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Three-color fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) combined detection method of enterovirus 71, Coxsackie virus A16 and other subtypes of enterovirus as well as kit thereof

A RT-PCR, Coxsackie virus technology, applied in biochemical equipment and methods, fluorescence/phosphorescence, microorganism-based methods, etc., can solve problems such as indistinguishable HFMD

Inactive Publication Date: 2010-11-17
BEIJING SUOAO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Often, HFMD caused by EV71 is clinically indistinguishable from CAV16

Method used

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  • Three-color fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) combined detection method of enterovirus 71, Coxsackie virus A16 and other subtypes of enterovirus as well as kit thereof
  • Three-color fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) combined detection method of enterovirus 71, Coxsackie virus A16 and other subtypes of enterovirus as well as kit thereof
  • Three-color fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) combined detection method of enterovirus 71, Coxsackie virus A16 and other subtypes of enterovirus as well as kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Collection and delivery of specimens

[0052] Applicable specimen types include suspected patient feces, throat swab specimens, herpes fluid, cerebrospinal fluid or virus isolation and culture specimens, etc. Pharyngeal swab specimens: Collect throat swab specimens within 3 days of the patient's onset, use a special sampling cotton swab, and wipe the posterior pharyngeal wall and tonsils on both sides with moderate force, avoiding touching the tongue; quickly put the cotton swab into a container containing 1-2ml In the sampling tube of preservation solution (maintenance solution or saline), break off the cotton swab rod near the top, tighten the tube cap and seal it for inspection. Herpes solution: Disinfect the skin around the herpes with 75% alcohol, then use a sterile needle to pierce the herpes, dip a cotton swab into the herpes solution, and quickly put the cotton swab in it with 1-2ml of preservation solution (maintenance solution or normal saline) In ...

Embodiment 2

[0053] Embodiment 2: the extraction of enterovirus RNA

[0054] Sample treatment: Take 500 μl of sample solution and add 500 μl of virus concentrated solution to fully shake, centrifuge in a refrigerated centrifuge at 13,000 rpm for 10 minutes at 4°C, remove the supernatant, keep about 50 μl of sample solution, add 150ul Trizolreagents (RNA extraction solution) to fully shake, and place at room temperature for 5 minutes . Add 100ul of chloroform, shake vigorously for 15s, let stand at room temperature for 5min, and centrifuge at 13,000rpm at 4°C for 10min. Carefully transfer the upper aqueous phase to a clean centrifuge tube, add an equal volume of isopropanol, mix well, and centrifuge at 13,000rpm for 10min. Discard the supernatant, add 500 μl of 75% DEPC ethanol, mix well, centrifuge at 13,000 rpm for 10 min, and carefully absorb most of the ethanol. Open the extraction tube and dry it in the air at room temperature for 5 min until the ethanol evaporates, and dissolve the ...

Embodiment 3

[0055] Embodiment 3: Applicable instruments for this detection method and kit

[0056] Applicable instruments mainly include ABI GeneAmp PCR System7700, ABI GeneAmp PCRSystem7500, ABI PRISM 7300, LightCycler, etc., capillary instruments such as LightCycler, MJOpticon series or qualified quantitative PCR instruments.

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Abstract

The invention provides a three-color fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) combined detection method of enterovirus 71, Coxsackie virus A16 and other subtypes of enterovirus as well as a kit thereof. The method can rapidly and accurately detect the enterovirus 71, the Coxsackie virus A16 and the other subtypes of nucleic acids of enterovirus in a sample. The method comprises the following steps of: (1) acquiring and conveying a sample of an infected patient or a suspected patient; (2) preprocessing the sample and extracting RNA; (3) detecting the sample by adopting a one-step PCR-three-color fluorescent probe in-vitro amplification method; and (4) analyzing the corresponding sample according to the fluorescence intensity of each amplification reaction after the amplification reaction is finished, thereby judging the existence of the enterovirus 71, the Coxsackie virus A16 and the other subtypes of nucleic acids of the enterovirus in the acquired sample and being capable of carrying out accurate quantitation (a figure 3) on the enterovirus 71, the Coxsackie virus A16 and the other subtypes of nucleic acids of the enterovirus. The invention realizes the aim of carrying out rapid and accurate combined detection of the enterovirus 71, the Coxsackie virus A16 and the other subtypes of nucleic acids of the enterovirus.

Description

1. Technical field [0001] The test invention belongs to the field of virus nucleic acid detection, relates to the detection method of enterovirus 71 type, Coxsackie virus A16 type and other subtypes of enterovirus RNA, and particularly relates to the use of three-color fluorescent probe quantitative PCR technology (PCR -Three-color fluorescent probe method) can quickly and accurately detect enterovirus 71, coxsackie virus A16 and intestinal Methods for other subtypes of viruses. The invention further relates to a kit for clinical detection of the virus, which is suitable for rapid laboratory detection of human enterovirus infection. 2. Background technology [0002] Hand, foot and mouth disease (HFMD) is an acute infectious disease caused by enteroviruses. Enterovirus 71 (EV71) and Coxsackievirus A16 (CAV16) are the most common viruses, and they mostly occur in Preschool children, especially those under the age of 3, have the highest incidence rate. The main symptoms are ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64C12R1/93
Inventor 史成军任美峰徐贵峰刘健翊
Owner BEIJING SUOAO BIOTECH
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