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Method and kit for detection of human enterovirus 71 RNA

An enterovirus and kit technology is applied in the field of enterovirus type 71 RNA detection and kits to avoid false negative results, good adsorption effect and high yield.

Active Publication Date: 2013-01-02
SANSURE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] The purpose of the present invention is to solve the defect of existing enterovirus 71 type nucleic acid detection kit, provide a kind of RNA extraction yield height, the enterovirus 71 type nucleic acid detection kit that detection sensitivity is high, apply this kit, can be to Rapid and accurate determination of EV71-RNA concentration in unknown samples such as throat swabs and feces, providing a reliable experimental basis for early diagnosis of enterovirus 71 infection

Method used

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  • Method and kit for detection of human enterovirus 71 RNA
  • Method and kit for detection of human enterovirus 71 RNA
  • Method and kit for detection of human enterovirus 71 RNA

Examples

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Embodiment 1

[0035] This embodiment provides an enterovirus type 71 nucleic acid detection kit (a kit for detecting EV71 RNA), which includes the following components:

[0036] ①RNA extraction solution I: It is composed of 0.6 (mass / volume)% sodium dodecyl sulfate, 3.5 (v / v)% triton, 0.8mol / L guanidine isothiocyanate and 300μg / ml magnetic beads;

[0037] ②RNA extraction solution II: including 4-hydroxyethylpiperazineethanesulfonic acid 100mmol / L, sodium chloride 200mmol / L, the pH value of solution II is 6.5±0.2;

[0038] ③RNA extraction solution III: Triton 0.5 (v / v)%, sodium chloride 300mmol / L;

[0039] ④RNA extraction solution Ⅳ: mineral oil;

[0040] ⑤ RNA eluent: Tris-HCl 1.2mol / L (pH8.0), EDTA 1.0mol / L (pH8.0) and purified water;

[0041] ⑥Internal standard (positive internal control): a recombinant DNA sequence of 78 base pairs inserted into the pUC18T vector, that is, a plasmid, the concentration is 1.00E+03copies / ml~1.00E+06copies / ml; 78 bases The sequence of pairs looks like th...

Embodiment 2

[0058] The present embodiment provides the operation steps that the kit described in the above-mentioned embodiment 1 is used to detect EV71-RNA in unknown samples such as throat swabs and feces:

[0059] 1. Reagent preparation

[0060] 1) Proportionately take the corresponding amount of RNA extraction solution I (200 μl to 1 ml / person) and internal standard (1 μl / person) and mix thoroughly to form RNA extraction solution 1-mix, and centrifuge briefly for later use.

[0061] 2) According to the quantity of the sample to be tested, EV71 negative control and EV71 positive control, take corresponding amounts of PCR reaction solution (43 μl / person) and EV71 enzyme mixture (2 μl / person) in proportion, and mix thoroughly to form PCR- mix and centrifuge briefly for later use.

[0062] 2. RNA extraction operation

[0063] 1) Split virus: add 200μl~1ml of RNA extraction solution 1-mix to each tube, then add 100μl~1ml of the sample to be tested (such as throat swab), cover the tube, s...

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Abstract

The invention provides a method for extraction and purification and detection of EV71 RNA (human enterovirus 71 RNA) and a corresponding kit for detection of EV71 RNA. The kit provided by the invention comprises an RNA extraction solution containing magnetic beads and a PCR reaction solution containing an upstream primer EV71-F1 and a downstream primer EV71-R1 used for amplification of target polynucleotide and a probe EV7-P1 used for detection of the target polynucleotide. The kit can detect an EV71 positive sample but can not detect non-EV71 pathogens, which proves the kit has good specificity; furthermore, a paramagnetic particle method with the advantages of a good adsorption effect and easy purification is selected for extraction of RNA in the invention, so high-purity high-yield nucleic acid can be obtained, detection sensitivity, accuracy and stability are greatly improved, the detection limit, i.e., sensitivity, of the kit, is up to 200 copies / ml, and a detecting range (the quantitative linear range of the kit) is 2. 00E + 02 to 2.00E + 08 copies / ml.

Description

technical field [0001] The present invention provides a method for extracting, purifying and detecting enterovirus 71 (Human enterovirus 71, EV71) RNA, and provides a corresponding kit for detecting EV71 RNA. Background technique [0002] EV71 is a common virus that causes hand, foot and mouth disease in infants and young children. Preschool children are generally susceptible to EV71, with acute onset and rapid progression. Adults are also infected, generally manifested as a recessive virus, which can be transmitted to infants and young children and cause disease. [0003] Similar to other enteroviruses, the method for diagnosing and classifying EV71 infection is generally through virus isolation and virus identification after collecting appropriate clinical specimens; if appropriate clinical specimens are not collected, or virus isolation cannot be performed, It can also be diagnosed by serological tests (such as neutralization assays) to detect neutralizing antibodies in ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/10C12R1/93
Inventor 戴立忠邓中平付亚成
Owner SANSURE BIOTECH
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