Primer probe composition capable of detecting five pathogenic bacteria at same time and multiplex real-time fluorescence PCR method
A primer probe, pathogenic bacteria technology, applied in the field of molecular biology, to achieve the effect of compensating for complicated steps, improving detection efficiency, and improving sensitivity
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Embodiment 1
[0030] The design of embodiment 1 primer and probe
[0031] Based on Salmonella invA gene sequence (geneID: 1254419), Vibrio vulnificus vvhA gene sequence (geneID: 2827959), Shigella ipaH gene sequence (gene ID: 3776622), Staphylococcus aureus nuc gene sequence (gene ID: 767621) , Listeria monocytogenes prfA gene sequence (gene ID: 2744465), using the primer design software PrimerExpress3.0, we designed the specific invA gene of Salmonella, the specific vvhA gene of Vibrio vulnificus, and the specificity of Shigella The ipaH gene, the specific nuc gene of Staphylococcus aureus, the upstream and downstream primers of the specific prfA gene of Listeria monocytogenes, and Taqman probes labeled with different fluorescein for each gene.
[0032] The designed primers were analyzed as follows: interference between primers, hairpin structure and dimer. And through the BLAST function of NCBI to preliminarily identify its specificity. After screening, the primers with complementary se...
Embodiment 2
[0038] Determination of the optimal reaction conditions of embodiment 2 multiplex fluorescent quantitative PCR
[0039] The method steps for detecting Salmonella, Vibrio vulnificus, Shigella, Staphylococcus aureus, and Listeria monocytogenes using primers, probes and multiple real-time fluorescent quantitative PCR detection methods in Table 1 are as follows:
[0040] (1) DNA extraction:
[0041] Bacterial DNA was extracted by boiling water bath method. The specific operation is to pick a single colony purely cultured on agar and add it to 100 μL sterilized double-distilled water, shake and mix well, boil in a boiling water bath for 10 minutes, let it stand at room temperature, and centrifuge at 12000 r / min for 2 minutes after it drops to room temperature, take the above Store at -20°C for later use.
[0042] (2) Establishment of single-plex real-time fluorescent quantitative PCR system and condition optimization
[0043] Using TAKARA's Premix Ex Taq reagent as the reaction ...
Embodiment 3
[0063] Embodiment 3 is detected to the specificity of five kinds of pathogenic bacteria
[0064] (1) In order to verify the performance of probes and primers, carry out specificity tests:
[0065] After rapidly amplifying 40 known bacterial strains, the target pathogenic bacteria were detected using single-plex and multiple real-time fluorescent quantitative PCR methods (using the PCR system and reaction conditions determined in Example 2). Use specific primers and probes to perform single-plex real-time fluorescent quantitative PCR reactions on the corresponding target pathogenic bacteria. The Ct values of the detected genes are all less than 36. Use mixed primer probes to perform multiple real-time fluorescent quantitative PCR reactions on these 40 known strains Detection, the detection results are completely consistent with the design: the five target pathogenic bacteria all have the amplification of the corresponding reporter group, and no amplification occurs for other kn...
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