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Primer probe for influenza B virus typing and identification and identification method

An influenza virus, typing and identification technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the difficulty of primer and probe design, reaction system configuration and identification result reporting time increase , multi-sample volume and other problems, to achieve the effect of reducing the configuration of the reaction system, convenient application, and simplifying the detection steps

Inactive Publication Date: 2018-06-19
SHANGHAI BIOGERM MEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When using the rRT-PCR method to detect viral nucleic acids, one of the difficulties is that the mutation of the virus brings difficulties to the design of primers and probes. In order to accurately identify the influenza viruses in circulation, it is necessary to regularly analyze the sequences of the epidemic strains. For comparison and analysis, if the primers and probes for typing identification are no longer suitable for popular strains, the primers and probe sequences need to be updated
The second is that the probes for identifying different subtypes or sublines use the same fluorescent label, and only one subtype or subline of influenza virus can be typed and identified in the same detection reaction system. If multiple For subtype or subline identification, more specimen volume is required, and the configuration of the reaction system and the time for reporting identification results increase accordingly

Method used

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  • Primer probe for influenza B virus typing and identification and identification method
  • Primer probe for influenza B virus typing and identification and identification method
  • Primer probe for influenza B virus typing and identification and identification method

Examples

Experimental program
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Effect test

Embodiment 1

[0039] The design and synthesis of embodiment 1, type B influenza virus Victoria line and Yamagata line primer and probe

[0040]Download GenBank database (http: / / www.ncbi.nlm.nih.gov / genomes / FLU / FLU.html) from 2013 to 2017 in China and GISAID (http: / / platform.gisaid.org / epi3 / frontend) The HA gene of influenza B virus in the database, the software compares and analyzes the consistency of each gene sequence of different viruses, and selects a relatively conserved region to design the genome-wide specific amplification primer sequence. In primer design, 4 or less degenerate bases are allowed at the same variable site. The extracted candidate primers were screened to meet the following requirements: ①The position of the probe is close to that of the same-strand primer, and the size of the PCR product is between 100bp and 150bp. ②The GC% of the primer and probe is between 25% and 75%; ③The length of the primer is about 20bp, the Tm value is between 58°C and 60°C, the length of ...

Embodiment 2

[0045] Embodiment 2, detect unknown virus

[0046] 1. Extraction of viral RNA:

[0047] Take 140 μL samples (throat swab, nasal swab, cell culture supernatant, chick embryo allantoic fluid, cell-free body fluid), add to 560 μL AVL lysate containing 5.6 μg Carrier RNA, press QIAamp Viral RNA MiniHandbook (Qiagen company, catalog#52904 / 52906) instructions to extract viral RNA, the elution volume is 50 μL.

[0048] 2. rRT-PCR reaction

[0049] 1) System configuration: using Bioline's SensiFAST TM Probe No-ROX One-Step Kit (BIO-76001) reaction solution, the configuration components are as follows in Table 1:

[0050] Table 1

[0051]

[0052] 2) rRT-PCR: Put the reaction tube with the above reaction system in the PCR machine for rRT-PCR. The reaction procedure is as shown in Table 2:

[0053] Table 2

[0054]

[0055] 4. Judgment of results: judge the results when the results of the negative and positive control are established, that is, the negative reference has no...

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Abstract

The invention discloses a primer probe for influenza B virus typing and identification and an identification method. According to the method, a group of primers and two probes as shown in SEQ ID No.1to SEQ ID No.4 are designed in a conserved region of an HA gene of an influenza B virus. The two probes select different fluorescent labels, thereby realizing simultaneous identification of two typesof the influenza B viruses in one tubular reaction system, and simplifying a definite diagnosis making process for a suspected influenza B virus infected case. Meanwhile, treatment of a sample to be detected, a fluorescent RT-PCR reaction system and reaction conditions are disclosed. The primer probe and the identification method can identify two types of epidemic influenza B viruses in the mainland of China, and are easy to operate and convenient to apply. A feasible technical method is provided for researches on respiratory tract infection etiology and clinical epidemiology.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a primer probe and an identification method for type identification of type B influenza virus. Background technique [0002] Influenza is an acute respiratory disease caused by influenza A, B or C viruses, which is characterized by seasonal epidemics or local outbreaks. Influenza virus belongs to the Orthomyxoviridae family and is a single-stranded negative-sense RNA virus with a segmented genome. Among them, type A influenza virus often causes influenza pandemics worldwide, while type B is a seasonal epidemic trend. Unlike influenza A, humans are the only host of influenza B virus. Influenza B viruses are not divided into HA and NA subtypes, but are divided into two branches with different antigens, namely, the Victoria line and the Yamagata line. [0003] Each year, one or two branches of influenza B virus co-circulate with seasonal H1N1 or seasonal H3N2 subtype influenza virus. Ab...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/701C12Q2561/113C12Q2563/107C12Q2545/114C12Q2561/101
Inventor 朱兆奎李晓丹赵百慧
Owner SHANGHAI BIOGERM MEDICAL TECH CO LTD