Quadruplex fluorescent quantitation PCR detection method and detection reagent kit for salmonella
A fluorescent quantitative and Salmonella technology, applied in the biological field, can solve the problems of mutual interference between primers and probes, and achieve high sensitivity, good repeatability, and good repeatability
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1 4
[0038] Embodiment 1 quadruple fluorescent PCR detection method
[0039] This embodiment adopts the primer probe group that is used for quadruple fluorescent PCR to detect Salmonella enteritidis, Salmonella pullorum, Salmonella gallinarum typhi, and Salmonella typhimurium to Salmonella enteritidis, Salmonella pullorum, Salmonella gallinarum typhi, and Salmonella pullorum. The detection of Salmonella typhi includes the following steps:
[0040] Step 1, prepare a PCR template based on the bacteria to be detected
[0041] Inoculate the strain of bacteria to be detected into LB liquid medium, and culture overnight at a temperature of 37°C to obtain a bacterial liquid, and then add 2 μL of the bacterial liquid as a template to the reaction system.
[0042] Step 2, prepare specific primer probe set
[0043] Preparation of primer probe sets for quadruple fluorescent PCR detection of Salmonella enteritidis, Salmonella pullorum, Salmonella gallinarum typhi, Salmonella typhimurium: whe...
Embodiment 2 4
[0068] Embodiment 2 Four-fold fluorescent PCR detection kit detects
[0069] This example is consistent with the steps of Example 1, except that this example directly uses the quadruple fluorescent PCR detection kit including the primer set mentioned in Example 1 to establish a reaction system.
[0070] The composition of the quadruple fluorescent quantitative PCR detection kit at this time includes: wherein, the volume of 2×Premix Ex Taq (ProbeqPCR) is 10 μL, and in the primer set, the volumes of the primer pair lygD-F and lygD-R are each 0.2 μL, The volume of probe lygD-P is 0.1 μL, the volume of primer pairs SGP-F and SGP-R is 0.1 μL each, the volume of probe SGP-P is 0.1 μL, and the volume of primer pairs SG-F and SG-R is 0.1 μL each. The volume of the probe SG-P is 0.1 μL, the volume of the primer pair SAT-F and SAT-R is 0.4 μL each, and the volume of the probe SAT-P is 0.8 μL.
[0071] Evaluation Example 1 Specificity Evaluation
[0072] In this embodiment, taking the ...
Embodiment 3
[0093] The application of embodiment 3 multiplex fluorescent quantitative PCR detection kits
[0094] Using the kit in Example 2 to detect 60 strains of Salmonella isolated from chicken farms, Salmonella enteritidis, Salmonella pullorum, Salmonella gallinarum typhi, and Salmonella typhimurium can be detected quickly and accurately. Specific steps are as follows:
[0095] Step 1, Isolation of Salmonella
[0096] Samples of sick and dead chickens were collected from chicken farms in Jiangsu, Anhui and other places, and inoculated aseptically on SS plates. A total of 60 strains of Salmonella were isolated and identified.
[0097] Step 2, fluorescent quantitative PCR method detection
[0098] Prepare the bacterial genome DNA of the Salmonella isolate according to the method of the fluorescent quantitative PCR detection kit in Example 2, then carry out the fluorescent quantitative PCR method detection, observe whether the fluorescent quantitative PCR amplification has a character...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More