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Method for detecting rhinovirus typing and enterovirus

A RT-PCR, specific technology, applied in the detection of rhinovirus typing and enterovirus, identification of HRVA, HRVB, EV, HRVC field, can solve the problems of complex operation, high mutation rate, indistinguishable, etc., to achieve throughput High, short time, simple operation effect

Active Publication Date: 2018-09-07
NINGBO HEALTH GENE TECHNOLOGIES CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, because HRV and EV are RNA viruses with high mutation rate, there are few genome conserved sequences among different strains of the same type, and the only conserved sequence IRES region is not subtype-specific, so HRVA, HRVB, HRVC and EV cannot be To distinguish, there is currently no convenient and effective method for HRV typing. Sequencing methods can be used for HRV typing, but the cost is high and the operation is complicated, which is not conducive to large-scale HRV typing.

Method used

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  • Method for detecting rhinovirus typing and enterovirus
  • Method for detecting rhinovirus typing and enterovirus
  • Method for detecting rhinovirus typing and enterovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1: Collection of Samples and Extraction of Nucleic Acids

[0066] 1. Sample collection:

[0067] 1. Sample type: throat swab or nasopharyngeal aspiration.

[0068] 2. Sampling method of throat swab:

[0069] (1) Collection equipment: use special sterile throat swabs (flocked nylon swabs are recommended, cotton swabs and wooden poles are not recommended).

[0070] (2) Sampling solution: Use a sampling solution containing protein stabilizers and antibiotics (bovine serum 5%, penicillin 200U / mL, streptomycin 200U / mL, nystatin 25U / mL), with 2% NaHCO 3 Adjust the pH value to 7.4 (or use isotonic phosphate buffer or normal saline if the above sampling solution is not available)

[0071] (3) Sampling tube: screw cap, frozen at -70°C.

[0072] (4) Sampling method: press the patient's tongue with the tongue depressor with the left hand, extend the swab to the pharynx with the right hand, wipe the posterior pharyngeal wall and tonsils on both sides several times with ...

Embodiment 2

[0079] Embodiment 2: Detection of HRV typing and EV

[0080] Samples C7659, B8460, C3604, and C5038 were selected. These four samples corresponded to HRVA, HRVB, HRVC, and EV-positive clinical samples (by virus isolation, culture, and sequencing identification). Throat swabs of patients with respiratory tract infection obtained by the hospital according to the sample collection method described in Example 1. Nucleic acid extraction was performed according to the nucleic acid extraction method in Example 1, and the extracted nucleic acid was used as a template for RT-PCR and electrophoresis analysis.

[0081] Specific steps are as follows:

[0082] 1. Sample pretreatment

[0083] Place the sampling tube on a vortex mixer and vortex fully for 10 seconds to wash off the virus and virus-containing cells attached to the swab, and absorb the corresponding volume of Samples are extracted.

[0084] 2. Nucleic acid extraction

[0085] Use "Nucleic Acid Extraction or Purification R...

Embodiment 3

[0112] Example 3: Verify the identified HRV typing and EV by sequencing

[0113] The nucleic acid extracted from the samples C7659, B8460, C3604, and C5038 in Example 2 was verified by sequencing.

[0114] 1. RT-PCR amplification

[0115] 1.1 Configure RT-PCR system

[0116] 1.1.1 In order to verify the detection accuracy of this method, the inventors used species conservative primers (primers designed based on the IRES region) for PCR amplification and sequencing.

[0117] 1.1.2 Thoroughly vortex the reaction master mix (including sequencing primers, PCR buffer, dNTP) and centrifuge for 10 seconds; Place upright on ice.

[0118] 1.1.3 Add premixed RT-PCR reagents (including the reaction master mix and RT-PCR enzyme solution in 1.1.2) to a 1.5mL centrifuge tube: 28 μL × (N+1) reaction master mix and 2 μL × (N +1) RT-PCR enzyme solution (N is the number of samples, 4 in this embodiment); the mixed solution in the centrifuge tube was inverted and centrifuged briefly, then pl...

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Abstract

The invention provides a method for rapidly detecting HRV typing and EV by single-tube multiplex-RT-PCR. The method comprises the following steps: a) collecting a sample; b) extracting nucleic acid from the sample; c) adding the extracted nucleic acid to a premixed RT-PCR reagent; d) running an RT-PCR amplification program; and e) performing fragment analysis on the obtained RT-PCR amplification product, wherein the premixed RT-PCR reagent contains a primer specifically for HRV typing and a primer specifically for EV. The method has the advantages of simplicity in operation, short time and high flux, so a result can be obtained in 4 h at the soonest, and the method is suitable for clinic detection and scientific researches. The HRV virus typing result detected by the method can be used forguiding clinicians to medicate, and also can be used for the pathological study of rhinovirus infection.

Description

technical field [0001] The present invention relates to the field of virus detection, in particular to a method for detecting rhinovirus typing and enterovirus; more specifically, the present invention relates to a method for identifying HRVA (rhinovirus type A), HRVB (rhinovirus type B), Methods for HRVC (rhinovirus type C), EV (enteroviruses other than rhinoviruses). Background technique [0002] Rhinovirus (Human Rhinovirus, HRV) originally belonged to the genus Rhinovirus of Parvoviridae. In 2008, the International Committee on Taxonomy of Viruses deleted the genus Rhinovirus and included Rhinovirus A under the genus Rhinovirus. (HRVA) and rhinovirus type B (HRVB) belong to the Enterovirus genus; in 2009, rhinovirus type C (HRVC) was added to the Enterovirus genus. [0003] HRV and EV (Enterovirus; when referring to EV here and below refer to non-rhinovirus enterovirus) have the same morphological structure and genome structure, both of which are spherical in shape with...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12R1/93
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2521/107C12Q2537/143
Inventor 吴勇张成余丁徐智
Owner NINGBO HEALTH GENE TECHNOLOGIES CO LTD
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