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Ebola virus typing fluorescence PCR detection kit

A technology of Ebola virus and detection kit, which is applied in the direction of microbial measurement/testing, biochemical equipment and methods, etc., to achieve the effect of protecting human health, simple method, rapid and accurate diagnosis

Inactive Publication Date: 2015-06-17
SANSURE BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0018] The purpose of the present invention is to solve the defects of the existing Ebola virus fluorescent quantitative PCR detection kit, and provide a kind of Ebola virus fluorescent PCR with fast operation, simple and convenient method, high detection sensitivity, wide detection range and the ability to distinguish specific types Detection kit

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  • Ebola virus typing fluorescence PCR detection kit
  • Ebola virus typing fluorescence PCR detection kit
  • Ebola virus typing fluorescence PCR detection kit

Examples

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Embodiment 1

[0047] The present embodiment provides a kind of Ebola virus fluorescent PCR detection kit, comprises the following independent components in the kit:

[0048] ① RNA extraction solution I: by sodium lauryl sulfate 0.2%~1.0% (mass / volume), triton 1.0%~4.0% (volume / volume), guanidine isothiocyanate 0.2mol / L~1.0mol / L. Composition of 100-400μg / ml magnetic beads;

[0049] ②RNA extraction solution II: 4-hydroxyethylpiperazineethanesulfonic acid 100-300mmol / L, pH6.5±0.2, sodium chloride 100-300mmol / L;

[0050] ③RNA extraction solution III: Triton 0.1%~1.0% (volume / volume), sodium chloride 100~300mmol / L;

[0051] ④ RNA extraction solution IV: mineral oil;

[0052] ⑤ RNA eluent: Tris-HCl 0.8~1.2mol / L, EDTA0.1~1.0mol / L;

[0053] ⑥ RT-PCR enhancer: a certain amount of Mn(OAc) 2 Mix well with purified water, Mn(OAc) 2 The final concentration is 100-500mmol / L.

[0054] ⑦Internal standard (positive internal control): It is a recombinant of a 105-base-pair artificially synthesized DNA...

Embodiment 2

[0075] This example provides the operation method of the Ebola virus nucleic acid detection kit described in Example 1 for detecting EBOV-RNA in samples such as plasma, serum, and urine.

[0076] 1 Reagent preparation:

[0077] 1) Take the corresponding amount of RNA extraction solution 1 and internal standard in proportion (RNA extraction solution 1 is 200 μl ~ 1ml / person + internal standard 0.4μl / person), mix well to form extraction solution 1-mix, and centrifuge instantaneously for backup use.

[0078] 2) According to the number of samples to be tested, negative controls, and positive controls, take each type of Ebola PCR reaction solution and RT-PCR enhancer in proportion (PCR reaction solution 49 μl / person + enhancer 1 μl / person) , mix thoroughly to form PCR-mix 1-4, centrifuge briefly and set aside.

[0079] 2RNA extraction operation

[0080] 1) Lysis virus: add 200μl~1ml RNA extraction solution 1-mix to each tube, then add 100μl~1ml of the sample to be tested (plasma, ...

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Abstract

The invention provides an ebola virus typing fluorescence PCR detection kit, the kit comprises a PCR reaction solution for detecting a primer probe sequence of Zaire ebola virus and a RNA extraction solution containing magnetic bead, the primer probe sequence is characterized in that an upstream primer is 5'-TGGTGATTTTCCGTTTGATGC-3'; a downstream primer is 5'-AAACGAGC TTGAGCCACTGAAT-3'; and a probe is 5'-FAM-TTGAAATCACAGCATCGTTGGCATC-BHQ13'. The kit has the advantages of rapid operation, simple method, high detection sensitivity (50 copie / ml), and wide detection scope; and can distinguish concrete types of ebola virus. The kit is used for rapidly detecting ebola virus nucleic acid in serum, blood plasma and urine samples and providing reliable experiment basis for diagnosis of ebola virus infection.

Description

technical field [0001] The invention provides a fluorescent PCR detection kit for Ebola virus typing. Background technique [0002] Ebola virus (EBOV) belongs to the Filiviridae family and is a single-stranded negative-sense RNA virus without segments. The virus is a long filament, which can be in various forms such as rod-shaped, filamentous, and "L"-shaped. Virus particles have an average length of 1000nm and a diameter of 70-90nm. Viruses have a lipid envelope with brush-like arrays of protrusions, mainly composed of viral glycoproteins. The Ebola virus genome is a non-segmented negative-strand RNA with a size of 18.9kb, encoding 7 structural proteins and 1 non-structural protein. [0003] EBOV can proliferate in mammalian cells such as humans, monkeys, and guinea pigs, among which Vero-98, Vero-E6, and Hela-229 cells are the most sensitive. Cytopathies appeared 6-7 hours after virus inoculation, manifested as rounded and shrunken cells, and inclusion bodies with fibr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/701C12Q1/686C12Q2561/101C12Q2531/113
Inventor 戴立忠李勃刘佳陈晓亮邓中平
Owner SANSURE BIOTECH INC
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