early diagnosis of cases.
However, the existing dengue virus in vitro detection kits or the provided primers and probes have low specificity and sensitivity, or there are false positive results, or the detection steps are cumbersome and the detection conditions are complicated. Therefore, the design of the detection steps is simple. A method with low cost, high sensitivity and specificity, and primers and probes with high specificity and sensitivity, which are conducive to the setting of experimental conditions, can improve the stability, accuracy and practicality of the method for detecting dengue virus and its typing Sex is important and has become one of the issues that have been in need of improvement

Method used

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  • Primers, probes and methods for detecting dengue virus and its typing
  • Primers, probes and methods for detecting dengue virus and its typing

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1: Design and synthesis of primers and probe sequences

[0039] Search the sequence of dengue virus target gene nonstructural protein NS5 from Genebank, use relevant bioinformatics software for homology analysis and sequence comparison analysis, screen out highly conserved regions, and use primer design software PrimerPremier5 for conserved sequences to design primers and probes Design; after the design of primers and probes, use NCBIBLAST to test the specificity, and use Primerpremier5 to detect whether there is a hairpin structure or dimer formation. The results show that the designed probe meets the specificity requirements and will not form a hairpin structure or dimer. The designed primers and probe sequences are listed in Table 1.

[0040] Table 1 Primers and probes for detecting dengue virus and its typing

[0041] name

Embodiment 2

[0042] Embodiment 2. Preparation of four kinds of serotype dengue virus-specific probe microsphere groups

[0043] 1. Probe synthesis

[0044] Various probe sequences were synthesized according to SEQ ID No: 13-16 in Table 1, and the 5' ends of all probes were modified by amination and connected to the C12 adjacent arm sequence.

[0045] 2. Coupling of probes and microspheres

[0046] Randomly select 4 different fluorescent microspheres. In this embodiment, the numbers of the selected fluorescent microspheres are 18, 30, 36, and 42. The coupling of the probe and the microspheres is carried out according to the following method:

[0047] 1. Put the 4 microsphere suspensions numbered 18, 30, 36, and 42 stored at 2-10°C and 2 bottles of unopened EDC powder (10mg / bottle) stored at -20°C at room temperature for 30- 60 minutes;

[0048] 2. Use ddH 2 O dissolved, the concentration is 0.1nmol / μl;

[0049] 3. Vortex the microsphere suspension at high speed for 30 seconds, then ult...

Embodiment 3

[0072] Embodiment 3: to the detection of 4 dengue virus clinical samples

[0073] 1. Sample preparation

[0074] The total RNA of No. 1-4 clinical samples was extracted by conventional methods, and the concentration was measured for future use. The clinical samples were serum or mosquitoes. In this embodiment, human serum was selected as the sample to be tested.

[0075] 2. Reverse transcription reaction

[0076] 1. Synthesize various reverse transcription primers according to SEQ ID No: 1-4 in Table 1.

[0077] 2. Prepare mixed reverse transcription primer working solution:

[0078] 1) Prepare each synthesized reverse transcription primer with TE (pH8.0) to prepare a 100 μmol / L stock solution;

[0079] 2) Add 10 μl of each reverse transcription primer storage solution to the same 1.5ml Eppendorf tube, add 460 μl TE (pH 8.0) to make up to a total volume of 500 μl, shake and mix well, and it is the mixed reverse transcription primer working solution.

[0080] 3. Sample tota...

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Abstract

The invention discloses primers, probes and method for detecting dengue virus and types of dengue virus and belongs to the field of biological detection and biological chemistry. According to the technical scheme, the invention provides a method for detecting dengue virus type I, II and III and types of dengue virus and also provides primers SEQ ID No:1-4 which are used in the method and are used for guiding reverse transcription of dengue virus genes, primers SEQ ID No:5-12 for amplifying target fragments of dengue virus, and probes SEQ ID No:13-16 for detecting the dengue virus. The invention has the beneficial effects that (1) specific primers and probe sequences are respectively designed for dengue virus of different serotypes, the application efficiency and sensitivity and the accuracy are high; (2) multiple reverse transcription polymerase chain reaction is combined with a liquid phase chip, thus achieving the advantages of high flux, high specificity and sensitivity, stable result, high repeatability, simple operation and high detection speed, and the dengue viruses can be subjected to rapid type detection.

Description

technical field [0001] The invention belongs to the fields of biological detection and biochemistry, in particular to a method for rapidly detecting dengue virus and its four serotypes, and provides specific primers and probes. Background technique [0002] Dengue fever is caused by dengue virus infection and is an acute infectious disease transmitted by mosquitoes. It is the most widely distributed and most common arbovirus disease in the world. The clinical manifestations are high fever, headache, severe pain in muscles, bones and joints, rash, enlarged lymph nodes, and decreased white blood cell count. It is widely prevalent in tropical and subtropical regions, especially in Southeast Asian countries bordering China. With the development of overseas tourism and business, the increase of population mobility, and the lack of effective vaccines and treatment measures, dengue fever has become a serious public health problem worldwide. Rapid, sensitive methods are therefore n...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/686C12Q1/70C12Q2537/143C12Q2563/131C12Q2563/149Y02A50/30
Inventor 韩伟史玲莉闫冀焕李云滑娜沈军吴志茹兰景李薇付晓昀
Owner 河北国际旅行卫生保健中心
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