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Multiplex DPO-PCR (dual-priming oligonucleotide-polymerase chain reaction) detection kit for sunflower white rust and black stem and application thereof

A technology of white rust bacteria and sunflower, applied in the direction of recombinant DNA technology, microbial measurement/inspection, DNA/RNA fragments, etc., can solve the problems of complex detection process, low specificity, long identification cycle, etc., and achieve high sensitivity, Good specificity and insensitive to annealing temperature

Inactive Publication Date: 2015-09-30
中华人民共和国伊犁出入境检验检疫局 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, it is very important to carry out accurate quarantine identification of these two pathogenic bacteria, while traditional morphological methods have a long identification cycle and poor timeliness.
Methods for identifying these two bacteria using serological and molecular biology techniques have been established, including PCR, RFLP, etc., but some of these methods require enzyme digestion, and the detection process is complicated; some are not specific or rely on more expensive equipment. Difficult to generalize between labs

Method used

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  • Multiplex DPO-PCR (dual-priming oligonucleotide-polymerase chain reaction) detection kit for sunflower white rust and black stem and application thereof
  • Multiplex DPO-PCR (dual-priming oligonucleotide-polymerase chain reaction) detection kit for sunflower white rust and black stem and application thereof
  • Multiplex DPO-PCR (dual-priming oligonucleotide-polymerase chain reaction) detection kit for sunflower white rust and black stem and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1, a kind of method that detects sunflower white rust bacterium and sunflower black stem bacterium

[0039] 1. Design of Multiplex DPO-PCR Primer Sets

[0040] According to the large subunit ribosomal RNA gene sequence of the sunflower white rust fungus and the ITS-5.8SrRNA gene sequence of the sunflower black stem fungus, the following multiplex DPO-PCR detection primer sets (primer sequences) for detecting the sunflower white rust fungus and the sunflower black stem fungus were designed The I in is hypoxanthine):

[0041] BS-DPO-F: CGAATTGTAGTCTATCGAGGCCAAGIIIIIIACGCAGGATCC (Sequence 1);

[0042] BS-DPO-R: GGAATGGACAGCGGACGCIIIIIGCTTCCCT (Sequence 2);

[0043] HJ-DPO-F: GATGCCGGTACTCTGGGTCTTTIIIIICATGTACC (Sequence 3);

[0044] HJ-DPO-R: ATTGTTTTGAGGCGAGTTTCCCCIIIIIGGAAACAT (SEQ ID NO: 4);

[0045] Among them, BS-DPO-F and BS-DPO-R are primers for detecting sunflower white rust, and the product size is 307bp; HJ-DPO-F and HJ-DPO-R are primers for detect...

Embodiment 2

[0062] Embodiment 2, the specific detection of multiplex DPO-PCR primer

[0063] 1. Extraction of DNA

[0064] Refer to the kit operation (Plant Genomic DNA Extraction Kit, Cat. No. DP305-02, Tiangen Biotechnology Co., Ltd.) to extract the genomic DNA of the susceptible plants or pure strains (stored in Yili Entry-Exit Inspection and Quarantine Bureau) in Table 2. The specific method is as follows: the non-culturable bacterial strains (numbered 5, 8, 9 in table 2) can directly collect susceptible plants, and the DNA is extracted after liquid nitrogen is fully ground; , 3, 4, 6, 7) After 5-7 days of pure culture, the hyphae were picked and freeze-dried, and then fully ground with liquid nitrogen to extract DNA.

[0065] Table 2. Tested strains

[0066]

[0067] 2. Multiplex DPO-PCR amplification

[0068] Adopt the method for detecting sunflower white rust bacterium and sunflower black stem bacterium of step 2 of embodiment 1, carry out multiple DPO-PCR amplification with ...

Embodiment 3

[0072] Example 3, Detection of Annealing Temperature Sensitivity of Multiplex DPO-PCR Primers

[0073] 1. Extraction of DNA

[0074] The kit method (Plant Genomic DNA Extraction Kit, Cat. No. DP305-02, Tiangen Biotechnology Co., Ltd.) was used to extract the genomic DNA of Helianthus albicans and Helianthus helianthus respectively.

[0075] 2. Multiplex DPO-PCR amplification

[0076] With the genomic DNA of the sunflower white rust fungus and the sunflower black stem fungus obtained in step 1 as a template, adopt the method for simultaneously detecting the sunflower white rust fungus and the sunflower black stem fungus in the step 2 of embodiment 1, with different annealing temperatures (45 ℃, 50°C, 55°C, 60°C, 65°C) for PCR amplification, respectively, and the rest of the reaction conditions were unchanged, to obtain multiple DPO-PCR amplification products respectively.

[0077] 3. Electrophoretic detection of multiplex DPO-PCR amplification products

[0078] After the mul...

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Abstract

The invention discloses a multiplex DPO-PCR (dual-priming oligonucleotide-polymerase chain reaction) detection kit for sunflower white rust and black stem and application thereof. The multiplex DPO-PCR detection kit comprises a primer set for detecting or auxiliarily detecting whether the detected strain is sunflower white rust or sunflower black stem, wherein the primer set is composed of a primer 1, a primer 2, a primer 3 and a primer 4. The test proves that the DPO primers and detection kit have the characteristics of favorable specificity, high accuracy and high sensitivity and can be used for detection, differentiation and identification of sunflower white rust and sunflower black stem.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a multiplex DPO-PCR detection kit for sunflower white rust fungus and black stem fungus and application thereof. Background technique [0002] Sunflower white rust (Albugo tragopogonis (Pers.) Gray) is the pathogen that causes sunflower white rust. This fungus has seriously occurred in a few foreign countries and regions, with great economic impact and high risk of introduction. It has become a quarantine pest of international concern. . Sunflower black stem fungus (Leptosphaeria lindquistii Frezzi) is the fungus that causes sunflower black stem disease. The incidence of serious diseased fields can reach 100%, causing devastating damage to sunflower production. It is an important quarantine pathogen. Therefore, it is very important to carry out accurate quarantine identification of these two pathogenic bacteria, while traditional morphological methods have a long identif...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 魏霜乾义柯袁俊杰张娜陈卫民尚爽梁巧玲赛铁尔汗刘中勇鄞杰平
Owner 中华人民共和国伊犁出入境检验检疫局
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