Campylobacter jejuni fluorescence quantitative PCR detection kit and detection method thereof
A technology for quantification of Campylobacter jejuni and fluorescence, which is applied in the direction of fluorescence/phosphorescence, measurement/inspection of microorganisms, biochemical equipment and methods, etc., can solve the cost of false positives, reduce the detection target of operation steps, increase the possibility of contamination and detection The difficulty and other issues, to achieve the effect of strong specificity, improved sensitivity and specificity, and avoid missed and false detections
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[0021] Example 1 Composition of the kit of the invention
[0022] 1. The composition of the basic kit: (1) PCR MIX reaction solution, its components are 5×FQ PCR buffer, a primer mixture with a concentration of 10μM (each primer concentration is 10μM), and a TaqMan probe mixture with a concentration of 10μM (each The probe concentration is 10μM), dNTPS and ddH at a concentration of 10mM 2 O, the volume mixing ratio of each component is 10:1:0.5:1:30.5; the primer mixture contains: upstream primer 16S rRNA-F, downstream primer 16S rRNA-R, upstream primer hipO-F and downstream primer hipO-R , TaqMan probe mixture: 16S rRNA-P and hipO-P; (2) Taq enzyme; (3) positive standard (1×10 7 copies / ml) and negative standards.
[0023] (1) Components in PCR MIX reaction solution:
[0024] I: 5×FQ PCR buffer composition: 250mM Tris-HCl (pH8.0), 35mM MgCl 2 , 250mM KCl and 10% (volume concentration) DMSO.
[0025] II: The design and preparation of primers and probes are as follows:
[0026] Select 1...
Example Embodiment
[0038] Example 2 Method for detecting Campylobacter jejuni by the kit of the present invention and its verification
[0039] Add 1~10CFU and 10~100CFU of bacteria to 225mL Bolton selective enrichment broth. Under microaerobic conditions, culture at 30℃ for 3h, then at 37℃ for 2h, and finally at 42±1℃ for 19~43h. Bacteria.
[0040] (1) Detect with the kit of the present invention
[0041] Use bacterial nucleic acid extraction kit to extract the DNA of the enrichment solution and the positive control strain, take the Campylobacter jejuni PCR MIX reaction solution and Taq enzyme system prepared above, melt at room temperature, shake and mix, and centrifuge at 10,000 rpm for 10 seconds. According to each detection reaction system, 24μL PCR MIX and 2μL Taq enzyme were added to a clean PCR amplification tube or well of appropriate volume, mixed well and centrifuged, and then added 4μL DNA of the enrichment solution (positive standard and negative control were added to the positive control...
Example Embodiment
[0045] Example 3 Sensitivity of detecting Campylobacter jejuni with the kit of the present invention
[0046] Dilute 0.1ml of the original enrichment solution, 0.1ml of 100-1,000 CFU gradient Campylobacter jejuni dilution, 0.1ml of 10-100 CFU gradient Campylobacter jejuni dilution, 0.1ml of 1-10 CFU gradient Campylobacter jejuni dilution Use a bacterial nucleic acid extraction kit to extract nucleic acid, and take 4μl of DNA extract for fluorescence real-time PCR reaction. The results confirmed that the Ct value of the nucleic acid extracted from 0.1ml of the original enrichment solution was 10.3; the Ct value of the nucleic acid extracted from 0.1ml of the 100-1,000 CFU gradient of bacterial dilution was 32.9; the Ct value of the nucleic acid extracted from 0.1ml of the 10-100 CFU gradient The Ct value was 36.9; the Ct value of the nucleic acid extracted from 0.1ml of the bacterial dilution with a gradient of 1-10 CFU was 39.8, and the blank control did not have any amplificatio...
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