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Method and kit for detecting EV RNA (enterovirus ribonucleic acid)

A kit and EV-R technology are applied in a field to achieve high purity and yield, high yield, and the effect of improving detection sensitivity

Active Publication Date: 2014-06-25
SANSURE BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to solve the defects of the existing enterovirus fluorescent quantitative PCR detection kit, provide a kind of RNA extraction yield high, high detection sensitivity, wide and accurate detection range EV nucleic acid fluorescent PCR detection kit, can be on the pharynx Rapid and accurate detection of EV-RNA in unknown samples such as swabs and feces, and the test results can be used for auxiliary diagnosis of EV infection

Method used

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  • Method and kit for detecting EV RNA (enterovirus ribonucleic acid)
  • Method and kit for detecting EV RNA (enterovirus ribonucleic acid)
  • Method and kit for detecting EV RNA (enterovirus ribonucleic acid)

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Embodiment 1

[0026] This embodiment provides a kind of enterovirus universal nucleic acid detection kit (kit for detecting EV RNA), which includes the following components:

[0027] ①RNA extraction solution Ⅰ: composed of sodium lauryl sulfate 0.2~1.0% (mass / volume), triton 1.0~4.0% (volume / volume), guanidine isothiocyanate 0.2~1.0mol / L and 100~400μg / ml of magnetic beads composition;

[0028] ②RNA extraction solution II: including 4-hydroxyethylpiperazineethanesulfonic acid 100~300mmol / L, sodium chloride 100~300mmol / L, solution II pH value is 6.5±0.2;

[0029] ③RNA extraction solution Ⅲ: Triton 0.1~1.0% (volume / volume), sodium chloride 100~300mmol / L;

[0030] ④ RNA extraction solution IV: mineral oil;

[0031] ⑤ RNA eluent: Tris-HCl0.8~1.2mol / L, EDTA0.1~1.0mol / L;

[0032] ⑥Internal standard (positive internal control): a 92-base-pair artificially synthesized DNA sequence inserted into the recombinant pUC18T vector, that is, a plasmid, with a concentration of 1.00E+03~1.00E+06copies / ml;...

Embodiment 2

[0047] This embodiment provides the operation steps of the kit described in the above-mentioned embodiment 1 for detecting EV-RNA in unknown samples such as throat swabs:

[0048] 1. Reagent preparation

[0049] 1) Take the corresponding amount of RNA extraction solution Ⅰ (200μl~1ml / person) and internal standard (1μl / person) in proportion and mix thoroughly to form RNA extraction solution 1-mix, centrifuge briefly and set aside.

[0050] 2) According to the number of samples to be tested, EV negative control, and EV positive control, take the corresponding amount of PCR reaction solution (43 μl / person) and EV enzyme mixture (2 μl / person) in proportion, and mix well to form a PCR- mix, centrifuged briefly for later use.

[0051] 2. RNA extraction operation

[0052] 1) Lyse the virus: add 200μl~1ml RNA extraction solution 1-mix to each tube, then add 100μl~1ml of the sample to be tested (such as a throat swab), cover the tube cap, shake and mix for 10 seconds, and centrifuge ...

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Abstract

The invention provides a method for extracting, purifying and detecting EV RNA (enterovirus ribonucleic acid) and a corresponding kit for detecting EV RNA. The kit provided by the invention comprises an RNA extraction solution containing magnetic beads, and a PCR (polymerase chain reaction) reaction solution which contains an upstream primer EV-F and a downstream primer EV-R for target polynucleotide amplification and a probe EV-P for target polynucleotide detection. The kit provided by the invention can detect EV RNA, including EV71, CA16 and other common EVs, but can not detect non-EV pathogens, which indicates that the kit has favorable specificity. Besides, the invention selects the favorable-adsorption-effect easy-purification magnetic bead method to extract the RNA to obtain high-purity high-yield nucleic acid, thereby greatly enhancing the detection sensitivity, accuracy and stability. The detection lower limit (sensitivity) is down to 200 copies / ml, and the detection range (quantitative linear range of the kit) is 2.00E+02-2.00E+08 copies / ml.

Description

technical field [0001] The present invention provides a method for extracting, purifying and detecting enterovirus (Enterovirus, EV) RNA. In the present invention, EV RNA includes Enterovirus 71 (Enterovirus71, EV71), Coxsackievirus A16 (Coxsackievirus A16, CA16) and other common enterovirus types, and provide a corresponding kit for detecting EV RNA. Background technique [0002] Real-time fluorescent PCR technology (FQ-PCR) integrates PCR, molecular hybridization and photochemistry, so that the whole process of PCR amplification and product analysis is carried out under single-tube closed conditions. Compared with other detection technologies, real-time fluorescent PCR technology has the following advantages: (1) Compared with immunological detection, it has higher sensitivity, and theoretically only one gene copy can be detected. (2) Primers are designed according to the unique conserved gene sequences of various pathogens, which can ensure the high specificity of PCR re...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/10C12R1/93
Inventor 戴立忠邓中平黄河
Owner SANSURE BIOTECH INC
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