Method and kit for detecting influenza A virus series by ligase

An influenza A virus and ligase technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of long time, low detection sensitivity, low throughput, etc., and achieve rapid detection and detection throughput. High, improve the effect of diagnosis efficiency

Inactive Publication Date: 2017-06-20
GUANGZHOU HEAS BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a method and a kit for using ligase to detect influenza A virus series. The kit can quickly detect four common subtypes of influenza A viruses that infect humans and are more harmful: H1, H3, H5 and H7 subtypes, no need for cumbersome nucleic acid extraction and reverse transcription processes, one tube and one amplification can realize the H1, H3, H5 and H7 subtypes of 4 common influenza A viruses, solving the problem of ordinary multiple fluorescence Problems of low detection sensitivity, long time-consuming and low-throughput of PCR method

Method used

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  • Method and kit for detecting influenza A virus series by ligase
  • Method and kit for detecting influenza A virus series by ligase
  • Method and kit for detecting influenza A virus series by ligase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1: the composition of kit of the present invention and the manufacturer and specification of main raw material

[0041] Composition Specification quantity main ingredient RNA extraction solution 1300ul / tube 1 tube Tris, EDTA Connect the reaction tube 10ul / tube 24 tubes Primers and ligases for upstream and downstream connection of influenza A virus H1, H3, H5, H7 subtypes Fluorescence PCR reaction tube 30ul / tube 24 tubes Hot start Taq enzyme, UDG enzyme, universal primers, specific fluorescent probes for each subtype of influenza A virus (H1, H3, H5, H7) negative control 100ul / tube 1 tube Sterilized purified water positive control 100ul / tube 1 tube Inactivated Influenza A Virus H1 Subtype Sample

[0042]Wherein, the connection primer includes the above-mentioned sequence of SEQ ID NO: 1-8, the universal primer includes the above-mentioned sequence of SEQ ID NO: 9-10, and the fluorescent p...

Embodiment 2

[0049] Embodiment 2: the using method of kit of the present invention

[0050] 1. Sample processing and RNA extraction

[0051] 1) Shake the throat swab preservation tube for 10-30 seconds (to make the cells fully fall into the liquid), and take 0.5ml-1.0ml of the exfoliated cell sample (if the number of cells is small, the volume can be increased to 2.0ml)

[0052] 2) Centrifuge the numbered samples at 10,000rpm for 3 minutes, discard the supernatant;

[0053] 3) Add 1ml sterilized saline to the sample pellet, wash the pellet, centrifuge at 10,000rpm for 3 minutes, and discard the supernatant;

[0054] 4) Add 50µl RNA extraction solution to the sample pellet, shake for 60 seconds, fully suspend the cell pellet, and set aside;

[0055] Extraction of RNA can also be performed using other known methods.

[0056] 5) Quality control treatment

[0057] Take the centrifuge tubes containing the positive quality control and negative quality control, centrifuge at 10,000rpm for 30 ...

Embodiment 3

[0076] Embodiment 3: The basis for setting the reference value of the kit of the present invention and the detection situation of clinical samples

[0077] Use this kit and the control kit to test the clinical samples, and use the re-check kit for detection if the two are inconsistent, and divide the samples into a positive group and a negative group according to the control and re-check test results, and compare the Ct values ​​detected by this kit , so as to determine the reference value of this kit. When the Ct value of this kit is greater than 37, it belongs to the negative group. When the Ct value of this kit is less than or equal to 37, it belongs to the positive group. Therefore, the Ct value greater than 37 is used as the reference value (CUTOFF value) of this kit.

[0078] The schematic diagram of the detection results using the detection kit of the present invention is as follows: Figure 4~6 shown. It can be clearly seen from the figure that the detection kit of t...

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Abstract

The invention relates to a kit for detecting influenza A virus series by ligase. The key point is that the method is characterized in that a special connecting primer is connected into a cDNA segment which is complementary with subtype RNA target sequences of influenza A viruses H1, H3, H5 and H7 under the action of ligase; a pair of universal primers in a later-stage fluorescent PCR reaction system can be amplified into successfully connected cDNA segments; and specific probes are marked by different fluorescence groups. The method applies the single fluorescent PCR technology to realize genotyping four common influenza A virus subtypes: H1, H3, H5 and H7 subtypes which infect people and generate relatively great harm in the same tube amplification system, so that the problems such as low amplification efficiency and low sensitivity caused by too many primers are effectively solved. Moreover, the kit is short in time consumption for detecting the whole period, realizes quick genotype detection for the influenza A virus subtypes H1, H3, H5 and H7, and is beneficial for epidemic surveillance and establishing effective prevent and control measures.

Description

technical field [0001] The invention relates to a method and kit for detecting influenza virus series, in particular to a method and kit for detecting influenza A virus series by using ligase. Background technique [0002] Influenza virus is a representative species of Orthomyxoviridae (Orthomyxoviridae), referred to as influenza virus, including human influenza virus and animal influenza virus. Human influenza virus is divided into A (A), B (B), and C ( C) Type III, which is the causative agent of influenza (flu). Influenza virus can cause acute upper respiratory tract infection and spread rapidly through the air, and there are often periodic epidemics around the world. Among them, influenza A virus is more likely to mutate, and an influenza pandemic is caused by the emergence of a new subtype or the reappearance of an old subtype of influenza A virus. [0003] Influenza A viruses can be identified by the subtypes of the HA and NA proteins on the surface of the virus. Inf...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 刘淑园陈华云肖湘文赵丽
Owner GUANGZHOU HEAS BIOTECH CO LTD
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