Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Multiplex fluorescent quantitative PCR primer, probe and kit for detecting clostridium difficile producing toxins

A multiple fluorescence quantitative and Clostridium difficile technology, applied in biochemical equipment and methods, microorganisms, recombinant DNA technology, etc., can solve the problems of limited popularization and application, inability to detect binary toxins, etc., to avoid mutual interference and shorten detection time , the effect of high sensitivity

Inactive Publication Date: 2020-11-03
ICDC CHINA CDC
View PDF2 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, PCR detection methods for toxigenic Clostridium difficile may rely on specific instruments (such as Xpert, etc.), which limits its clinical application, or are only for the detection of toxins A and B, and cannot detect binary toxins , so it cannot be highly virulent strains (A + B + CDT + ) for early warning and prompting (Jin Dazhi, Luo Yun, Luo Li, etc. Multiplex fluorescent PCR combined with Allglo probe technology to detect Clostridium difficile-related toxin genes [J]. Chinese Journal of Laboratory Medicine, 2014, 37(01): 32- 35. Xie Le, Xu Yuqiao, Liu Chengcheng, et al. Direct detection of Clostridium difficile tcd B gene in stool samples by real-time fluorescent quantitative PCR [J]. Journal of Clinical Laboratory, 2016,3:211-214)

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
  • Multiplex fluorescent quantitative PCR primer, probe and kit for detecting clostridium difficile producing toxins
  • Multiplex fluorescent quantitative PCR primer, probe and kit for detecting clostridium difficile producing toxins
  • Multiplex fluorescent quantitative PCR primer, probe and kit for detecting clostridium difficile producing toxins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Design of Multiplex Fluorescent Quantitative PCR Primers and Probes for Detecting Toxigenic Clostridium difficile

[0051] The present invention takes the tcdA, tcdB and cdtB genes of toxigenic Clostridium difficile as detection targets, and through the tcdA, tcdB and cdtB gene sequences of different molecular types of toxigenic Clostridium difficile reported in the NCBI database, as well as the complete clinical isolates The genome sequencing sequence was analyzed and compared to clarify the genetic characteristics and sequence mutations of the genes. The primer probe design regions of the tcdA, tcdB and cdtB genes were respectively determined, and several pairs of specific primers and probes were designed for the design regions. BLAST was performed online by NCBI to exclude its non-specific binding to other species, and it was further verified and screened through experiments. Among them, the tcdA gene sequence needs special emphasis. At present, the common ...

Embodiment 2

[0067] Example 2 Multiplex fluorescent quantitative PCR detection method for toxigenic Clostridium difficile

[0068] The optimal primers and probes determined in Example 1 optimize the reaction conditions of multiplex fluorescent quantitative PCR, wherein, in order to simultaneously and efficiently amplify the toxin coding genes tcdA, tcdB and cdtB, the optimization of the annealing temperature of the PCR reaction conditions The process is as follows:

[0069] The reaction conditions of multiplex fluorescent quantitative PCR are as follows: the first step, pre-denaturation, 95°C, 20s, one cycle; the second step, PCR reaction, 95°C, 3s, 58°C, 30s, 40 cycles.

[0070] The annealing temperatures were set at 56°C, 58°C and 60°C, respectively, and the results showed that the amplification efficiencies at each annealing temperature were shown in Table 1. The results showed that the three genes had better amplification efficiencies at 58°C ( figure 2 ), therefore, 58°C was chosen...

Embodiment 3

[0080] Example 3 Sensitivity of fluorescent quantitative PCR to the detection of three Clostridium difficile toxin genes

[0081] Utilize embodiment 1 to determine specific primers and probes and the detection method determined in embodiment 2 to analyze the sensitivity of three kinds of Clostridium difficile toxin gene detection, take the lg value of standard concentration template as the abscissa, and take the corresponding Ct value as The vertical axis is to establish the detection standard curve of the detection method against the three toxin coding genes. details as follows:

[0082] 1. Toxin A gene (tcdA)

[0083] The concentration of recombinant plasmid DNA was 123 μg / μL, and the calculated plasmid copy number concentration was 5.53×10 10 copies / μL, the plasmid was diluted 10 times to obtain 5.53×10 0～ 5.53×10 9 Copies / μL total of 10 gradient standard plasmids. These 10 gradient standards were used as templates for fluorescence quantitative PCR amplification, and t...

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 relates to the technical field of detection of clostridium difficile producing toxins, in particular to a multiple fluorescent quantitative PCR primer, probe and kit for detecting clostridium difficile producing toxins. The invention provides a primer probe group and kit for multiplex fluorescence quantitative PCR detection of clostridium difficile producing toxins, and a multiplex fluorescence quantitative PCR detection method of the clostridium difficile producing toxins. The detection method has the advantages of high specificity, high sensitivity and good repeatability, can realize simultaneous and efficient detection of three toxin genes of enterotoxin A, cytotoxin B and binary toxin CDT through one-time loading, and significantly improves the detection efficiency of infection with the clostridium difficile producing toxins.

Description

technical field [0001] The invention relates to the technical field of detection of toxigenic Clostridium difficile, in particular to multiple fluorescent quantitative PCR primers, probes and kits for detecting toxigenic Clostridium difficile and applications thereof. Background technique [0002] Clostridium difficile (Clostridium difficile) is an important pathogen of antibiotic-associated diarrhea. It is a strictly anaerobic, spore-forming Gram-positive bacillus, and its cultivation is relatively difficult and requires anaerobic equipment. The main pathogenic factors of Clostridium difficile infection are enterotoxin A, cytotoxin B, and binary toxin CDT. Therefore, rapid and accurate identification of toxin-producing Clostridium difficile can be achieved without culturing. Diagnosis and timely and accurate treatment are of great significance. [0003] The nucleic acid-based Clostridium difficile detection method (NAAT) has been listed as a rapid detection method recommen...

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
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/06C12N15/11C12R1/145
CPCC12Q1/6851C12Q1/689C12Q2600/16C12Q2531/113C12Q2537/143C12Q2561/101
Inventor 吴媛卢金星贾筱溪王媛媛李文革张文竹
Owner ICDC CHINA CDC
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com