A Multiplex PCR Primer Set for Detecting Main Tomato Viruses and Its Application
A primer set, RT-PCR technology, applied in the field of plant virus detection and biological detection, can solve the problem of large consumption of detection reagents
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0056] Example 1: RT-PCR specific detection primer design
[0057] Virus sequences were downloaded from the NCBI database, TMV accession number: HE818416; ChiVMV accession number: KC711055; PeVYV accession number: KP326573; ToCV accession number: KY618797; TZSV accession number: NC_010490. Primer design was performed using primer 5.0 software, and the primer sequences and amplification lengths were shown in Table 1.
[0058]
Embodiment 2
[0059] Example 2: Singleplex RT-PCR validation.
[0060] 1. Information about the sample to be tested.
[0061] A total of 12 tomato samples with virus disease symptoms collected in Yuanmou, Yunnan were selected, including 17YV1381, 17YV1365-H, 17YV1385, 17YV1367, 17YV1372-T, 17YV1377, 17YV1383, 17YV1370-T, 17YV1382, 17YV1366, 17YV1367 and 17YV1372. The results of metagenomic sequencing showed that these samples were multi-infected by multiple viruses, so specific primers were designed for RT-PCR verification analysis.
[0062] 2. Reverse transcription.
[0063] In a 0.2 mL RNase Free centrifuge tube, add 1 μL random primer (oligo(dT)Primer), 5 μL RNA, denature at 70°C for 10 min, and quickly ice bath for 5 min; then add 4 μL 5 × M-MLV Buffer, 1 μL 10mM dNTP, 0.5 μL RNase Inhibitor (TaKaRa), 1 μL RTase-MLV (TaKaRa), 3.5 μL RNase Free H 2 O, 60 min at 42°C, 15 min at 70°C.
[0064] 3. PCR condition system.
[0065] PCR system was 30 μL: Premix Taq (TaKaRa Taq Version 2.0...
Embodiment 3
[0069] Example 3: Optimization of multiplex RT-PCR detection system.
[0070] 1. Information of the sample to be tested.
[0071] Seven samples 17YV1365-H, 17YV1366, 17YV1367, 17YV1368, 17YV1377, 17YV1381 and 17YV1385 identified by single-plex RT-PCR in Example 2 and confirmed by sequencing analysis were selected as samples for multiplex RT-PCR detection.
[0072] 2. Reverse transcription system optimization.
[0073] Synthesis of cDNA for multiplex PCR: Add 5 μL random primers (oligo dTPrimer), 4 μL total RNA, 2 μL dNTP Mixture (10 mmol / L each) to a 0.2 mL centrifuge tube, denature at 65°C for 5 min, and quickly ice bath for 5 min ; Add 8 μL of 5 × Prime Script TM Buffer, 1 μL RNase Inhibitor (40U / μL), 1 μL Prime Script TM RTase, 4 μL RNase Free H 2 O, 60 min at 42°C, 5 min at 95°C.
[0074] 3. PCR system optimization.
[0075] The primer concentrations were adjusted according to the band brightness of each virus. After optimization, the concentrations of each primer ...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More