Specific sequence characterized amplified region (SCAR) marker and rapid polymerase chain reaction (PCR) detection method for Heterodera filipjevi

A technology for the detection of Phillips spores, which is applied in the field of rapid PCR detection of Phillips cyst nematode SACR specificity, and can solve the problems of reporting and lack of specific primers for Phillips cysts nematode

Inactive Publication Date: 2012-01-25
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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Problems solved by technology

[0012] Although there are research reports on the ITS region of the ribosomal DNA of Phillips cyst nematode at home and abroad, there is no report on the specific primers of Phillips cyst nematode, let alone the detection of Phillips spp. Research Reports on Cyst Nematode

Method used

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  • Specific sequence characterized amplified region (SCAR) marker and rapid polymerase chain reaction (PCR) detection method for Heterodera filipjevi
  • Specific sequence characterized amplified region (SCAR) marker and rapid polymerase chain reaction (PCR) detection method for Heterodera filipjevi
  • Specific sequence characterized amplified region (SCAR) marker and rapid polymerase chain reaction (PCR) detection method for Heterodera filipjevi

Examples

Experimental program
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Effect test

Embodiment 1

[0045] Example 1: DNA fragments, specific primers for Phillips cyst nematode specific SCAR markers

[0046] 1.1 Extraction of DNA from Philip's cyst nematode

[0047] Pick one cyst from each group of Phillips cyst nematode, cereal cyst nematode, and soybean cyst nematode by hand, put it into 10 μl sterilized redistilled water, and freeze it in a -20°C refrigerator for 1 hour. A glass rod sterilized with 75% alcohol was rotated in an eppendorf tube until the ice melted, 7 μl of 10X PCR buffer and 3 μl of proteinase K solution (600 μg / ml) were added, and then frozen at -20°C for at least 2 hours. After 2 hours, take the eppendorf tube out of the refrigerator and incubate at 65°C for 1.5h; then denature proteinase K at 95°C for 10 minutes, centrifuge at 1000 rpm for 1 minute, and take the supernatant DNA suspension and store it at -20°C for later use.

[0048] 1.2 Screening of Phillips cyst nematode-specific RAPD markers (SCAR markers)

[0049] Drawing on the results of genetic...

Embodiment 2

[0060] Example 2: Molecular detection of Phillips cyst nematode-specific SCAR marker primers

[0061]Phillips cyst nematode-specific SCAR marker primers HfF1 and HfR1 constructed in the present invention were synthesized by Shanghai Sangon Biology Co., Ltd. The total volume of the specific SCAR marker amplification reaction system was 50 μl, which contained 5 μl 10XPCR buffer (containing Mg++); 4 μl 2.5mM dNTP; 1.0μl HfF1 (0.1μg / μl); 1.0μl HfR1 (0.1μg / μl); 1U Taq DNA polymerase (5U / μl); 2μl template DNA (Philips cyst nematode and other cyst nematode population extraction DNA); ddH 2 O 36.5 μl.

[0062] The applied Phillips cyst nematode-specific SCAR marker primer sequences are as follows:

[0063] HfF1: 5'-AATCCTTACACCGTTGTTTATTA-3'

[0064] HfR1: 5'-ATTCTTCCTCCTTCTCCCACTAT-3'

[0065] The PCR amplification program was as follows: pre-denaturation at 94°C for 4 min, followed by 35 cycles of 94°C for 30S, 56°C for 30S, 72°C for 1 min, 72°C for 10 min, and storage at 4°C. ...

Embodiment 3

[0067] Embodiment 3: One-step double PCR method of specific SCAR marker primers and universal primers (TW81 and AB28) detects Phillips cyst nematode

[0068] In the present invention, the constructed philip cyst nematode specific SCAR marker primers HfF1 and HfR1 and the universal primers TW81 and AB28 are placed in the same PCR reaction, and HfF1 and HfR1 specifically amplify the SCAR of philip cyst nematode genomic DNA The marker fragment, primers TW81 and AB28 were used to amplify the DNA fragment of the rDNA gene ITS extension region. The one-step double PCR method adopted in the present invention is the world's first molecular diagnosis method of Philip's cyst nematode, and the molecular detection method contains two fragments of genomic DNA and ribosomal DNA.

[0069] The molecular detection method of Phillips cyst nematode of the present invention contains a 25 μl PCR reaction system, and the proportion is as follows: 10X PCR-buffer (containing Mg++) 2.5 μl, dNTPs 2 μl,...

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Abstract

The invention discloses a specific sequence characterized amplified region (SCAR) marker and a rapid polymerase chain reaction (PCR) detection method for Heterodera filipjevi, and belongs to the field of biotechnology. A deoxyribonucleic acid (DNA) fragment of the specific SCAR marker for the Heterodera filipjevi is characterized in that: the DNA fragment has a nucleotide sequence shown as SEQ ID NO1. Primers, namely HfF1 and HfR1 which are designed according to the DNA fragment of the specific SCAR marker for the Heterodera filipjevi can amplify a fragment of 313bp in the rapid PCR detection method for the Heterodera filipjevi; meanwhile, universal primers, namely TW81 and AB28 can also be added into a PCR reaction system to serve as interior labels, and a fragment of 1000bp can be synchronously amplified. The sensitivity of molecular detection is improved, and the method is quick and accurate, and has high application value in the fields of the detection of the Heterodera filipjevi and the early diagnosis of Heterodera filipjevi disease.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a complete sequence of a philip cyst nematode specific SCAR marker, a specific SCAR marker primer sequence and a rapid PCR detection method for a philip cyst nematode SACR specificity. Background technique [0002] Wheat cyst nematode (CCN) is a worldwide important pathogenic nematode on temperate cereal crops. Since it was discovered in Germany in 1874, it has subsequently spread in the United Kingdom, Russia, Norway, Australia, Canada, Japan, India, the United States, New Zealand, and China. More than 40 countries, including the United States, have suffered damage to varying degrees, seriously affecting the yield and quality of cereal crops, especially wheat crops. In my country, since Peng Deliang and others first discovered that cereal cyst nematodes damaged wheat in Tianmen County, Hubei, China in 1989, it has been found in Hubei, Hebei, Henan, Beijing, Shanxi, Inner Mongo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 彭德良亓晓莉黄文坤彭焕贺文婷
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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