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A kind of group A rotavirus nucleic acid detection standard substance and its preparation method and application

A rotavirus and detection standard technology, which is applied in the field of group A rotavirus nucleic acid detection standard substances and its preparation, can solve the problem of no quantitative standard samples of rotavirus RNA fragments, etc., and achieve the effect of easy preparation and good uniformity

Active Publication Date: 2022-01-18
BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU INSPECTION & QUARANTINE TECH CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no quantitative standard samples using rotavirus RNA fragments as raw materials at home and abroad.

Method used

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  • A kind of group A rotavirus nucleic acid detection standard substance and its preparation method and application
  • A kind of group A rotavirus nucleic acid detection standard substance and its preparation method and application
  • A kind of group A rotavirus nucleic acid detection standard substance and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1 is used for developing the specific primer probe of group A rotavirus nucleic acid detection standard substance

[0043]Select the target sequence of binding protein VP2 in the genotyping region of group A rotavirus, and use the NCBI online tool for sequence analysis and alignment, and use Prime Express software V4.0 (ABI, Foster City, CA, USA) to design more than 10 The combination of primers and probes was screened to finally obtain a highly specific standard substance for the preparation of group A rotavirus nucleic acid detection standard substances and subsequent reverse transcription micro-droplet digital polymerase chain reaction (Reverse Transcript-droplet digital Polymerase Chain Reaction, RT-ddPCR (RT-ddPCR) detection method with 1 set of primers and probe combinations, and the sequences are shown in Table 1. The 5' end of the probe is labeled with FAM, and the 3' end is labeled with BHQ. Primers and probes were synthesized by Beijing Liuhetong E...

Embodiment 2

[0046] Example 2 Preparation of Group A Rotavirus Nucleic Acid Detection Standard Substance Candidates

[0047] Extract viral RNA from the biological sample (mostly stool sample) that is identified as A group rotavirus positive, utilize the specific primer that screens out to amplify the DNA fragment as shown in SEQ ID No.5; Then this DNA The fragment is connected with a plasmid vector to construct a recombinant plasmid, and the recombinant plasmid is transformed into Escherichia coli competent cells to construct Escherichia coli containing the recombinant plasmid; through the proliferation of Escherichia coli, a large amount of recombinant plasmids of the above-mentioned DNA fragments can be produced; extraction Recombinant plasmid, and then use HamH Ⅰ restriction endonuclease to single-enzyme digest the recombinant plasmid to form an in vitro transcription template, and obtain group A rotavirus nucleic acid detection standard substance candidates through in vitro transcriptio...

Embodiment 3

[0084] Example 3 Establishment of RT-ddPCR method

[0085] The RT-ddPCR method is mainly used for the research on the uniformity, stability and value determination of the standard substance preparation process for rotavirus nucleic acid detection. The optimal detection range of RT-ddPCR is 20-2000 copies / μL. Use the balance weighing method to carry out gradient dilution of the RNA standard substance. Refer to the method of the One-Step RT-ddPCR Kit for Probes kit to prepare the standard substance above. For RT-ddPCR detection, the primers and probes used are the same as shown in Table 1. Refer to the kit method. The specific operation method is as follows:

[0086] 1. PCR reaction system

[0087] 2×one-step RT-ddPCR supermix 10μL, 25mM manganese acetate solution0.8μL, forward primer and reverse primer (as shown in SEQ ID No.2 and SEQ ID No.3) each 1.0-1.8μL, fluorescent probe (As shown in SEQ ID No.4) 0.5 μL, RNA template 2.0 μL, DEPC water to make up to 20 μL. The sequence...

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Abstract

The invention discloses a group A rotavirus nucleic acid detection standard substance, a preparation method and application thereof. The RNA sequence of the standard substance for detection of group A rotavirus nucleic acid of the present invention is shown in SEQ ID No.1. The invention also specifically discloses the preparation method of the standard substance for detecting group A rotavirus nucleic acid and the application as the standard substance in the detection of group A rotavirus nucleic acid. The standard substance for detection of group A rotavirus nucleic acid of the present invention has no biological infectivity, is easy to prepare, has good uniformity and sufficient stability, and the standard substance has accurate and traceable characteristic values; it can be used as a group A rotavirus The positive control for the qualitative detection of viral nucleic acid can also be used as an external standard for the quantitative detection of group A rotavirus nucleic acid, and can also be used to evaluate new detection methods for group A rotavirus nucleic acid. Provide material basis, can be widely used in various laboratories.

Description

technical field [0001] The invention relates to the technical field of biological detection. More specifically, it relates to a group A rotavirus nucleic acid detection standard substance and its preparation method and application. Background technique [0002] With the continuous improvement of living standards, food safety has become a key issue of public health in the world, and the factors that affect food safety have attracted more and more attention. In recent years, emergencies in food safety have occurred frequently, among which foodborne diseases caused by pathogenic microorganisms are one of the most important factors affecting food safety, such as hemorrhagic Escherichia coli O157:H7 food poisoning in Japan, European The spread of mad cow disease, Listeria poisoning in France, bird flu outbreak in Thailand, dioxin incident in Belgium, etc. The globalization of world trade has also brought food safety risks. Therefore, establishing scientific and efficient food t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6806C12N15/11
CPCC12Q1/6806C12Q1/701C12Q2600/166
Inventor 徐蕾蕊魏咏新李丹曾静
Owner BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU INSPECTION & QUARANTINE TECH CENT
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