Primers for detecting sugarcane rust and detection method thereof

A detection method and technology for rust, which are applied in the fields of biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., which can solve the actual needs of difficult to meet the rapid and high-throughput diagnostic detection, time-consuming and labor-intensive, etc. problem, to achieve the effect of rapid detection method and good primer specificity

Inactive Publication Date: 2012-03-07
SUGARCANE RES INST OF YUNNAN ACADEMY OF AGRI SCI
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  • Claims
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Problems solved by technology

[0003] For a long time, the detection of sugarcane rust has mainly relied on field symptom observation and indoor microscope or electron microscope observation to confirm. These detection methods are not only time-consuming and labor-intensive, but also the accuracy and reliability of the results depend largely on the researchers. It is difficult to meet the actual needs of rapid and high-throughput diagnostic testing

Method used

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  • Primers for detecting sugarcane rust and detection method thereof
  • Primers for detecting sugarcane rust and detection method thereof
  • Primers for detecting sugarcane rust and detection method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0014] Example 1 Design and synthesis of primers

[0015] According to the sugarcane rust genome sequence (sequence accession number JN035303) reported in the GenBank database, the specific primer PM1 / PM2 for amplifying sugarcane rust was designed, and the target fragment size of the amplification was 593bp, and the primer sequence was as follows:

[0016] PM1: 5'-ATCATCTGTGGGTCAAAC-3'

[0017] PM2: 5'-AATAAAGCCAGAGTAAGC-3'

Embodiment 2

[0018] Example 2 Sugarcane leaf tissue total DNA extraction:

[0019] (1) Take by weighing 0.5g leaves with obvious symptoms of sugarcane rust and put them into a mortar, add an appropriate amount of liquid nitrogen to grind to powder and then transfer to a 2ml centrifuge tube;

[0020] (2) Add 600ul of CTAB extract preheated at 65°C to the centrifuge tube containing the grinding material, mix it upside down and put it in a constant temperature bath at 65°C to incubate for 2 hours, and invert the centrifuge tube several times every 20 minutes;

[0021] (3) Take out the centrifuge tube containing the grind and CTAB extract and place it at -20°C for 2 min;

[0022] (4) Add 600ul of chloroform:isoamyl alcohol (24:1), mix well, and centrifuge at 12000rpm / min for 10min;

[0023] (5) Take the supernatant and add 2 / 3 volume (340ul) of isopropanol, mix gently; place overnight at -20°C;

[0024] (6) Centrifuge at 12000rpm / min for 10min at room temperature;

[0025] (7) The supernata...

Embodiment 3

[0029] The establishment of embodiment 3 PCR amplification method:

[0030] Using the total DNA as a template, carry out PCR reaction. 25ul reaction system for PCR reaction, that is, add in a 200ul PCR reaction tube: 2×Taq PCR MasterMix 6.0ul, PM1 (20umol / L) 1.0ul, PM2 (20umol / l) 1.0ul, DNA template 1.0ul, sterilized wxya 2 O 16ul, short-speed centrifugation for 8-10 seconds after adding, put into PCR instrument, pre-denaturation at 94°C for 5min; denaturation at 94°C for 1min, annealing at 53°C for 50s, extension at 72°C for 1min, 34 cycles; extension at 72°C for 5min, 4 cycles Store at ℃.

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Abstract

The invention provides primers for detecting sugarcane rust and a detection method thereof. The nucleotide sequences of the primers are represented by PM1 and PM2 in a sequence table. The invention further provides the method for detecting sugarcane rust; according to the method, total DNA of a sample is used as a template, the above-mentioned primers are used for PCR amplification, and determination is carried out according to the position of specifically amplified segments after amplification is finished. The method provided in the invention can effectively overcome defects in conventional detection methods like field symptom observation and indoor microscope or electron microscope observation, can rapidly, accurately, specifically and sensitively detect sugarcane rust and can be used for large scale sugarcane rust PCR detection.

Description

Technical field: [0001] The invention relates to a primer for detecting sugarcane rust and a detection method thereof. Background technique: [0002] Sugarcane rust is a disease of sugarcane caused by fungi, which has generally occurred in countries such as Java, the Caribbean (Cuba, Jamaica, etc.), Australia, the United States, Mexico, India, Thailand, and Mauritius in Africa, often causing huge Economic losses. At present, due to the frequent introduction of varieties, single species, diverse planting periods and adverse climate, sugarcane rust has spread to many major sugarcane planting areas in my country, such as Guangxi, Yunnan, Fujian, Sichuan, Jiangxi and Guangdong. Therefore, it is of great significance to strengthen the early detection of sugarcane rust and cut off the seriously diseased leaves in time to reduce the spread of the disease. [0003] For a long time, the detection of sugarcane rust has mainly relied on field symptom observation and indoor microscope...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 王晓燕李文凤黄应昆罗志明尹炯
Owner SUGARCANE RES INST OF YUNNAN ACADEMY OF AGRI SCI
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