Combined gene detection kit and detection method for potato yellow dwarf virus
A yellow dwarf virus, potato technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. PYDV detection, time-consuming and other problems, to avoid non-specific amplification, high sensitivity, easy to operate
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Embodiment 1
[0088] Embodiment 1, potato yellow dwarf virus combined gene detection kit and detection method
[0089] 1. A complete set of specific primers and fluorescent probes for the detection of Potato Yellow Dwarf Virus
[0090] The invention designs and screens two sets of specific primers and fluorescent probes according to the conserved region of the RdRp gene of the potato yellow dwarf virus and the conserved region of the NP gene of the potato yellow dwarf virus.
[0091] The primers and fluorescent probes designed in the present invention for detecting the RdRp gene of potato yellow dwarf virus are composed of primer PYDV-f1, primer PYDV-r1 and fluorescent probe PYDV-Probe1. The sequences of primers and fluorescent probes are as follows:
[0092] PYDV-f1: 5'-GGTCAAATCGGAAATGAG-3' (SEQ ID NO: 1);
[0093] PYDV-r1: 5'-CTGGTTACAGTGATCAGA-3' (SEQ ID NO: 2);
[0094] PYDV-Probe1: 5'-FAM-TAAGCGGCAACCAACTGTCG-TAMRA-3' (SEQ ID NO: 3);
[0095] The primers and fluorescent probes desig...
Embodiment 2
[0117] Embodiment 2, the detection method of potato yellow dwarf virus combined gene detection kit
[0118] 1. Establishment of real-time fluorescent quantitative RT-PCR reaction system and optimization of reaction conditions for potato yellow dwarf virus combined gene
[0119] The sample infected with Potato Yellow Dwarf Virus was used as the sample to be tested, and the kit in Example 1 was used to detect the Potato Yellow Dwarf Virus in the sample to be tested. Specific steps are as follows:
[0120] 1) Reverse transcription: Add 3 μL of total RNA of the sample to be tested, RNase-free ddH to the PCR tube 2 7 μL of O and 1 μL of random primers with a concentration of 100 μmol / L, mix well, bathe in water at 70°C for 10 minutes, then ice-bath for 5 minutes, add 5 μL of 5×RT Buffer, 1 μL of reverse transcriptase at a concentration of 200 U / μL, and a concentration of 10 mmol / L 2 μL of dNTPs in L and 1 μL of RNase inhibitor at a concentration of 40 U / μL, bathed in water at 42°...
Embodiment 3
[0134] Embodiment 3, detect the detection method of potato yellow dwarf virus for single gene
[0135] One, be used for detecting the method for the primer of potato yellow dwarf virus RdRp gene, fluorescent probe detection potato yellow dwarf virus
[0136] A sample infected with Potato Yellow Dwarf Virus was used as a test sample, and the Potato Yellow Dwarf Virus in the test sample was detected by real-time fluorescent quantitative RT-PCR for the Potato Yellow Dwarf Virus RdRp gene. Specific steps are as follows:
[0137] 1) Reverse transcription: Add 3 μL of total RNA of the sample to be tested, RNase-free ddH to the PCR tube 2 7 μL of O and 1 μL of random primers with a concentration of 100 μmol / L, mix well, bathe in water at 70°C for 10 minutes, then ice-bath for 5 minutes, add 5 μL of 5×RT Buffer, 1 μL of reverse transcriptase at a concentration of 200 U / μL, and a concentration of 10 mmol / L 2 μL of dNTPs in L and 1 μL of RNase inhibitor at a concentration of 40 U / μL, ...
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