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Nest PCR (polymerase chain reaction) detection method for peanut pseudomonas solanacearum

A R. solanacearum and detection method technology, which is applied in the direction of biochemical equipment and methods, microbe determination/inspection, etc., can solve the problem of high sensitivity, and achieve the effect of high sensitivity, good practicability, simple and fast operation

Inactive Publication Date: 2014-03-05
INST OF PLANT PROTECTION FAAS
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The present invention compares the 16S-23S rDNA ITS sequence of Ralstonia solanacearum of peanut and the genus of closely related species to select different sites to design specific primers, and uses nested PCR to detect Rsalcum wilt of peanut, which has high sensitivity and strong specificity. see the report

Method used

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  • Nest PCR (polymerase chain reaction) detection method for peanut pseudomonas solanacearum
  • Nest PCR (polymerase chain reaction) detection method for peanut pseudomonas solanacearum
  • Nest PCR (polymerase chain reaction) detection method for peanut pseudomonas solanacearum

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1 Nested PCR Detection of Ralstonia solanacearum in Peanut

[0030] 1. Sample DNA extraction

[0031] 1.1 Extraction of cell DNA

[0032] Inoculate peanut solanacearum in NA liquid medium, culture overnight at 28°C and 200rpm, and extract bacterial genomic DNA by CTAB method. The specific operation is as follows:

[0033] 1) Take 1.5ml culture in a 2.0ml centrifuge tube and centrifuge at 12000rpm for 2min;

[0034] 2) Discard the supernatant, add 567μl TE buffer to resuspend the cells, add 30μl 10% SDS solution and 3μl 20mg / ml proteinase K, mix gently, and incubate at 37℃ for 1h;

[0035] 3) Add 100 μl 5mol / L NaCl solution, mix thoroughly, then add 80 μl CTAB / NaCl solution, mix and incubate at 65°C for 10 minutes;

[0036] 4) Add 800μl of phenol / chloroform / isoamyl alcohol solution (the volume ratio of the three is 25:24:1), mix well and centrifuge at 12000rpm for 5min;

[0037] 5) Transfer the supernatant to a new 1.5ml centrifuge tube, add 0.6-0.8 times th...

Embodiment 2

[0063] Example 2 Conventional PCR Sensitivity Test of Peanut Solanacearum

[0064] Genomic DNA of R. solanacearum was diluted to 8 concentration gradients of 10ng / μl, 1ng / μl, 100pg / μl, 10pg / μl, 1pg / μl, 100fg / μl, 10 fg / μl and 1 fg / μl respectively. Take 1 μl of DNA of each concentration as a template, and only use specific primers W1 / W2 to carry out PCR amplification once respectively, and the same results are obtained after 3 repetitions.

[0065] figure 2 It is the amplification result of conventional PCR sensitivity detection of R. solanacearum. Among them, M is the 2000 bp DNA molecular weight marker, 1-8 are different concentrations of R. solanacearum DNA, the concentrations are 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, 1 fg / μl , 9 is the negative control.

[0066] figure 2 The results showed that a single PCR amplification using specific primers W1 / W2 alone amplified a 374bp specific band in lanes 1-5, indicating that the minimum detection concentration of pat...

Embodiment 3

[0067] Example 3 Nested PCR Sensitivity Test of Ralstonia solanacearum in Peanut

[0068] As in Example 2, the genomic DNA of Ralstonia solanacearum was diluted to 8 samples of 10ng / μl, 1ng / μl, 100pg / μl, 10pg / μl, 1pg / μl, 100fg / μl, 10fg / μl and 1fg / μl respectively. Concentration gradient. Take 1 μl of DNA at each concentration as a template, and perform the first and second rounds of PCR amplification sequentially according to Step 2 and Step 3 of Example 1, respectively.

[0069] The electrophoresis results of the products amplified by two rounds of PCR are shown in image 3 . In the figure, M is a 2000bp DNA molecular weight marker, 1-8 are different concentrations of peanut R. solanacearum DNA, the concentrations are 10ng, 1ng, 100 pg, 10 pg, 1pg, 100 fg, 10 fg, 1 fg / μl, 9 is a negative control.

[0070] image 3 The results showed that after two rounds of PCR amplification, a specific band of 374bp was amplified in lanes 1-7, indicating that the minimum detection concen...

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Abstract

The invention relates to a nest PCR (polymerase chain reaction) detection method for peanut pseudomonas solanacearum, which comprises the following steps of performing a first round of PCR amplification by use of a universal primer L1 / L2 for a bacterial 16S-23S rDNA ITS sequence by adopting a sample DNA as a template; performing a second round of PCR amplification by use of a designed pair of specific primers W1 / W2 for identifying the peanut pseudomonas solanacearum by adopting the product of the first round of PCR as a template; performing electrophoresis detection on the amplification product, wherein if a 374bp specific strip appears, the detected pathogenic bacterium is determined to be peanut pseudomonas solanacearum. In the nest PCR detection method for peanut pseudomonas solanacearum, provided by the invention, the detection sensitivity on the germ genome DNA is 10fg, and the shortcomings of the methods such as biological assay, enzyme linked immunosorbent assay technology, conventional PCR and the like can be overcome. The invention provides a quick, sensitive and specific detection method for peanut pseudomonas solanacearum, which can be applied to the early diagnosis of field peanut pseudomonas solanacearum and is of great significance to the early detection and timely prevention of the disease.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a nested PCR detection method for R. solanacearum of peanut. Background technique [0002] Peanut bacterial wilt is caused by Ralstonia solanacearu A bacterial vascular disease caused by The disease was first reported in Indonesia in 1905, and subsequently occurred or reported in more than 20 countries and regions. At present, China, Indonesia and Vietnam are mainly at risk. Reports on peanut bacterial wilt in my country first appeared in the late 1930s, and are now mainly distributed in the central and southern regions. 2 Above, the degree of harm ranks first in the world. The incidence of peanut bacterial wilt is generally about 10%-30%, and the yield is reduced by more than 20%; the incidence of severe disease areas can be more than 50%, and even lead to complete crop failure. In recent years, reports have shown that peanut bacterial wilt has shown a tendency to s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/04C12Q1/6848C12Q1/686C12Q2549/119C12Q2531/113
Inventor 游泳谢世勇李本金陈庆河丁雪玲刘裴清
Owner INST OF PLANT PROTECTION FAAS
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