Real-time fluorescent PCR detection kit for Nosema bumblebee and detection method thereof
A detection kit and technology for microsporidia, which is applied in the field of animal health, can solve the problems that the detection method of bumblebee microsporidia has not been reported, and achieve the effects of high sensitivity, good repeatability, and low fluorescence background
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Benefits of technology
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0035] A real-time fluorescent PCR detection kit for Microsporidium bumblebee, comprising forward primer N.bombi-F, reverse primer N.bombi-R and TaqMan probe N.bombi-P, wherein forward primer N.bombi-F The sequence of the reverse primer N.bombi-R is 5'-GTCTCTCAGGCTCCTTCTCC-3', the sequence of the reverse primer N.bombi-R is 5'-ATGTGCACCGCAGATAACTA-3'; the sequence of the TaqMan probe N.bombi-P is 5'-FAM-ACCGTTACCCGTCACTGCCTTGT-BHQ1-3'. The kit includes the following reagents (50 reactions):
[0036] (1) Positive standard: Use primers N.bombi-F and N.bombi-R to amplify the genomic DNA of No. bumblebee, clone the product into pMD-18T vector, transform DH5α Escherichia coli, and extract positive clones by alkaline lysis Plasmid, after calculating the plasmid purity and concentration, 10-fold serial dilution to 10,000 copies / μL as a positive standard, 100 μL / cartridge, stored at -20 °C;
[0037] (2) Negative control: 1 negative control with 100 μL concentration of 100 ng / μL, the ...
Embodiment 2
[0042] Optimization of the annealing temperature for the detection method of the real-time fluorescent PCR detection kit for No. bumblebee
[0043] (1) PCR reaction system: Add 16.5 μL of PCR reaction solution, 0.5 μL of 5 U / μL ExTaq enzyme, and 2.0 μL of positive control to each PCR tube, and then add 60. μL of sterilized water to make the total reaction volume 25.0 μL µL;
[0044] (2) PCR optimization reaction program: pre-denaturation at 95°C for 5 min; then denaturation at 95°C for 5 s, annealing at 55°C-65°C (gradient temperature) for 30 s, and 3 repetitions for each gradient temperature, a total of 40 cycles;
[0045] (3) Selection of annealing temperature: the optimization results of annealing temperature are as follows: figure 2 As shown, the fluorescence intensity of the amplified product was higher when the annealing temperature was 59.0°C and 61.4°C than at other temperatures, so 60°C was selected as the annealing temperature for the reaction program of the kit. ...
Embodiment 3
[0047] The detection method using the real-time fluorescent PCR detection kit of Bombus bumblebee comprises the following steps:
[0048] (1) PCR reaction system configuration: Add 16.5 μL of PCR reaction solution, 0.5 μL of 5 U / μL ExTaq enzyme, 2.0 μL of sample DNA to be tested into the PCR tube, and then add 6.0 μL of sterilized water to make the total reaction volume 25.0 μL ;
[0049] (2) PCR reaction program: pre-denaturation at 95°C for 5 min; then denaturation at 95°C for 5 s, annealing and extension at 60°C for 30 s, a total of 40 cycles, and single-point fluorescence detection at 60°C;
[0050] (3) Judgment of results: The negative control has no Ct value and no amplification curve, while the positive control has a Ct value ≤ 35 and a typical amplification curve, indicating that the experiment is valid, otherwise the experiment is invalid. If the experiment is valid, the sample to be tested has no Ct value and no amplification curve, indicating that the sample does n...
PUM
Abstract
Description
Claims
Application Information
- R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com