RT-LAMP reagent kit for detecting yellow fever viruses, and special primer of RT-LAMP reagent kit

A technology of RT-LAMP and yellow fever virus, applied in the direction of microorganism-based methods, DNA/RNA fragments, microorganisms, etc., can solve the problems of high environmental requirements for virus isolation, failure to meet detection needs, and many influencing factors, etc., and reach a broad market Foreground, suitable for large-scale promotion and application, and the effect of high specificity detection

Active Publication Date: 2020-05-12
首都儿科研究所 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The sensitivity and specificity of serological detection are not as high as nucleic acid detection, which cannot meet the detection needs in some grassroots units; virus isolation has high environmental requirements, a long cycle, and many influencing factors, so it is not suitable for grassroots laboratories; nucleic acid detection mainly includes PT-PCR and Real-time RT-PCR, but special instruments are required, and some basic units with relatively simple equipment conditions cannot carry out

Method used

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  • RT-LAMP reagent kit for detecting yellow fever viruses, and special primer of RT-LAMP reagent kit
  • RT-LAMP reagent kit for detecting yellow fever viruses, and special primer of RT-LAMP reagent kit
  • RT-LAMP reagent kit for detecting yellow fever viruses, and special primer of RT-LAMP reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Primer Design for RT-LAMP Detection of Yellow Fever Virus

[0038] This embodiment retrieves 43 yellow fever virus genome sequences altogether from the GenBank database, carries out homology analysis by BLAST, screens, intercepts and finally determines that a section of specific conserved fragment of yellow fever virus is the target gene sequence (SEQ ID in the sequence listing NO: 1), then according to the target gene sequence, use the software Primer design V5 (http: / / primerexplorer.jp / lampv5e / index.html) to design primers for RT-LAMP detection of yellow fever virus, a total of Four sets of yellow fever virus detection primers were designed, and the nucleotide sequences of each set of primers are shown in Table 1 below.

[0039] Table 1: Nucleotide sequence information of yellow fever virus universal detection primers

[0040]

[0041] Utilize above-mentioned four groups of detection primers respectively to yellow fever virus positive plasmid pGSI-YF-NS (...

Embodiment 2

[0043] Embodiment 2: the establishment of yellow fever virus RT-LAMP detection method

[0044] In this embodiment, under the same reaction system (described in detail below), the NS2a-4 primer set of Example 1 was used at 58-65° C. The sample (yellow fever virus culture, derived from the Wuhan Institute of Virology, Chinese Academy of Sciences) was extracted and obtained) for RT-LAMP detection to determine the optimum reaction temperature, including the following steps:

[0045] 1) Preparation of reaction system: using yellow fever virus genomic RNA (final concentration>1ng / μl) as template, 25μl RT-LAMP reaction system includes: genomic RNA 2μl, 2.5μl 10×ThermoPol Reaction Buffer, 5mM betaine 4μl, 100mM MgSO 4 1.5μl, 10mM dNTP 3.5μl, Bst DNA polymerase 1μl, WarmStart RTxreverse transcriptase (New England Biolabs Inc., Massachusetts, USA, add when the template is RNA, that is, add when RT-LAMP) 0.5μl, the amount of primer added: 100μM FIP 0.8 μl of each BIP primer (final conc...

Embodiment 3

[0049] Embodiment 3: the specificity, the sensitivity of the RT-LAMP detection method of yellow fever virus

[0050] In this example, the NS2a-4 primer set obtained in Example 1 was used to detect yellow fever virus by using the yellow fever virus RT-LAMP detection method to test the specificity and sensitivity of the method.

[0051] 3.1. Specificity detection

[0052] This step uses yellow fever virus positive plasmid pGSI-YF-NS, hepatitis C virus HCV genome full-length plasmid, hepatitis B virus HBV genome full-length plasmid, human acquired immunodeficiency virus HIV genome full-length plasmid, SARS coronavirus Envelope protein S1 C-terminal deletion of 19 AA plasmids pCAGGS-SARS-S1Δ-19, Middle East respiratory syndrome virus MERS spike protein particle, coronavirus OC43 envelope protein S1 C-terminal deletion of 17 AA plasmids pCAGGS-OC43-S1Δ-17, Ebola virus Ebola envelope glycoprotein particle, type A influenza virus Influenza A virus, Neisseria meningitidis group B B C...

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Abstract

The invention discloses an RT-LAMP reagent kit for detecting yellow fever viruses, and a special primer and application of the RT-LAMP reagent kit, and belongs to the field of biological technique detection. The provided RT-LAMP primer can realize specificity detection of the yellow fever viruses, the provided RT-LAMP reagent kit is simple in detection operation, high in specificity and high in sensitivity, the yellow fever viruses can be detected quickly and efficiently, and the RT-LAMP primer and the RT-LAMP reagent kit can be used for screening and detecting the yellow fever viruses in production units of livestock industry, basic-level medical health unit and disease preventing and controlling centers.

Description

technical field [0001] The invention belongs to the molecular biology detection method of virus in the field of biotechnology, in particular to a kind of RT-LAMP detection special primer and detection kit for detecting yellow fever virus, and the application of the detection kit in detection of yellow fever virus . Background technique [0002] Yellow fever virus is a single-stranded positive-sense RNA virus belonging to the family Flaviridae and the genus Flavivirus. The virus is transmitted by mosquitoes, and the main vector is Aedes aegypti. It is currently prevalent in Africa and South America. The population is more susceptible to yellow fever virus, and the fatality rate can reach 20%-40%. It is one of the three major quarantine infectious diseases in the world. Although there is no large-scale epidemic of yellow fever in my country at present, the geographical and climatic conditions in some areas of my country are similar to areas with high incidence of yellow feve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6844C12Q2531/119Y02A50/30
Inventor 袁静刘翟陈晨马莉萍崔晶花李少丽曾辉蒋栋
Owner 首都儿科研究所
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