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Quality control material for detecting respiratory tract pathogen nucleic acid and preparation method thereof

A respiratory and pathogenic technology, applied in the field of medical testing, can solve the problems of different accuracy and traceability of measurement values, lagging development of related quality control substances, difficulty in ensuring the validity and consistency of measurement results, etc., to reduce biosafety risks, Effects of enhanced specificity and yield, improved stability

Pending Publication Date: 2021-04-06
GUANGZHOU BDS BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The development of relevant quality control substances is relatively lagging behind, making the testing of many products impossible, and we have to rely on foreign imports to meet laboratory testing requirements
Due to the different management systems implemented by the producers of these imported quality control substances, the accuracy and traceability of the measurement values ​​are also different, so it is difficult to ensure the validity and consistency of the measurement results in my country

Method used

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  • Quality control material for detecting respiratory tract pathogen nucleic acid and preparation method thereof
  • Quality control material for detecting respiratory tract pathogen nucleic acid and preparation method thereof
  • Quality control material for detecting respiratory tract pathogen nucleic acid and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047]Example 1 Get coronavirus culture

[0048]First, obtain coronavirus genome sequences

[0049]1. Download the human coronavirus HCOV-NL63 gene sequence from the National Center for Biotechnology Center (NCBI) https: / / www.ncbi.nlm.nih.gov / ; human coronavirus HCOV-229E gene sequence; HCOV-HKU1 gene sequence; human coronary virus HCOV-OC43 gene sequence; SARS-COV gene sequence; MERS-COV gene sequence; new coronavirus gene sequence.

[0050]2, divide the full-length genome sequence into 6 target genes: according to the disclosed coronavirus gene sequence, divide the full-length sequence into 6 segments, using gene synthesis, synthesized 6 target genes, at the end of each fragment Overlap is designed with a length of about 70bp. The six destination gene information is as follows:

[0051]Table 1 6 Purpose gene information

[0052]

[0053]

[0054]Second, the construction of the coronavirus slow virus

[0055]1. Connect the purpose gene and the expression vector according to the seamless clone connection t...

Embodiment 2

[0067]Example 2 Take the culture of other respiratory pathogens

[0068]First, prepare an influenza virus H1N1 (ATCC VR-1520), anhydrotective virus H5N1, an influenza virus H7N9, an influenza virus H9N2, a fluvosis (ATCC VR-1931), respiratory syncytic virus (ATCC) VR-1540), subflu virus (ATCC VR-3), adenovirus (ATCC VR-846), nasal virus (ATCC VR-1177) Culture:

[0069](a) take the cell line from the liquid nitrogen tank, immediately put the bottle quickly in the 37 ° C water bath and quickly shake until completely dissolved, to complete the temperature within 2 minutes; after complete dissolution, quickly take out from the water bath The jar is wiped with 70% alcohol.

[0070](b) Transfer the cell line to 15 ml of centrifuge tube, flat, 1000 rpm from intracellular suspension cells for 5 minutes, discarded; add 5-10 ml of culture solution, gently blow it, transfer cell suspension into T-25 cell culture flask Culture in 37 ° C, 5% carbon dioxide incubator; the growth of the cells was observed ...

Embodiment 3

[0087]Example 3 Inactivation of pathogen cultures

[0088]The above-mentioned cultured pathogen was inactivated:

[0089]1. Physical inactivation: first place the pathogen culture in a constant temperature water bath, soak for 30 minutes to perform physical inactivation;

[0090]2, chemical inactivation: then adding a nucleic acid diluent in the above pathogenic culture for chemical inactivation, wherein the isothiocyanate and SDS can enable protein to inactive effect.

[0091]3, formulation of nucleic acid dilution:

[0092](a) Take 40ml sterilized pure water to add sterilization beaker;

[0093](b) Pickup 5 mLDMSO to add the beaker and stir well;

[0094](c) Weigh 0.19GEGTA to add in the beaker and stir it to dissolve;

[0095](d) Weigh 0.1 g of SDS to add in the beaker, stirred and dissolve;

[0096](e) Weigh 1GBSA to add a beaker and stir it;

[0097](f) Weigh 1 g of guanidin guanidium isocyanate to add a beaker, stirred and dissolve;

[0098](g) Weigh 1.21 g of TRIS-HCl to add a beaker and stirred and dissolve...

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Abstract

The invention provides a quality control material for detecting respiratory pathogen nucleic acid. The quality control material comprises any one or more of the following respiratory pathogens: coronavirus, influenza virus, adenovirus, mycoplasma pneumoniae, streptococcus pneumoniae, rhinovirus or / and legionella pneumophila. A full-length genome sequence of the coronavirus is divided into six target fragments, and the length of each fragment ranges from 4000 bp to 5500 bp. The quality control material disclosed by the invention covers all detection target sequences (or target spots) of the coronavirus, the coverage is wide, the detection target spots are comprehensive, and the phenomenon of missing detection is avoided. The quality control material provided by the invention contains main respiratory tract infection pathogens, has a wide coverage range, and has more accurate detection targets by taking a real virus sample and a slow virus sample as raw materials. According to the coronavirus quality control material prepared by the invention, a target gene sequence is integrated into a host genome through a lentiviral vector, an autonomously replicated gene is knocked out in the preparation process, and the coronavirus quality control material has a self-inactivation capability, so that the recombinant lentivirus cannot be replicated in a target cell and infects other cells.

Description

Technical field[0001]The present invention belongs to the technical field of medical test, and more particularly to a mass control for detecting a respiratory pathogen nucleic acid and a preparation method thereof.Background technique[0002]Respiratory infection is the most common infectious disease, caused by respiratory infectious diseases, mainly pathogens causes respiratory infection by respiratory system such as nasal cavity, trachea. Respiratory viruses refers to a large class of viruses caused by acute respiratory tissue infection or other tissue organ lesions in respiratory mucosa epithelial cell proliferation. According to statistics, 90% of the original acute upper respiratory tract infection is caused by respiratory viruses, and the virus triggers the lower respiratory tract infection with 62%. Studies have shown that some respiratory pathogens, such as Middle Eastern respiratory syndrome viruses, avian influenza viruses, etc., due to the spread of the infection of these v...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/689C12Q1/6806C12R1/93C12R1/35C12R1/46C12R1/01
CPCC12Q1/701C12Q1/689C12Q1/6806C12Q2523/308C12Q2527/125
Inventor 李尔华高旭年钟凤然吴淑贤
Owner GUANGZHOU BDS BIOLOGICAL TECH CO LTD
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