Real-time fluorescence PCR detection reagent for detecting Verticillium alboatrum
A black and white Verticillium, real-time fluorescence technology, applied in microorganism-based methods, microbial determination/inspection, microorganisms, etc., can solve the problems of false negatives, false positives, cross-contamination, etc. The effect of eliminating pollution
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Examples
Embodiment 1
[0010] Embodiment 1, the design of the special primer and probe that detects Verticillium black and white
[0011] The gene sequences of Verticillium black and white and its close species β-tubulin were retrieved from GenBank, and DNAMAN 6.0.40 software was used for comparison and analysis to find out the conserved gene sequence of Verticillium black and white. According to the design of primers and probes In general, use the software Primer Express 3.0 to design primers and TaqMan-MGB probes, then compare the designed primers and probes on NCBI, and finally determine a pair of primers and a probe through screening. The sequence is: VA3F:5 '- GAG TCC CGA ACC ACG TTG TC -3'; VA3R: 5'- GAG GTG TGT AGT ATG GTC AGG TTT GA -3'; VAMGB3: 5'- AAG ACG ACG ACA CGC G -3', probe 5' The 3' end contains a FAM reporter fluorescent dye, and the 3' end contains a non-fluorescent quencher group and has an MGB molecule.
Embodiment 2
[0012] Embodiment 2, real-time fluorescent RT-PCR detects the specificity test of Verticillium black and white
[0013] The specific process includes the following steps:
[0014] 1. Sample source
[0015] 6 samples of pure culture of Verticillium black and white (VAA2, VAA27, VAA39, VAA40, VAA42, VAA43), V. chlamydosporium 1 copy （ VCH1), V. dahliae 2 copies （ VD1, VD2 ), V. fungicola 1 copy （ VFU1), V. griseum 1 copy (VGR1), V. lecanii 1 copy (VLE1), V. lindauianum 1 copy (VLI1), V. longisporum 2 copies （ VDL46, VDL44), V. nigrescens 2 copies （ VNI1, VNI2), V. nubilum 1 copy (VNU1), V. psalliotae 1 copy (VPS1), V. theobromae 2 copies （ VTH1, VTH4), V. tricorpus 1 copy (VTR1).
[0016] 2. Extraction of sample DNA
[0017] DNA was extracted from the sample in step 1 with a DNA extraction kit (purchased from Qiagen). For details, see the kit instruction manual.
[0018] 3. Specific detection
[0019] Use the DNA obtained in step 1 and step 2 as ...
Embodiment 3
[0020] Embodiment 3, optimization of real-time fluorescent PCR reaction system
[0021] Using the DNA of VAA2 obtained in step 2 of Example 2 as a template, the primer concentration and probe concentration in the real-time fluorescent PCR detection system were respectively optimized. The specific steps are as follows:
[0022] 1. Primer concentration optimization
[0023] In the system of Step 3 in Example 2, the concentration of other components was kept constant, and the final concentration of the primer was increased by 0.1 μM from 0.1 μM to 1.0 μM. The reaction conditions were carried out according to the conditions of Step 3 in Example 2. The results showed that when the final primer concentration was 1.0 μM, the △Rn value reached the maximum and the Ct value reached the minimum. After three repeated experiments, 1.0 μM was finally determined as the optimal concentration of primers.
[0024] 2. Probe concentration optimization
[0025] In the system of Step 3 in Examp...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More