LAMP primer group and method for identifying Me toxic meloidogyne spp
A root-knot nematode and primer set technology, applied in the field of molecular biology, can solve the problem of no relevant research and reports on virulent nematodes, and achieve accurate and stable detection results, easy operation, and good specificity
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Embodiment 1
[0030] Example 1 DNA Extraction of Metotoxic Meloidogyne incognita and Optimization of LAMP Reaction System
[0031] 1. DNA Extraction of Metotoxic Meloidogyne incognita
[0032]The DNA extraction method of Metotoxic M. incognita refers to the method of Wang Jiangling et al. (2011) with a slight modification. Pick a single Metotoxic M. 10 μL ddH 2 In the 0.2mL PCR tube of O, add 7μL nematode lysis solution (20mM Tris-HCl (pH8.3), 1.5mM MgCl 2 , 60mM KCl, 10mg / mL TritonX-100, 0.2mM DDT) and 3μL of proteinase K (2mg / mL) were incubated at 65°C for 45min, at 95°C for 10min, then cooled to room temperature for 5min, and then aspirated the supernatant That is, it can be used as a DNA template.
[0033] 2. Design of specific LAMP primers
[0034] Using primers MeF (5'-CTTACAACGCCGCTCTGAAT-3') and MeR (5'-CGACTCCTATAGGGGCGAAT-3'), the unique sequence of Metotoxic root-knot nematode was amplified. The reaction system is: DNA template 1μL (50ng), 10×PCR buffer (+Mg 2+ )50μL, 250μm...
Embodiment 2
[0048] Example 2 LAMP reaction sensitivity and specificity detection
[0049] With reference to the method in Example 1, the DNA of a single Metotoxic root-knot nematode was extracted, diluted successively, and the gradient was 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 , respectively take 1 μL of the above-mentioned diluted DNA as a template, perform LAMP detection according to the optimized system in Example 1, and use double-distilled water as a negative control. At the same time, the same amount of DNA was used as a template to carry out PCR detection according to the reaction procedure in Example 1, and the amplified band was detected by gel electrophoresis. The results show that the template concentration level detected by the LAMP method is 10 -4 Level, while the reaction concentration detected by PCR method is 10 -2 , which means that the detection sensitivity of the LAMP method is 100 times that of the PCR reaction.
[0050] With reference to the method ...
Embodiment 3
[0051] Example 3 Field Application Verification of Me-toxic Meloidogyne incognita LAMP Detection Kit and Technology
[0052] We went to Langfang, Hebei, Shunyi, Beijing, Xinxiang, Henan and other places to collect root knots on the roots or egg masses in the soil for root-knot nematode occurrence fields. The root knots and egg masses collected from tomato roots in Shunyi, Beijing were tested (sample number SY-1\2\3); the root knots and egg masses collected from peppers in Langfang, Hebei were tested (sample number LF-1\2\ 3) Root knots and egg masses on cucumber roots were collected in Xinxiang, Henan (sample number XX-1\2\3). Samples from each location were replicated three times.
[0053] According to the LAMP detection method and kit, the samples were tested on-site, and double distilled water was used as a negative control for detection. The operating procedure is as follows:
[0054] (1) Extract DNA samples from root knots or egg masses of diseased plants
[0055] Rin...
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