Primers and probe for detecting meloidogyne incognita chitwood through real-time fluorescent quantitative polymerase chain reaction (PCR) and application
A real-time fluorescence quantitative technology for root-knot nematode incognita, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., and can solve the problems of high professional knowledge background requirements and slow detection speed of detection personnel , to achieve a good linear relationship
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Embodiment 1
[0025] Embodiment 1 Designs primers
[0026] According to the genomic DNA of M. incognita, the present invention designs and detects the nucleotide sequences of primers and probes for M. incognita,
[0027] Forward primer sequence MiF2: 5'-CTACCCTTATCGGTGGATCACTA-3',
[0028] Reverse primer MiR2: 5'-AATGACCCTGAACCAGACGTT-3';
[0029] TaqMan probe sequence MiP2: 5'-AGTTTGACCTCAATGCGGCCATTTGCGT-3', its 5' end is labeled with a fluorescein FAM group, and its 3' end is labeled with a TAMAR quencher group.
[0030] The primers of the present invention are compared with 2 groups of primers with higher scores designed by software, the results are shown in Table 1, figure 1 .
[0031] Table 1 qPCR detection results of the same template, different primers and probes
[0032]
[0033]
[0034] The results are shown in Table 1. Under the conditions of the same template and different primers, the amplification Ct value of Mi-F2 / R2 was smaller than that of other primers and the f...
Embodiment 2
[0040] Embodiment 2 examines the specificity of primer of the present invention
[0041] The present invention uses oat cereal cyst nematodes, soybean cyst nematodes, Phillips cyst nematodes, sugar beet cyst nematodes, stem nematodes, root-knot nematodes incognita, brevigrain nematodes, Verticillium dahliae, and Rhizoctonia solani AG-3, Rhizoctonia solani AG-4HGⅢ, Fusarium oxysporum wilt-specific FOV7, Fusarium oxysporum watermelon-specific Fon, Fusarium oxysporum cucumber-specific FOC1-2-11, Glucosporum anthracnose Bacteria as a control, with ddH 2 O negative control. Extract the DNA of the above-mentioned nematodes or soil-borne disease pathogens, and perform PCR amplification with primers MiF2 / MiR2. The PCR conditions are: rTaq 0.5 μL, MgCl 2 3 μL, 10xPCR (Mg 2+ free) Buffer 2.5 μL, dNTPs (25mm) 2 μL, MiF2 (10 μM) 1 μL, MiR2 (10 μM), 1 μL, 0.1% BSA 5 μL, MiP2 1 μL, H 2 O 4 μL, template 5 μL. PCR amplification program: 95°C for 30s; 94°C for 5s, 60°C for 30s, 45cycles....
Embodiment 3
[0047] Embodiment 3 examines the sensitivity of primer
[0048]The M. incognita specific primer Mi-F2 / R2 was used as a primer and the M. incognita genome was used as a template for PCR amplification. The amplified product was cloned into pMD18-T to construct the plasmid recombination vector pMD18-MiT. The extracted plasmid pMD18-MiT was digested with the endonuclease EcoRI, and the digested product was electrophoresed on agarose gel, and then the gel was cut to recover the corresponding fragments. The fragments were recovered by using the GelExtraction Kit, and the DNA of the recombinant plasmid was determined by a Nanodrop 2000 spectrophotometer. Concentration, and the copy number of recombinant plasmid DNA was calculated according to the formula (Whelan et al., 2003).
[0049] with ddH 2 O Dilute the above plasmid vector gradient to 1 × 10 8 , 1×10 7 , 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 , 1×10 2 , 1×10 1 , 1×10 0 copies / μL of dilution.
[0050] The real-time fluore...
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