Method for detecting southern bean mosaic virus and tobacco ringspot virus by using multiple DPO-RT-PCR
A DPO-RT-PCR, DPO-PCR technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganism determination/inspection, etc., can solve the problems of long time and low throughput, and achieve strong specificity. , The effect of strong concealment and difficulty in interception
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Embodiment 1
[0027] Embodiment 1 Materials and methods
[0028] 1.1 Materials and equipment
[0029] Detection reagents: Plant Total RNA Extraction Kit (DP432), FastKing One-Step RT-PCR Kit (KR123), Marker II DNA Marker (MD102) were purchased from TIANGEN Company; primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. ; See Table 1 for the sources of experimental samples.
[0030] Main equipment: Veriti PCR instrument, Nanodrop 2000 nucleic acid and protein analyzer, American Thermofisher Company; electrophoresis instrument, Geldoc XR+ gel imaging system, American Bio-rad Company.
[0031] Table 1 Sample source
[0032]
[0033] 1.2 Experimental method
[0034] 1.2.1 Extraction of total RNA
[0035] Total RNA was extracted from the samples in Table 1 according to the requirements of plant total RNA extraction kit DP432, and placed in an ultra-low temperature refrigerator for later use.
[0036] 1.2.2 Primer design
[0037] Referring to the DPO primer design method ...
Embodiment 2
[0053] Embodiment 2 result analysis
[0054] 2.1 Establishment of one-step double DPO-RT-PCR detection method
[0055] By adjusting and optimizing the one-step DPO-RT-PCR reaction system, the amplification effect is the best when the final primer concentration is 0.2 μmol / L. The results are as follows figure 1 As shown, through agarose gel electrophoresis, there are specific bands at 609bp and 1060 bp, which are consistent with the target bands, indicating that the one-step double DPO-RT-PCR established by the present invention can be applied to SBMV and TRSV two kinds of viruses detection.
[0056] 2.2 One-step dual DPO-RT-PCR specificity evaluation
[0057] like figure 2 , only samples containing SBMV and TRSV were amplified to obtain the target band, and the other 8 samples and the blank control did not amplify specifically, indicating that the established one-step double DPO-RT-PCR detection method has good specificity and can be used In the detection of SBMV and TRSV...
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