Dual RT-PCR detection method for corn lethal necrosis
A technology of RT-PCR and detection method, which is applied in the field of double RT-PCR detection of lethal necrosis of corn to achieve the effects of good reproducibility, improved detection efficiency and stable method
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Embodiment 1
[0030] The detection effectiveness of primers MCMV3333F / 4173R and SCMV1F / 2R were verified respectively.
[0031] Test samples: samples of lethal necrosis of corn and healthy corn leaves stored in the laboratory have been tested and verified by DAS-ELISA.
[0032] RNA extraction: 500 mg of leaves from positive samples and healthy leaves were ground with liquid nitrogen, and total RNA was extracted with an RNA extraction kit (Axygen).
[0033] Take 2-5 μl of total RNA, add primer 20 μM random primer 1 μl and 20 μM oligodT 18 1μl, denatured at 75°C for 5min, immediately placed on ice for 5min; then added 5×RT buffer 5μl, 5mM dNTP 2μl, 40U / μl PRI 0.5μl and 200U / μl 0.5μl, added RNase-free water or DEPC-treated Make up the total volume to 25 μl with water, mix well and perform reverse transcription reaction. The reaction conditions are 30 min, 42 °C, 45 min, 70 °C, 15 min, and quickly cool on ice. The synthesized cDNA is immediately used in the next reaction.
[0034] The cDNA was...
Embodiment 2
[0044] Using the reverse transcription product in Example 1 as a template, the annealing temperature determination experiment of primers MCMV3333F / 4173R and SCMV1F / 2R was carried out.
[0045] The PCR reaction system is as described in Example 1, the annealing temperature is set to 48.1°C, 49.5°C, 52.0°C, 53.5°C, 56.1°C and 58.1°C respectively, and the reaction conditions are: 94°C for 5min; 94°C for 30sec, annealing for 30sec, and 72°C for 40sec , 30 cycles; 72°C for 10 min.
[0046] Carry out electrophoresis with 1% agarose gel, photograph and analyze electrophoresis result: primer MCMV3333F / 4173R can amplify the band of 843bp to positive sample, and primer SCMV1F / 2R can amplify the band of 468bp to positive sample ( See image 3 with Figure 4 ).
[0047] image 3 Legend: Lane M: D2000DNA Marker, the reference fragments are 2000, 1000, 750, 500, 250 and 100bp from top to bottom; Detection results of primer MCMV3333F / 4173R at 56.1°C and 58.1°C.
[0048] image 3 Spect...
Embodiment 3
[0053] Using the reverse transcription product in Example 1 as a template, the experiment of the mixing ratio of primers MCMV3333F / 4173R and SCMV1F / 2R during double PCR was carried out.
[0054] The cDNA was amplified by PCR using two primer mixing ratios of 2 μl each for MCMV3333F / 4173R, 0.5 μl each for SCMV1F / 2R, 0.5 μl each for MCMV3333F / 4173R, and 2 μl each for SCMV1F / 2R; the reaction system was as follows:
[0055]
[0056]
[0057] The reaction conditions are: 94°C for 5 min; 30 cycles of 94°C for 30 sec, 52°C for 30 sec, and 72°C for 40 sec; 72°C for 10 min.
[0058] Electrophoresis was performed with 1% agarose gel, and the electrophoresis results were photographed and analyzed: during double PCR, two bands of two viruses could be amplified from positive samples. When the primer MCMV3333F / 4173R was used in a high amount, the two bands were similar in amount. When the primer SCMV1F / 2R was used in a high amount, the 843bp band of the maize chlorotic mottle virus wa...
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