LAMP primer group and method for identifying Mi 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, strong practicability, and good specificity
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Embodiment 1
[0028] Example 1 DNA Extraction of Mi Toxic Meloidogyne incognita and Optimization of LAMP Reaction System
[0029] 1. DNA Extraction of Mitotoxic Meloidogyne incognita
[0030] The DNA extraction method of Mi-toxic root-knot nematode incognita is referred to the method of Wang Jiangling et al. 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 Triton X-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 the supernatant was drawn That is, it can be used as a DNA template.
[0031] 2. Specific LAMP primer design
[0032] Using primers Mi F / R (5'-AGGGAGCTCGTGTGTGTGT-3'; 5'-GTCAAGCAGGCAGGCAGT-3'), the unique sequence of Mi virulent root-knot nematode was amplified. The reaction system is 1 μL (50ng) of DNA template, 10×PCRbuffer (+Mg 2+)50μL, 250μmol L -1 2 μL of dNTPs, 1 μL of each primer BX1 F / R (0.3 μmol...
Embodiment 2
[0045] Example 2 LAMP reaction sensitivity and specificity detection
[0046] With reference to the method of embodiment 1, extract the DNA of single Mi poisonous root-knot nematode, dilute successively, gradient is 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 of the example, 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.
[0047] With reference to the method of embodim...
Embodiment 3
[0048] Example 3 Mi Toxic Meloidogyne incognita LAMP Detection Kit and Technical Field Application Verification From Langfang, Hebei, Shunyi, Beijing, Xinxiang, Henan and other places, the root knots or soil on the root system were collected for the field plots where root-knot nematode occurred egg mass in. 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.
[0049] 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:
[0050] (1) Extract DNA samples from root knots or egg masses of diseased plants
[0051] ...
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