Single tube nested PCR (polymerase chain reaction) detection method for wheat stripe rust bacteria and primer thereof

A technology of stripe rust single and wheat, which is applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., to achieve the effect of reducing detection cost, simple method and improving sensitivity

Inactive Publication Date: 2011-08-17
CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there are no reports and applications based on the single-tube

Method used

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  • Single tube nested PCR (polymerase chain reaction) detection method for wheat stripe rust bacteria and primer thereof
  • Single tube nested PCR (polymerase chain reaction) detection method for wheat stripe rust bacteria and primer thereof
  • Single tube nested PCR (polymerase chain reaction) detection method for wheat stripe rust bacteria and primer thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1: PCR detection specificity verification of wheat stripe rust

[0033] 1 Specific primers for detecting wheat stripe rust

[0034] Pst-stnInF: 5'-CCTAAG GTGTCT GATACC GTT-3'

[0035] Pst-stnInR: 5'-GGCATC AAACAT TTGCGA-3'

[0036] 2 Common PCR amplification reaction system and reaction procedure of wheat stripe rust DNA

[0037] 25 μL reaction system for general PCR amplification of wheat stripe rust DNA, including TaqpreMix 12.5 μL, 10 μM primers Pst-stnInF, Pst-stnInR 1.0 μL each, DNA template 1.0 μL, ddH 2 O 9.5 μL. Perform PCR amplification on a PCR instrument according to the following procedures: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec, extension at 72°C for 60 sec, and 35 cycles.

[0038] 3 Required DNA preparation

[0039] Genomic DNA of wheat stripe rust, leaf rust, stalk fungus, powdery mildew, root rot, gibberella, and leaf blight was extracted by CTAB method, the DNA quality and DNA c...

Embodiment 2

[0043] Embodiment 2: Comparison of the detection sensitivity of ordinary PCR and single-tube nested PCR of wheat stripe rust ureterospore

[0044] 1 Specific primers for detecting wheat stripe rust

[0045] Pst-stnOutF: 5'-TATGAT GGCCAC CTTCTC CGTTGT CC-3';

[0046] Pst-stnOutR: 5'-AAGTAT CGGCCG TGTCTC GGGTCA GA-3';

[0047] Pst-stnInF: 5'-CCTAAG GTGTCT GATACC GTT-3';

[0048] Pst-stnInR: 5'-GGCATC AAACAT TTGCGA-3';

[0049] 2 Gradient concentration DNA preparation for sensitive detection

[0050] DNA of known concentration was treated with ddH 2 O was serially diluted 10 times to 10 -8 , as a DNA template for ordinary PCR and single-tube nested PCR.

[0051] 3 common PCR sensitivity test

[0052] 25 μL reaction system for common PCR amplification of wheat stripe rust DNA, including TaqpreMix 12.5 μL, 10 μM primers Pst-stnInF, Pst-stnInR 1.0 μL each, DNA template 1.0 μL, ddH 2 O 9.5 μL.

[0053] Perform PCR amplification on a PCR instrument according to the following ...

Embodiment 3

[0060] Embodiment 3: Single-tube nested PCR detection on wheat leaves infected by wheat stripe rust and still in incubation period

[0061] 1 Wheat seedling inoculation and preparation of sample DNA to be tested

[0062] Spray method was used to inoculate wheat leaves at the one-leaf stage, the concentration of uredospores of wheat stripe rust was 0.15 mg / mL, and the inoculated wheat leaves were kept moist in the dark, and 5 wheat leaves were cut each at 12h, 24h, 2d, 4d, and 6d, respectively. Extract DNA.

[0063] 2 Single-tube nested PCR detection of wheat leaves not yet diseased

[0064] Using the DNA prepared in 1 as a template, use 25 μL stn PCR amplification system: Ex TaqMix 12.5 μL, 0.1 μM forward and reverse outer primers (Pst-stnOutF / PS-stnOutR) 1 μL each, 10 μM forward and reverse inner primers (Pst -stnInF / Pst-stnInR) each 1.5μL, template DNA 1.5μL, with ddH 2 O to make up to 25.0 μL)

[0065] Perform PCR amplification on the PCR instrument according to the fol...

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Abstract

The invention relates to a single tube nested PCR (polymerase chain reaction) detection method for wheat stripe rust bacteria and a primer thereof. The method comprises the following steps: in a single PCR reaction tube, finishing the nested PCR amplification once by an inner pair and an outer pair of wheat stripe rust bacteria specific primers; and judging that wheats or unknown pathogen spores to be detected are wheats infected by the wheat stripe rust bacteria or the wheat stripe rust bacteria by a target segment, wherein the sequences of the nucleotide residues of the inner pair and the outer pair of wheat stripe rust bacteria specific primers are disclosed in SEQ ID No.1, 2, 3 and 4. The method disclosed by the invention has good specificity, is simple and easy to realize, and can beused for early detection for field wheat stripe rust and used for monitoring diseases, and the detection sensitivity is 20fg.

Description

technical field [0001] The invention relates to the field of plant disease epidemic monitoring, in particular to a single-tube nested one-step PCR detection method and primers for wheat stripe rust. Background technique [0002] Wheat stripe rust is the most important disease of wheat, the main food crop in my country, and its occurrence directly affects the yield and quality of wheat, threatening national food security. Since 1949, the four major epidemics of wheat stripe rust in my country have caused losses of 6.0, 3.0, 180, and 130 million kilograms of wheat production respectively. Wheat stripe rust is an airborne large-scale epidemic disease. Longnan and Longdong are recognized as oversummer bases of wheat stripe rust in my country. The occurrence of wheat stripe rust in autumn seedlings in this area is very important for the development of spring disease in other endemic areas. Therefore, the early monitoring and control of wheat stripe rust in Longnan and Longdong is...

Claims

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Application Information

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IPC IPC(8): C12Q1/04C12Q1/68C12N15/11
Inventor 马占鸿黄冲孙振宇王海光
Owner CHINA AGRI UNIV
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