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Fluorescent quantitative PCR primer composition for detecting tigecycline drug-resistant gene tet(X) and variants thereof and application of fluorescent quantitative PCR primer composition

A technology of primer composition and drug-resistant gene, which is applied in the direction of recombinant DNA technology, microbial measurement/inspection, biochemical equipment and methods, and can solve the problems of time-consuming and labor-intensive

Pending Publication Date: 2021-05-07
CHINA AGRI UNIV
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  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, traditional detection methods, such as conventional PCR and Sanger sequencing, are time-consuming and labor-intensive

Method used

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  • Fluorescent quantitative PCR primer composition for detecting tigecycline drug-resistant gene tet(X) and variants thereof and application of fluorescent quantitative PCR primer composition
  • Fluorescent quantitative PCR primer composition for detecting tigecycline drug-resistant gene tet(X) and variants thereof and application of fluorescent quantitative PCR primer composition
  • Fluorescent quantitative PCR primer composition for detecting tigecycline drug-resistant gene tet(X) and variants thereof and application of fluorescent quantitative PCR primer composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Embodiment 1, design and preparation of primers

[0085] A large number of sequence analyzes and comparisons were carried out to obtain 5 variants (tet(X), tet(X1), tet(X2), tet(X3), tet (X4)) several primers. Each primer was subjected to a pre-experiment to compare performances such as sensitivity and specificity, and finally a primer set for detecting five variants of the tigecycline resistance gene tet(X) was obtained, as shown in Table 1.

[0086] Table 1 Primer information of 5 variants of tigecycline resistance gene tet(X) and reference gene 16S rRNA

[0087]

[0088] Primer tet(X / X2)-F and primer tet(X / X2)-R form primer pair I;

[0089] Primer tet(X1)-F and primer tet(X1)-R constitute primer pair II;

[0090] Primer tet(X3)-F and primer tet(X3)-R constitute primer pair III;

[0091] Primer tet(X4)-F and primer tet(X4)-R constitute primer pair IV.

Embodiment 2

[0092] Embodiment 2, the construction of plasmid standard

[0093] 1. Tet(X / X2) plasmid standard product: insert the double-stranded DNA molecule shown in sequence 14 of the sequence table into the pDM19-T vector (pDM19-T vector: Takara Bao Bioengineering (Dalian) Co., Ltd., catalog number: 6013) EcoR Ⅴ site, get the tet(X / X2) plasmid standard product, and name it pDM19-T-tet(X / X2), and the pDM19-T-tet(X / X2) is pDM19-T in EcoR Insert sequence 14 into site V and keep the other parts of the pDM19-T plasmid unchanged. pDM19-T-tet(X / X2) contains the common sequence of tigecycline resistance genes tet(X) and tet(X2) (ie, the nucleotide sequence shown in sequence 14).

[0094] 2. Tet(X1) plasmid standard product: Insert the double-stranded DNA molecule shown in sequence 15 of the sequence table into the EcoR V site of the pDM19-T vector to obtain the tet(X1) plasmid standard product, which is named pDM19-T -tet(X1), the pDM19-T-tet(X1) is pDM19-T inserting sequence 15 at the EcoRV...

Embodiment 3

[0097] Embodiment 3, establishment of standard curve

[0098] Standard curves were respectively established using the five plasmid standard products prepared in Example 2.

[0099] 1. Calculate the copy number of the plasmid standard, the calculation method is as follows:

[0100] copies / μl=(L×C) / (N×M×10 9 );

[0101] L: Avogadro constant (6.02x10 23 / mol);

[0102] C: the concentration (ng / μl) of plasmid DNA;

[0103] N: the length of the recombinant plasmid, in bp;

[0104] M: The average amount of each base pair (660 / bp).

[0105] 2. The plasmid standard products pDM19-T-tet(X), pDM19-T-tet(X1), pDM19-T-tet(X2), pDM19-T-tet(X3) and pDM19-T-tet( X4) EASYDilution (purchased from Takara Bao Biological Engineering (Dalian) Co., Ltd., item number: 9160) was used to dilute to different gradient concentrations (10 - 1 ng / μL, 10 -2 ng / μL, 10 -3 ng / μL, 10 -4 ng / μL, 10 -5 ng / μL, 10 -6 ng / μL, 10 -7 ng / μL, 10 -8 ng / μL, 10 - 9 ng / μL and 10 -10 ng / μL).

[0106] 3. Use...

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Abstract

The invention discloses a fluorescent quantitative PCR primer composition for detecting a tigecycline drug-resistant gene tet(X) and variants thereof and an application of the fluorescent quantitative PCR primer composition. A corresponding fluorescent quantitative PCR method is established for five variants tet(X), tet(X1), tet(X2), tet(X3) and tet(X4) of the tigecycline drug-resistant gene tet(X), the detection method is suitable for culturable bacteria and non-culturable bacteria, and the PCR reaction conditions are the same, so that the five variants of the tigecycline drug-resistant gene tet(X) in a sample can be detected at the same time under the same PCR condition, and the fluorescent quantitative PCR primer composition has important significance on monitoring the drug resistance of antibiotic tigecycline in the environment.

Description

technical field [0001] The present invention relates to the molecular biological detection method of drug-resistant gene in the field of biotechnology, in particular to the SYBRGreen fluorescent quantitative PCR primer composition for detecting high-level drug-resistant gene tet(X) and tet(X) variants of tigecycline and its application. Background technique [0002] Tigecycline is a new type of glycylcycline antibacterial drug specially used for human medicine. It has good effect and low side effects in the treatment of multi-drug resistant "super bacteria" infection, and has a good application prospect. However, recently, the applicant's team discovered for the first time the tet(X) gene variants tet(X3) and tet(X4) carried by plasmids from Acinetobacter and Escherichia coli in my country's food animals and animal foods. Oxygen-modifying enzymes can not only mediate the high-level drug resistance of bacteria to tigecycline (MIC=32-64mg l -1 ), and can also be leveled throu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12N15/11
CPCC12Q1/689C12Q1/6851C12Q2600/106C12Q2531/113C12Q2545/114C12Q2563/107
Inventor 汪洋付玉林刘德俊宋黄威郝玉欣翟卫帅刘志海吴聪明沈建忠
Owner CHINA AGRI UNIV
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