Primer, kit and method for rapid detection of machilus nanmu leaf blight
A technology for rhizoma rhizome and leaf blight, which is applied in the field of primers for rapid detection of rhizoma rhizome leaf blight, can solve the problems of time-consuming, easy to miss the best control period, consumables, etc., and achieves the effect of high sensitivity
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Embodiment 1
[0023] Example 1: Primer Design and Screening
[0024] Using DNAMAN software, multiple homology comparisons were performed between the sequencing results of the PCR amplification products of P.microspore strain DNA and the ITS sequences of other species registered in the GenBank database, and the differential sites were selected to design primers with Primer premier5.0, and then used Oligo7.0 performs primer specificity comparison and comprehensive evaluation supplemented by manual design, and selects primers through pre-experiment:
[0025] F: 5'-GAACTTACCATTGTTGCCTCG-3';
[0026] R: 5'-CGCCGTTGTATTTCAGGAG-3', the expected amplified fragment is 578bp.
Embodiment 2
[0027] Embodiment 2: Nested PCR amplification and specificity detection
[0028] Using the DNA of the P. microspore tested strain and other strains as reaction templates, the specific primers screened in Example 1 were used for PCR amplification.
[0029] Nested PCR amplification system 25 μL: DNA template 1 μL, specific upstream and downstream primers F / R 1 μL each, TaqDNA polymerase 22 μL (Chengdu Qingke Zixi Biotechnology Co., Ltd.), and ddH 2 O was used as a negative control instead of DNA template. Amplification program: pre-denaturation at 98°C for 2min, denaturation at 98°C for 10s, annealing at 63°C for 10s, extension at 72°C for 20s, a total of 34 cycles, and extension at 72°C for 2min. After amplification, 3 μL of the product was taken for 2% agarose gel electrophoresis, and then analyzed on a gel imaging system. Each sample was analyzed at least three times. For specific results, see figure 1 .
[0030] figure 1 The middle marks are: 1: Marker; 2: N, negative c...
Embodiment 3
[0034] Embodiment 3: Sensitivity detection of nested PCR
[0035] The P. microspore DNA extracted from the leaves of Runnan was subjected to 2% agarose gel electrophoresis and purified to obtain a standard product, and its content and purity were detected by an ultra-differential spectrophotometer. Dilute the genomic DNA of P.microspora into 100 ng / μL, 10ng / μL, 1ng / μL, 100pg / μL, 10pg / μL, 1pg / μL, 100fg / μL, 10fg / μL, 1fg / μL9 in a 10-fold concentration gradient concentration. Take 1 μL each time as template DNA for conventional PCR and nested PCR reactions. Referring to the above reaction system and reaction procedure, the sensitivity detection was repeated at least three times. For specific results, see Figure 2-3 .
[0036] figure 2 The winning number: 1. M.DL2000 DNA Marker; 2-10. Conventional PCR reaction gradients are 100, 10, 1ng / μL, 100, 10, 1pg / μL, 100, 10, 1fg / μL; 11. Negative control.
[0037] pass figure 2 It is known that the lowest concentration that can be ...
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