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Primer pairs for detecting germs of paddy bacterial glume blight and detection method

A kind of rice blight bacteria, bacterial technology, applied in the field of primers and detection for the detection of rice bacterial rice blight, can solve the problems of lack of detection methods and standards for rice bacterial blight, achieve high accuracy, high detection sensitivity, Good effect of primer specificity

Inactive Publication Date: 2010-05-26
湖南出入境检验检疫局检验检疫技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, my country lacks detection methods and standards for bacterial rice blight

Method used

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  • Primer pairs for detecting germs of paddy bacterial glume blight and detection method
  • Primer pairs for detecting germs of paddy bacterial glume blight and detection method
  • Primer pairs for detecting germs of paddy bacterial glume blight and detection method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Primer design and synthesis

[0027] According to the reported ITS region sequence (genebank D87080) of bacterial rice blight of rice, the primer sequence was designed as follows:

[0028] SEQ ID NO.1 (forward): 5'-ACACGGAACACCTGGGTA-3';

[0029] SEQ ID NO. 2 (reverse): 5'-TCGCTCTCCCGAAGAGAT-3'.

[0030] According to the reported gryB gene (genebank AB207074) of bacterial rice blight of rice, primers and probes were designed, and the primer sequences are:

[0031] SEQ ID NO.3 (forward): 5'-GCAGCGGCAAGGAAGACG-3';

[0032] SEQ ID NO. 4 (reverse): 5'-GTCGTCGCCCGACGTCTC-3'.

Embodiment 2

[0034] Isolation of Rice Bacterial Rice Blight from Hybrid Rice Seeds

[0035]The semi-selective isolation medium S-PG agar medium reported in the literature was used to isolate and cultivate the pathogen in rice materials. The bacterial grain blight showed two growth forms on S-PG, one colony was reddish-brown, round, smooth, and raised; the other colony was lavender, round, smooth, and raised.

[0036] Sample handling method

[0037] After washing a large number of seed samples with distilled water, weigh 10g or 400 seeds and put them into 90mL 0.001% Tween-phosphate buffer (pH7.0) for 4-6 hours at low temperature (5°C-15°C), The instrument was smashed to prepare the sample extract. If it is to detect a small amount of seed samples, take 20 seeds and add 10 mL of sterilized 0.001% Tween-phosphate buffer (pH 7.0), fully grind the seeds with a sterilized mallet and mortar or agitator, and prepare into an extract.

[0038] Place the sample extract in an environment of 22°C-...

Embodiment 3

[0041] Bacterial DNA Extraction

[0042] 1) Directly pick at least 5 typical or suspected colonies grown on the S-PG plate into a 1.5mL centrifuge tube, add 1mL sterilized water to shake fully, centrifuge at 12000g for 3min, discard the supernatant, and then add 1mL sterilized Water, repeat three times, take the precipitate for DNA extraction;

[0043] 2) Add 600 μL of TE buffer, 30 μL of 10% SDS solution, and 15 μL of 20 mg / mL proteinase K to the precipitate, mix well, and incubate in a water bath at 37° C. for 1 h.

[0044] 3) Add an equal volume of chloroform-isoamyl alcohol (24:1), and mix well. Centrifuge at 10,000 g for 5 min, and transfer the supernatant to a new centrifuge tube.

[0045] 4) Add an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1), mix well, centrifuge at 10000 g for 5 min, and transfer the supernatant to a new centrifuge tube.

[0046] 5) Add 0.6 times the volume of isopropanol, mix gently, precipitate at -20°C for 1 hour, centrifuge at 10...

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Abstract

The invention provides two pairs of primers for detecting germs of paddy bacterial glume blight. The nucleotide sequences of the primers are SEQ ID NO.1: 5'-ACACGGAACACCTGGGTA-3', SEQ ID NO.2: 5'-TCGCTCTCCCGAAGAGAT-3', SEQ ID NO.3: 5'-GCAGCGGCAAGGAAGACG-3', and SEQ ID NO.4: 5'-GTCGTCGCCCGACGTCTC-3'. The invention also provides a method for detecting germs of paddy bacterial glume blight, which comprises the following steps: separating the germs of paddy bacterial glume blight in samples, using the bacterial DNA as a template, carrying out dual PCR amplification by using the primers, carrying out DHPLC analysis on the amplification products, and amplifying the obtained positive results by the primers BGF3 and BGR3 to obtain positive fragments as molecule specimens for carrying out identification. The invention has good primer specificity and high accuracy and high sensitivity of the detection method, thereby ensuring safety in import and export.

Description

technical field [0001] The invention relates to biological detection technology, in particular to a primer and a detection method for the detection of rice bacterial rice blight. Background technique [0002] Bacterial grain rot of rice is caused by Burkholderia glumae. It is a serious rice seed-borne disease in recent years. It not only damages grains, but also causes rice Seedlings rot, causing severe yield reduction and loss of rice. Seed carrier is the main way for the long-distance transmission of the pathogen. When sowing the disease-carrying grains, there are suitable conditions for the disease, such as high temperature and sunshine, and less rainfall. The germs can invade through the stomata and wounds, the germs multiply in the plant, spread among the cells, and cause the decomposition of the parenchyma of the leaf sheath. [0003] In the late 1970s, due to the implementation of mechanized production and large-scale industrial seedling cultivation, rice seedling r...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12R1/01
Inventor 朱金国莫瑾
Owner 湖南出入境检验检疫局检验检疫技术中心
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