Method for detecting nucleic acid of respiratory pathogen

A detection method and respiratory technology, applied in the field of molecular biology, can solve problems such as insufficient sensitivity, inability to realize point-of-care testing, bedside diagnosis, and inability to meet the needs of primary inspections.

Active Publication Date: 2019-09-10
GUANGZHOU PLUSLIFE TECH CO LTD
View PDF4 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main disadvantage of this method is that it needs to rely on a PCR machine or an expensive real-time quantitative PCR machine, and other supporting equipment, as well as a dedicated PCR laboratory and professional operators for PCR testing.
PCR detection cannot realize instan...

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for detecting nucleic acid of respiratory pathogen
  • Method for detecting nucleic acid of respiratory pathogen
  • Method for detecting nucleic acid of respiratory pathogen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Discovery of nucleic acid detection sites for respiratory pathogens based on the CRISPR / Cas12a system

[0033] The pathogenic bacteria of common respiratory infections in this study are Mycobacterium complex, Bordetella pertussis, and Chlamydophilapneumoniae. We obtained the genome sequences of these three respiratory pathogens, and compared them through bioinformatics analysis to find the specific identification regions of each strain of the three pathogens. The specific operation method is as follows: search the whole genome sequences of these three pathogens in the NCBI nucleic acid sequence database, obtain all the existing whole genome sequences and screen out the reference genome according to the completeness of the sequence; conduct homology analysis on the above genome sequences, find The target gene sequence of the pathogen is conserved among different genome sequences but specific to the human genome (hg19). In units of 20 bp bases, search for seque...

Embodiment 2

[0039] Example 2 Detection of Respiratory Tract Pathogenic Nucleic Acids Based on CRISPR / Cas12a System

[0040] 1. CRISPR / Cas12a gene cloning and protein expression

[0041] The Cas12a protein gene derived from Lachnospiraceae bacterium is used, and the codon is optimized to make the gene more suitable for expression in mammalian cells. The optimized Cas12a protein gene was cloned into the pET28a plasmid with a 6-His histidine tag to facilitate protein purification and expression. The Cas12a protein recombinant expression vector was transformed, and the expression strain was BL21star (DE3).

[0042] The specific protein expression conditions are: in culture solution OD 600 When =0.6, add 0.5mMIPTG and cultivate for 4 hours. Bacteria were collected for protein purification. The purification conditions are: resuspend the bacteria in the lysate (50mM Tris, pH8.0, 300mM NaCl, 5% glycerol, 20mM imidazole), and perform sonication (70% amplitude, 2s On / 4s Off, 3 minutes, Sonics 7...

Embodiment 3

[0069] Example 3 Multiple Respiratory Pathogen Nucleic Acid Detection Method Based on CRISPR / Cas12a System

[0070] In order to realize the simultaneous detection of multiple respiratory pathogens, we have developed a multiplex detection system for the above pathogenic targets. First, analyze the genome identification regions of the three selected respiratory pathogens and the corresponding gRNA sequences. According to the similarity of these sequences, GC content, base homogeneity, whether there is a secondary hairpin structure, whether there is cross-reaction in the same reaction and other parameters , to optimize the reaction system and gRNA combination.

[0071] 1. Specific operation: After preparing gRNA according to the method of step 3 in Example 2, take 4 kinds of gRNA and mix in equal proportions (SEQ ID NO.18, SEQ ID NO.21, SEQ ID NO.25 and SEQ ID NO. 27), and then prepare the CRISPR / Cas12a detection system according to the method of step 4 in Example 2, and detect ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a method for detecting a nucleic acid of a respiratory pathogen. The method obtains a specific nucleic acid sequence site for detecting three respiratory pathogens based on CRISPR/Cas12a technology by research, and for the site, qualitative detection of the three pathogens can be achieved by utilizing a CRISPR/Cas12a system, and different types of the three pathogens can bespecifically distinguished; and a respiratory pathogenic nucleic acid detection system and detection kit based on CRISPR/Cas12a are constructed. The technology is good in specificity and is high in sensitivity for detection of the nucleic acid of the respiratory pathogen, and can achieve simultaneous detection of multiple sites, and at the same time, the technology can be performed at room temperature, is convenient and rapid to detect, is low in detection cost, is of great significance for the detection and screening of the respiratory pathogens, and has good application prospects.

Description

technical field [0001] The invention belongs to the technical field of molecular biology. More specifically, it relates to a method for detecting nucleic acid of respiratory pathogens. Background technique [0002] Respiratory tract infectious diseases often have an acute onset, with dry cough, chest tightness, dyspnea and other respiratory symptoms as the main manifestations, and may be accompanied by systemic symptoms such as fatigue, headache, muscle and joint pain, and are the second most common cause of morbidity and death worldwide. Diagnostic methods for respiratory pathogens include microscopic examination, bacterial culture, antigen and antibody detection, biochemical reactions, and molecular biology methods. Commonly used diagnostic methods are often based on empirical judgment or the detection of a single pathogen, and there are few products for the overall detection of common pathogens based on syndromes. Common pathogenic bacteria of respiratory tract infectio...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12Q1/689C12Q1/04C12N15/113C12N9/22
CPCC12Q1/689C12N15/113C12N9/22C12Q2600/16C12N2310/10C12N2310/20Y02A50/30
Inventor 刘华勇陈翀杨嘉玉
Owner GUANGZHOU PLUSLIFE TECH CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap