Kit and detection method for rapidly detecting three flaviviruses in combined manner

A combined detection and kit technology, applied in the field of medical biological detection, can solve the problems of time-consuming and laborious detection, and there is no flaviviridae virus at all.

Inactive Publication Date: 2013-10-23
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0010] The applicant has applied for the Chinese invention patent CN200910047186.5 on March 6, 2009, and the title of the invention is "a combined rapid detection kit for Japanese encephalitis virus, dengue virus and West Nile virus and its detection Method", the publication number is CN101629215, selected some Japanese encephalitis virus primers, dengue virus primers and West Nile virus primers reported in the literature, when using this kit, due to the lack of Flaviviridae virus primers, the detection For samples with negative results, it can only be confirmed that there is no Japanese encephalitis virus, dengue virus or West Nile virus. Whether there are other flaviviruses or no flaviviruses is still unknown. The detection will be time-consuming and laborious; and the kit detects Japanese encephalitis virus, dengue virus and West Nile virus one by one, not simultaneously and quickly

Method used

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  • Kit and detection method for rapidly detecting three flaviviruses in combined manner
  • Kit and detection method for rapidly detecting three flaviviruses in combined manner
  • Kit and detection method for rapidly detecting three flaviviruses in combined manner

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Embodiment 1: kit of the present invention

[0103] Make the LAMP kit for the combined rapid detection of three yellow fever viruses according to the following formula:

[0104] (1) 16μl main reaction solution A: containing 2.5μl 10×Buffer, 1.0μl AMV (5U / μl), 0.25μl Rnasin (40U / μl), 2.0μl Bst DNA polymerase (8U / μl), 3.5μl 10mM dNTP, 1.5μl 100mM MgSO4, 4.0 μl 5M betaine and 1.25 μl DEPC-treated water.

[0105] Among them: the 10-fold isothermal reaction buffer contains trihydroxymethylaminomethane hydrochloride (200mM Tris-HCl, pH8.8) with a concentration of 200mM and a pH value of 8.8, potassium chloride (100mM KCl) with a concentration of 100mM, The concentration is 100mM sulfuric acid press (100mM (NH 4 ) 2 SO 4 ), a concentration of 20mM magnesium sulfate (20mM MgSO 4 ) and a concentration of 1% Triton X-100 (1% Triton X-100).

[0106] (2) Specific primer P: including LAMP-specific primers PFVP, JEV, PDV (including PDV1, PDV2, PDV3, PDV4) that specifically ampl...

Embodiment 2

[0156] Example 2: Correlation Analysis of LAMP Reaction Product Turbidity Detection Value (OD Value) and LAMP Real-time Fluorescence Monitoring Value

[0157] According to the principle that a large amount of magnesium pyrophosphate (Magnesium Pyrophosphate) is produced while the DNA is amplified in the LAMP reaction, the reaction system presents a certain degree of turbidity, so the turbidity, that is, the absorbance of the magnesium pyrophosphate salt (absorption wavelength 400nm) can be indirectly reflected by detecting the DNA. Amplification situation.

[0158] 1) Detection method for the amount of magnesium pyrophosphate produced in the LAMP reaction:

[0159] RNA of known viruses was used as 10 2 Doubling dilution (10 －1 、10 －3 、10 －5 、10 －7 、10 －9 、10 － 11 、10 －13 、10 －15 ), take 16 μl of the main reaction solution A and 7 μl of the specific primer P to mix, and add to each tube after 10 2 2 μl of RNA with different concentrations diluted in a ratio, the tota...

Embodiment 3

[0172] Embodiment 3: detect with kit of the present invention

[0173] 1).Materials and methods

[0174] 1.1 Source of virus strain

[0175] The international standard strains of dengue virus types 1-4 (type I Hawaii strain, type II New Guinea C strain, type III H87 strain and type IV H241 strain) were provided by the Center for Disease Control and Prevention of Guangzhou Military Region. The above-mentioned international standard strains were revived by seeding in suckling mouse brain and culturing C6 / 36 cells.

[0176] JE virus (SA-14, SH-103, FJ-03) was donated by the Institute of Viral Diseases, Beijing Center for Disease Control and Prevention, and JE virus (SHJ0701, SHJ0708) was donated by the Microbiology Department of Shanghai Center for Disease Control and Prevention.

[0177] West Nile virus uses the method of artificially synthesizing the target gene. Referring to the sequence of WN virus strain NY99 (accession number AF196835) in GeneBank, the 220bp sequence of t...

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Abstract

The invention relates to the technical field of medical biological detection, and provides a kit for rapidly detecting three flaviviruses in a combined manner. The kit is characterized in that firstly, a loop-mediated isothermal amplification (LAMP) is utilized to detect whether the flaviviruses are positive, then, Japanese encephalitis virus (JEV), dengue pathogen (DEV) and west nile virus (WNV) are simultaneously detected and distinguished. The invention also provides a method for detecting by utilizing the kit for rapidly detecting three flaviviruses in the combined manner. The kit provided by the invention has the advantages that the detection is simple and convenient, rapid and sensitive, and the combined rapid detection for the three flaviviruses is realized.

Description

technical field [0001] The invention relates to the technical field of medical biological detection, in particular to a kit and a detection method for rapid joint detection of three flaviviruses. Background technique [0002] A variety of viruses in the Flaviviridae family can cause severe diseases in humans and animals, especially encephalitis and hemorrhagic fever, with high mortality. The clinical manifestations of this type of virus infection are relatively complicated, and are often misdiagnosed or dubbed "unknown fever". At present, the common flavivirus infections in my country include dengue fever and Japanese encephalitis. With the reform and opening up and the development of tourism, new flaviviruses may appear. For example, West Nile virus is still spreading around the world. the trend of. As an international metropolis, Shanghai has increasing international and domestic goods and personnel exchanges, which makes the risk of West Nile virus being introduced throu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCY02A50/30
Inventor 曹广文殷建华张宏伟李淑华李自雄
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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