Rapid lentinus edodes virus detection kit and detection method thereof
A technology for detection kits and detection methods, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of low detection efficiency, single detection of mushroom virus, etc., and achieve the effect of suitable size
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Embodiment 1
[0060] Embodiment 1: Establish multiple RT-PCR detection system
[0061] The test strain was selected as Y76 (carrying LePV1, LeV-HKB, LeTyLV1, and LeNSRV1 viruses at the same time), and a multiple RT-PCR detection method was established, which specifically included the following steps:
[0062] S1. Synthesize target primers: According to the reference Tm value of the primer synthesis list, finally determine a pair of primers for LePV1, LeV-HKB, LeTyLV1 and LeNSRV1. All primers are synthesized by Beijing Qingke Xinye Biotechnology Co., Ltd. The details of the synthetic primers are as follows :
[0063] LePV1-1F is: 5′-CTCCGAAGTTTCGCTCAGAA-3′
[0064] LePV1-1R is: 5′-GCCAGGTAGGTAGTGGATAGA-3′
[0065] LeV-HKB-1F is: 5'-TAGTACCCGGCGTCGTCTTA-3'
[0066] LeV-HKB-1R is: 5′-ACCAAAGAACCGATGTGGCA-3′
[0067] LeTyLV1-1F is: 5'-GTGTTCCGTTATGCGAGAGTAG-3'
[0068] LeTyLV1-1R is: 5′-CCAGATCAGACCACGAGAGATA-3′
[0069] LeNSRV1-1F is: 5′-CTGCCCTGCTTAGGCTATTT-3′
[0070] LeNSRV1-1R is: 5...
Embodiment 2
[0091] The multiple RT-PCR detection sensitivity that embodiment 2 test establishs
[0092] S1: Extract the RNA of the test strain: select the test strain Y76, and the extraction method is the same as that in Example 1.
[0093] S2: Prepare different concentrations of RNA templates: take 10ng of the total RNA of the Y76 strain, and carry out 10-fold serial dilutions to a total of 6 dilutions (10 1 ~10 -4 ), serially diluted from 10ng / μL to 0.1pg / μL, numbered 1-6 from high to low, "-" is the negative control.
[0094] S3: Single-plex RT-PCR detection:
[0095] Reaction system: 1.25 μL of One Step Enzyme Mix, 12.5 μL of 2×One Step Mix (Dye Plus), 1 μL of RNA template of the test strain Y76 (RNA concentration setting 10 1 ~10 -4 ), a pair of primers in S1 0.5 μL (primer concentration 0.25 μmol / L), complement RNaes Free dH 2 0 to 25 μL.
[0096] Reaction program: the first step is 50°C for 30 min, 94°C for 3 min, the second step is 94°C for 30 sec, the annealing temperature ...
Embodiment 3
[0100] Embodiment 3: the multiple RT-PCR detection method specificity that tests establish
[0101] S1: Extraction of total RNA from strains to be tested: 22 mushroom strains were used as test materials, and the extraction method was the same as in Example 1.
[0102] S2: multiplex RT-PCR amplification: the annealing temperature was 57.5°C, the concentration of each primer was 0.25 μmol / L, and the RNA concentration was 1 ng / μL.
[0103] S3: Identification by gel electrophoresis
[0104] 2% gel electrophoresis to observe whether there is cross-amplification and non-specific amplification, the amplification results are shown in Figure 4 A. The one-step multiplex RT-PCR amplification product was sent to Beijing Qingke Xinye Biotechnology Co., Ltd. for sequencing and identification. The sequencing result was consistent with the corresponding virus sequence.
[0105] Further, only the primers were replaced, and the Y76 strain was used as the test material to detect the Lentinus ...
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