Nucleic acid test standard substance for rotavirus group A and preparation method and application thereof

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 problems of lack of quantitative standard samples of rotavirus RNA fragments, etc., to avoid biological safety, sufficient stability, The effect of good uniformity

Active Publication Date: 2018-05-29
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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  • Nucleic acid test standard substance for rotavirus group A and preparation method and application thereof
  • Nucleic acid test standard substance for rotavirus group A and preparation method and application thereof
  • Nucleic acid test standard substance for rotavirus group A and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0043]Select the target sequence encoding binding protein VP2 in the genotyping region of group A rotavirus, conduct sequence analysis and alignment through NCBI online tools, 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 specific reference material for the preparation of group A rotavirus nucleic acid detection and subsequent reverse transcription droplet digital polymerase chain reaction (Reverse Transcript-dropletdigital Polymerase Chain Reaction, RT-ddPCR) detection method has 1 set of primer and probe combination, and the sequence is 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 Economic and Trade Co., Ltd.

[0044] Table 1 ...

Embodiment 2

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

[0047] Extract viral RNA from biological samples (mostly fecal samples) identified as group A rotavirus positive, and amplify the DNA fragment shown in SEQ ID No. 5 using the screened specific primers; then the DNA The fragment is connected with the plasmid vector to construct a recombinant plasmid, and the recombinant plasmid is transformed into E. coli competent cells to construct an E. coli containing the recombinant plasmid; through the proliferation of the E. coli, a large number of recombinant plasmids of the above DNA fragments can be prepared; extracting The recombinant plasmid was then single-enzyme digested with HamHI restriction endonuclease to form an in vitro transcription template, and a group A rotavirus nucleic acid detection standard substance candidate was obtained by in vitro transcription and synthesis of the T7 promoter. Among them, the sequence of T7 (...

Embodiment 3

[0084] Example 3 Establishment of RT-ddPCR method

[0085] RT-ddPCR method is mainly used for the homogeneity, stability and quantitative research in the preparation process of rotavirus nucleic acid detection standard material. The optimal detection range of RT-ddPCR is 20-2000 copies / μL. The balance weighing method is used to dilute the RNA standard material in a gradient manner. Refer to the One-Step RT-ddPCR Kit for Probes kit method for the above prepared standard material. For RT-ddPCR detection, the primers and probes used are the same as in Table 1, with reference to the kit method, and the specific operation methods are as follows:

[0086] 1. PCR reaction system

[0087] 2×one-step RT-ddPCR supermix 10μL, 25mM manganese acetate solution 0.8μL, 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, and DEPC water to make up to 20 μL. The primer probe seque...

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Abstract

The invention discloses a nucleic acid test standard substance for rotavirus group A and a preparation method and application thereof. The nucleic acid test standard substance for rotavirus group A has an RNA sequence shown in SEQ ID No. 1. The invention also particularly discloses a preparation method of the nucleic acid test standard substance for rotavirus group A and application of the nucleicacid test standard substance for rotavirus group A in rotavirus group A nucleic acid test. The nucleic acid test standard substance for rotavirus group A is free of biological contamination, easy toprepare and good in uniformity and stability, and has accurate and traceable traits; the nucleic acid test standard substance for rotavirus group A can serve as a positive control for qualitative nucleic acid test of rotavirus group A, may also serve as an external standard for quantitative nucleic acid test of rotavirus group A and may also serve for evaluating novel nucleic acid test methods ofrotavirus group A, material basis can be provided for the certification and accreditation of nucleic acid test capacity for rotavirus group A, and the nucleic acid test standard substance for rotavirus group A is widely applicable to various labs.

Description

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

Claims

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Application Information

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Patent Type & Authority Applications(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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