Fluorescence quantitative PCR method for fast detecting Campylobacter jejuni macrolide drug resistant mutational site

A technology of Campylobacter jejuni and macrolides, which is applied in the fields of fluorescence/phosphorescence, biochemical equipment and methods, and microbial measurement/inspection, can solve the problems of cumbersome experiments, high technical requirements, and many influencing factors, and achieve Effects of inhibiting PCR amplification, improving amplification efficiency, and high amplification efficiency

Active Publication Date: 2011-06-01
HUAZHONG AGRI UNIV
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Problems solved by technology

However, this method involves processes such as PCR amplification, preparation of linear probe gel strips, and reverse hybridization, which requires high technical requirements for operation, and there are too many influencing factors in the reaction process, poor stability, and inconvenient. fast
[0010] 2005, Vacher (Vacher S, Menard A, Bernard E, Santos A, Megraud F. Detection of mutations associated with macrolide resistance in thermophilic Campylobacter spp. by real-timePCR. Microb Drug Resist. 2005 Spring; 11(1): 40- 7) et al established a real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) method to detect the macrolide resistance mutation of Cam

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  • Fluorescence quantitative PCR method for fast detecting Campylobacter jejuni macrolide drug resistant mutational site
  • Fluorescence quantitative PCR method for fast detecting Campylobacter jejuni macrolide drug resistant mutational site
  • Fluorescence quantitative PCR method for fast detecting Campylobacter jejuni macrolide drug resistant mutational site

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Experimental program
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Effect test

Embodiment 1

[0057] Example 1. Selection of target gene sequences related to Campylobacter macrolide drug resistance, design and synthesis of primers and probes

[0058] Using the BLAST tool in NCBI, the differences between the 23S rDNA gene sequence of Campylobacter jejuni and other intestinal bacteria were compared. A pair of primers were designed by using Beacon Designer 2.1 (molecular beacon design software 2.1) to specifically amplify the target genes related to the resistance to macrolides in Campylobacter jejuni. The length of the amplified target gene is 147bp, and its gene sequence is not highly homologous with other intestinal bacteria, which can ensure the specific amplification of the macrolide resistance-related gene of Campylobacter jejuni by PCR reaction. The specificity of the primers was checked by ordinary PCR amplification. From figure 1 It can be seen from the results that the designed primers can only specifically amplify the Campylobacter jejuni bacterial strain, bu...

Embodiment 2

[0065] Embodiment 2, the construction of three kinds of control plasmids

[0066] 1. Construction of wild-type control plasmid (plasmid WT)

[0067] Referring to Amera Gibreel (Gibreel, A., and D.E.Taylor.2006.Macrolide resistance inCampylobacter jejuni and Campylobacter coli.J.Antimicrob.Chemother.58:243-255.), PCR amplification of Campylobacter jejuni standard strain ATCC 33291 (purchased A 308bp nucleic acid sequence from the 23S rDNAV region of the American Standards Depository. After the gel of the amplified PCR product was recovered, the PCR product was connected to the pMD18-T carrier (the carrier was provided by the kit itself) using the TA cloning kit produced by Dalian Bao Biological Co., Ltd., according to the method described in the kit instruction manual. band), and transformed into Escherichia coli DH5α to construct a wild-type recombinant plasmid. The PCR product of the recombinant plasmid was sequenced, and it was determined that the inserted sequence was a w...

Embodiment 3

[0089] Embodiment 3, TaqMan fluorescent quantitative PCR reaction system and reaction condition optimization

[0090] Selection of annealing temperature for TaqMan fluorescent quantitative PCR reaction: In order to reduce non-specific amplification and ensure high amplification efficiency, we combined the Tm values ​​of primers and probes, and set three annealing temperatures of 58°C, 62°C and 64°C temperature.

[0091] Optimization of the ratio of primers and probes in TaqMan fluorescent quantitative PCR reactions: In order to avoid non-specific binding of probes and templates, referring to the concentration settings of primers and probes in other related TaqMan fluorescent quantitative PCR reactions, using the principle of square array design, we selected Three ratios of 10:3, 5:3 and 5:1 were determined for comparison to determine the optimal ratio of primers and probes (see Table 1-4).

[0092] The TaqMan fluorescent quantitative PCR reaction system is 25 μl, which includ...

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Abstract

The invention belongs to the biotechnical field, relating to a method for detecting drug-resistant related gene mutation of campylobacter jejuni macrolide drugs. The method makes use of a realtime fluorescence quantitative PCR TaqMan probe technology to design a specific primer and a probe according to a nucleic acid sequence of a V zone inside a campylobacter jejuni macrolide drug-resistant mutation zone 23S rDNA; moreover, through optimizing a TaqMan realtime fluorescence quantitative PCR reaction system and conditions, related drug-resistant mutational sites of target genes are accurately detected. The method can realize quick, accurate and simultaneous detection of two main drug-resistant mutational sites of campylobacter jejuni macrolide type, and has better specificity and higher sensitivity than a related molecular biology detection method reported at present; moreover, the method has simple operation, high accuracy and quick speed compared with the prior sequencing and drug sensitive experimental method. The method provides an effective technical means for molecular epidemiological monitoring of macrolide campylobacter jejuni resistance.

Description

technical field [0001] The invention belongs to the technical field of veterinary drug residue detection in animals, and specifically relates to a method for detecting gene variation related to resistance to macrolides in Campylobacter jejuni by using fluorescent quantitative polymerase chain method, specifically detecting the 2074 and 23S rDNA in Campylobacter jejuni Whether the gene at position 2075 is mutated. Background technique [0002] Campylobacteriosis is a general term for a series of diseases caused by bacterial infection of the genus Campylobacter. There are many species of Campylobacter, among which Campylobacter jejuni and Campylobacter coli are Gram-negative, highly motile, microaerophilic and thermophilic bacteria, which are now recognized as the The main pathogens of human acute bacterial enteritis in various places. Studies have shown that in some developed countries, the diarrhea caused by Campylobacter jejuni infection is even 2-7 times higher than that...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
CPCY02A50/30
Inventor 袁宗辉郝海红戴梦红王玉莲黄玲利彭大鹏王旭陈冬梅陶燕飞魏亚静郭文韬
Owner HUAZHONG AGRI UNIV
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