Kit for rapid joint detection of epidemic JEV, DEV and WNV and detection method thereof

A technology of Japanese encephalitis virus and West Nile virus, applied in biochemical equipment and methods, microbial-based methods, microbial determination/inspection, etc., can solve the problems of rapid detection and large-scale screening, and early monitoring of WNV issues such as impact and time-consuming

Inactive Publication Date: 2010-01-20
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, a series of detection methods for WNV have been initially established, such as PCR, TaqMan RT-PCR, NASBA (Nucleic acid sequence-based amplification), SYBR Green RT-PCR and other nucleic acid detection, virus isolation, serological analysis, etc., nucleic acid detection , Live virus isolation and culture methods are relatively mature, but these methods take a long time, and cannot perform rapid detection and large-scale screening in a short period of time, which affects the early monitoring of WNV

Method used

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  • Kit for rapid joint detection of epidemic JEV, DEV and WNV and detection method thereof
  • Kit for rapid joint detection of epidemic JEV, DEV and WNV and detection method thereof
  • Kit for rapid joint detection of epidemic JEV, DEV and WNV and detection method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0141] The LAMP kit for joint rapid detection of Japanese encephalitis virus, dengue virus and West Nile virus was made according to the following formula:

[0142] (1) 16μl main reaction solution: 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 for a total volume of 16 μl.

[0143] in:

[0144] The 10× isothermal reaction buffer contains tris-hydrochloride (200 mM Tris-HCl, pH 8.8) at a concentration of 200 mM, pH 8.8, potassium chloride (100 mM KCl) at a concentration of 100 mM, and a concentration of 100 mM Sulfuric acid (100 mM (NH4)2SO4), magnesium sulfate (20 mM MgSO4) at a concentration of 20 mM and Triton X-100 (1% Triton X-100) at a concentration of 1%.

[0145] (2) Specific primers: including specific amplification of Japanese encephalitis virus, dengue virus, West Nile virus LAMP-specific primers PJEV, PDV (including PDV1, PDV2, PDV3, ...

Embodiment 2

[0196] Detection of Japanese encephalitis virus, dengue virus and West Nile virus with the kit of the present invention

[0197] 1. Extraction of RNA from samples

[0198] 1) Extract Japanese encephalitis virus, dengue virus, and West Nile virus RNA from patient serum samples, cell culture supernatant, and mouse cerebrospinal fluid:

[0199] Add 750 μl to a 1.5 ml Eppendorf tube containing 250 μl cell culture supernatant LSReagen (invitrogen) was mixed with a vortex mixer; 200 μl chloroform was added to the Eppendorf tube, shaken vigorously for 15 s, left at room temperature for 2-15 min, and centrifuged at 12000 r / min at 4 °C for 15 min; carefully transfer the upper layer of the colorless aqueous phase to another fresh Add 500μl isopropanol to a 1.5ml Eppendorf tube, mix with a vortex mixer, and let stand at room temperature for 10 minutes; centrifuge at 12,000r / min at 4°C for 10 minutes, discard the supernatant, and keep the white RNA precipitate at the bottom of the tube;...

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Abstract

The invention relates to a kit for the rapid joint detection of epidemic JEV (Japanese Encephalitis Virus), DEV (Dengue Virus) and WNV (West Nile Virus). The kit consists of a main reaction solution and specific primers, wherein, the main reaction solution comprises a reaction buffer, AMV (Avian Myeloblastosis Virus) reverse transcriptase, a nuclease inhibitor, Bst (Bacillus stearothermophilus) DNA polymerase, dNTP (deoxyribonucleoside triphosphate), magnesium sulfate, betaine and DEPC (diethylpyrocarbonate) and conditioning water; and the specific primers comprise a specific amplification primer of JEV (PJEV), a specific amplification primer of DEV (PDV) and a specific amplification primer of WNV (PWNV). The invention is capable of rapidly detecting three viruses of flaviviridae by employing the LAMP (Loop-mediated Isothermal Amplification) technology and designing high-degree specific primers, thereby achieving the purpose of highly-efficient specific amplification. The invention further provides a detection method in which an ultraviolet-visible spectrophotometer is utilized for detecting the absorbance value of the reaction system and for indirectly reflecting the DNA amplification of target genes. Therefore, the invention is applicable to rapid detection at primary and field levels and is of greater application value.

Description

technical field [0001] The invention relates to a biological reagent, in particular to a method and a kit for detecting the presence of Japanese encephalitis virus (JEV), dengue virus (DEV) and West Nile virus (WNV) in clinical samples. In particular, it relates to a combined rapid detection kit for Japanese encephalitis virus, dengue virus and West Nile virus and a detection method thereof. 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 are dengue fever and Japanese encephalitis, and new flaviviruses may appear, such as West Nile virus, which is still showing a trend of continuing to spread around the world, posing a serious threat of sudden in...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCY02A50/30
Inventor 曹广文李淑华刘世建常文军张宏伟
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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