Primer and kit for detecting aminoglycoside drug resistance genes of aeromonas hydrophila
A technology of Aeromonas hydrophila and aminoglycosides, applied in the field of microbial detection, can solve the problems of poor detection accuracy, low specificity, and low efficiency, and achieve the effect of simple operation and low cost
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Embodiment 1
[0052] Example 1: Synthetic Primer Pairs
[0053] The nucleotide sequences of two aminoglycoside-resistant genes aac(6')-Iz and aph of Aeromonas hydrophila were detected. 2 pairs of specific primers were commissioned by Sangon Bioengineering (Shanghai) Co., Ltd. to synthesize:
[0054] Primer pair for amplifying the aac(6')-Iz drug resistance gene:
[0055] F1 (SEQ ID NO: 1): 5'-CCCATCTCAACGCCTTTC-3';
[0056] R1 (SEQ ID NO: 2): 5'-AAGAAGACGACCCGCTCC-3';
[0057] Primer pairs for amplifying the aph drug resistance gene:
[0058] F2 (SEQ ID NO: 3): 5'-GCGATTCCCTCTTGTTG-3';
[0059] R2 (SEQ ID NO:4): 5'-GCAGGCGAAGGTCTCA-3'.
Embodiment 2
[0060] Embodiment 2: the preparation method of kit.
[0061] (1) PCR reaction solution: including DNA polymerase 1U, 10×PCR reaction buffer 2μL, Mg 2+ 1.5mM, dNTP200μM, stored at -20°C;
[0062] (2) Primer mixture: After the nucleotide sequences shown in SEQIDNO:1-4 were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., they were mixed in a tube and mixed with ddH 2 Dissolve in O, the final concentration of each primer is 2.5 μM, store at -20°C;
[0063] (3) Positive control: the aqueous solution of the Aeromonas hydrophila genomic DNA containing aac (6')-Iz, aph drug resistance gene respectively;
[0064] (4) Negative control: the aqueous solution of Escherichia coli genomic DNA; at the same time, the aqueous solution of the genomic DNA of Aeromonas salmonicida, Aeromonas caviae, Aeromonas sobria, and Aeromonas victoria was used as a template to detect hydrophile gas Specificity of Primers for Aminoglycoside Resistance Genes in Monascus.
Embodiment 3
[0065] Embodiment 3: detection method.
[0066] Instruments: BioRad PCR detector (S1000), Sigma low-temperature high-speed centrifuge (3K15), IKA vortex mixer (labdancer), BioRad electrophoresis instrument (PowerPacHV), BioRad gel imager (UniversalHoodII);
[0067] (1) Preparation of the genomic DNA template of Aeromonas hydrophila: referring to the published literature, a relevant commercial bacterial genomic DNA extraction kit was used, and the bacterial genomic DNA was prepared according to the kit instructions, and used as a PCR reaction template for future use.
[0068] (2) Using the genomic DNA described in step (1) as a template, use 2 pairs of specific primers to carry out the amplification detection of the aminoglycoside drug-resistant genes aac(6')-Iz and aph of Aeromonas hydrophila, specifically including Follow the steps below:
[0069] (2a) Preparation of PCR reaction solution: Take out the components of the kit from the -20°C refrigerator, melt at room temperatu...
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