Multiple real-time fluorescent PCR method for identifying the drug-resistant mutation of macrolides and for identifying Campylobacter jejuni

A technology of macrolides and Campylobacter jejuni, which is applied in the fields of fluorescence/phosphorescence, biochemical equipment and methods, and microbial determination/inspection, etc. It can solve complex operations, inability to distinguish and identify Campylobacter jejuni, and low detection throughput and other issues to achieve high resolution, increase hybridization stability, and ensure specificity and sensitivity

Active Publication Date: 2013-12-04
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is a new type of DNA sequencing technology with high accuracy, but it also has disadvantages, such as complicated operation and low detection throughput.
However, this technology still has some shortcomings, for example, ① this technology cannot distinguish Campylobacter jejuni from other Campylobacter (such as Campylobacter coli and Campylobacter fetalis); ② this technology can only detect A2074C and A2075G in 23SrDNA Two site mutations, and this method is a single-plex fluorescent quantitative PCR method, the probes for detecting two site mutations are separated in two independent reaction systems

Method used

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  • Multiple real-time fluorescent PCR method for identifying the drug-resistant mutation of macrolides and for identifying Campylobacter jejuni
  • Multiple real-time fluorescent PCR method for identifying the drug-resistant mutation of macrolides and for identifying Campylobacter jejuni
  • Multiple real-time fluorescent PCR method for identifying the drug-resistant mutation of macrolides and for identifying Campylobacter jejuni

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Embodiment 1: Design and performance investigation of primers and probes

[0082] According to the VS1 gene sequence (GI: 296939), 23S rRNA sequence (GI: 3245050) and rplD gene sequence (GI: 905980) of the whole genome (NC_002163.1) of the standard strain of Campylobacter jejuni (Campylobacter jejuni NCTC11168=ATCC700819) published by NCBI , using Primer Premier 5 and Primer Express 2.0 software to design three pairs of primers, which were used to amplify the 214bp fragment of the VS1 gene unique to Campylobacter jejuni (position 931-1144 of the VS1 whole gene), the 96bp fragment of the 23S rRNA gene (the 23S rRNA whole gene 2020-2115) and a 134bp fragment of the rplD gene (120-253 of the rplD gene). Primer Premier 5 and Primer Express 2.0 software were used to evaluate the designed primers, and the primers with the strongest binding ability and the lowest probability of dimer formation were selected. In addition, the BLAST tool in NCBI was used to evaluate the specifi...

Embodiment 2

[0086] Embodiment 2, the construction of seven kinds of control plasmids

[0087] 1. Construction of wild-type 23S rDNA and rplD control plasmids

[0088] According to the 23S rRNA sequence (GI: 3245050) and the rplD gene sequence (GI: 905980) of the whole genome (NC_002163.1) of the standard strain of Campylobacter jejuni (Campylobacter jejuni NCTC11168=ATCC700819) published by NCBI, use Primer Premier 5 and Primer Express 2.0 The software designed two pairs of primers, which were used to amplify the 147bp fragment at positions 1995-2141 of the unique 23S rRNA gene of Campylobacter jejuni and the 270bp fragment at positions 120-389 of the rplD gene. site. The designed primers were entrusted to Nanjing GenScript Biotechnology Co., Ltd. for synthesis. The forward and reverse sequences of the two pairs of primers are shown in Table 2, and the binding base sequences of the two pairs of primers in the 23S rDNA and the rplD whole gene are shown in Table 2. figure 1 B and figure...

Embodiment 3

[0104] Embodiment 3: Optimization of multiple real-time fluorescent PCR reaction system and reaction conditions

[0105] Primers, probes, Mg in the multiplex real-time fluorescent PCR reaction system 2+ Concentration, concentration of Taq enzyme, etc. are the key parameters affecting the effect of fluorescent PCR amplification. In the present invention, 3 pairs of primers and 4 probes that have been designed and synthesized are placed in the same reaction system for multiple real-time fluorescent PCR, and the concentration of each component in the reaction system is optimized: the concentration of the primers is increased from 0.2 to 0.6uM by 0.05μM. The needle concentration is increased from 0.1μM to 0.45μM by 0.05μM, the concentration of dNTP is increased by 0.025mM from 0.075mM to 0.25mM, the concentration of Mg2+ is increased by 0.25mM from 0.75mM to 2.5mM, and the amount of Taq enzyme is increased by 0.5U from 1U to 5U . Only one variable is set for each test and the sa...

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Abstract

The invention belongs to the field of bacterial drug-resistant molecular detection, and relates to a multiple real-time fluorescent PCR method for identifying the drug-resistant mutation of macrolides and for identifying Campylobacter jejuni. The method is characterized in that an idiocratic primer, MGB probe, and combination of both are designed, not only is the specificity identification performed for the Campylobacter jejuni, but also the mutation of nucleotide of 2074th and 2075th of the 23S rRNA gene of Campylobacter jejuni and that of nucleotide of 170th and 221st of L4 flagellum gene rp1D of ribosome protein can be detected at the same time. The quick detection for drug-resistant mutation points of the various macrolides and the specificity identify for the Campylobacter jejuni are realized, which is first realized in the same reaction system, so that the identification and drug tolerance detection for the Campylobacter jejuni in clinical trials is greatly reduced, and as a result, a novel method is provided for Campylobacter jejuni drug-resistant monitoring and medicines for clinical trials infection treatment.

Description

technical field [0001] The invention belongs to the technical field of drug resistance detection of animal source bacteria. It is related to the multiplex fluorescent quantitative PCR method. The invention specifically relates to a fluorescent quantitative PCR method for identifying Campylobacter jejuni and its macrolide resistance-related mutations. In the same reaction system, not only can the species identification of Campylobacter jejuni be carried out, but also the detection of Campylobacter jejuni can be performed simultaneously. Mutations of 23S rDNA and rplD genes, the target genes of Bacillus resistance to macrolides. Background technique [0002] Campylobacter spp, including Campylobacter jejuni, Campylobacter coli and Campylobacter.lari, etc., are Gram-negative, highly motile, microaerophilic and thermophilic bacteria. Among them, Campylobacter jejuni is widely present in various animals, and can infect humans through contaminated water sources, raw milk, and i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/10G01N21/64
CPCY02A50/30
Inventor 袁宗辉刘杰郝海红王玉莲戴梦红黄玲利程古月王旭彭大鹏陈冬梅陶燕飞刘振利谢长清
Owner HUAZHONG AGRI UNIV
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