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A double PCR detection method and application of cereal cyst nematode and Phillips cyst nematode

A technology for Cyst Nematode and Philips spore, which is applied in the fields of biochemical equipment and methods, recombinant DNA technology, and microbial determination/inspection, etc., can solve problems such as the inability to achieve the detection effect, and achieve a small demand for samples and a reduction in the number of samples. , the effect of simple operation

Active Publication Date: 2019-01-04
NANJING AGRICULTURAL UNIVERSITY
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  • Description
  • Claims
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Problems solved by technology

Double PCR requires specific amplification of different target sites in the same reaction system at the same time, so there are many factors that affect the amplification effect, such as whether dimers are formed between different primers, the difference in annealing temperature, and the ratio of primers. Different parameters such as temperature and reaction temperature have high requirements. Toumi and others tried to mix specific primers for the detection of cereal cyst nematodes and Phillips cyst nematodes respectively, and performed double PCR detection of the two nematodes, but failed to meet expectations. The detection effect, the primers used can only detect the corresponding cyst nematodes alone [13]
At present, there is no report on the detection of cereal cyst nematodes and Phillips cyst nematodes using double PCR technology at home and abroad

Method used

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  • A double PCR detection method and application of cereal cyst nematode and Phillips cyst nematode
  • A double PCR detection method and application of cereal cyst nematode and Phillips cyst nematode
  • A double PCR detection method and application of cereal cyst nematode and Phillips cyst nematode

Examples

Experimental program
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Effect test

Embodiment 1

[0045] Example 1 Extraction of Cereal Cyst Nematode and Phillips Cyst Nematode DNA, mtDNA COI Gene Amplification and Sequence Analysis

[0046] 1.1 DNA extraction of cereal cyst nematodes and Phillips cyst nematodes

[0047] Pick single mature and full cysts of different two kinds of nematodes, put them on a glass slide that has been dripped with 20 μL of lysis buffer, and grind them thoroughly, and recover them into PCR tubes; freeze them in liquid nitrogen for 1 min, and put them in a water bath at 65°C for 2 min, repeat 3 times; finally incubated at 65°C for 1.5h, treated at 95°C for 10min, centrifuged at 14,000r / min for 1min, and the supernatant was used as a nematode DNA template for COI gene amplification and double PCR reaction.

[0048] 1.2 Amplification and sequence analysis of the mtDNA COI gene of Cereal cyst nematode and Phillips cyst nematode

[0049] Universal primers JB3 (5'-TTTTTTGGGCATCCTGAGGTTTAT-3', SEQ ID NO.6) and JB5 (5'-AGACCTAAACTTAAAACATAATGAAAATG-3',...

Embodiment 2

[0050] Example 2 Establishment of double PCR detection method for cereal cyst nematode and Phillips cyst nematode

[0051] 2.1 Duplex PCR primer design

[0052] According to the amplified sequencing results of the COI genes of Cereal cyst nematode and Phillips cyst nematode, and by comparing the sequences with the reported COI genes of other plant parasitic nematodes, the specific upstream primers HaF8 and Phillips cyst nematode were designed and screened. The cyst nematode-specific upstream primer HfF9 and the common downstream primer HafR8 of two kinds of nematodes were synthesized by Shanghai Handsome Biotechnology Co., Ltd. The primer sequences are as follows:

[0053] ①HaF8: 5'-GCTCATCATATATTTGTTGTTGGT-3'(SEQ ID NO.3);

[0054] ②HfR9: 5'-ATTTTGCCGGCATTTGGTCTGG-3'(SEQ ID NO.4);

[0055] ③HafR8: 5'-CCCTTAAACCTCCAACAGTA-3'(SEQ ID NO.5);

[0056] 2.2 Double PCR reaction system configuration:

[0057] 1) Primer mixture: 0.24 μmol / L for HaF8, 0.16 μmol / L for HfF9, and 0.4 ...

Embodiment 3

[0063] Example 3 Double PCR Specific Detection of Cereal Cyst Nematode and Phillips Cyst Nematode

[0064] Collect barley cyst nematode, soybean cyst nematode, root-knot nematode incognita, root-knot nematode incognita, root-knot nematode java, pine wood nematode, potato rot nematode, rice stem apical nematode and other plant parasitic nematodes, and extract their DNA as templates The specificity of the dual PCR detection system was tested by PCR amplification with the DNA templates of cereal cyst nematode and philip cyst nematode respectively.

[0065] After the above primer mixture and reaction buffer mixture are mixed evenly, add 1 μl of template DNA and proceed according to the reaction conditions in 2.3. After the reaction, 5 μl of the product is electrophoresed on 2% agarose gel, stained with EB, observed under ultraviolet light and take pictures (such as figure 2 shown), a specific band of about 200bp can be seen in the first lane (cereal cyst nematode), a specific ba...

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Abstract

The invention discloses primer composition for duplex PCR (polymerase chain reaction) detection of heterodera avenae and heterodera filipjevi and an application of the primer composition. The detection primer comprises HaF8:SEQ ID NO.3, HfF9:SEQ ID NO.4 and HafF8:SEQ ID NO.5. A duplex PCR technology is used, the heterodera arenaria and the heterodera filipjevi are detected synchronously through a single reaction, and the detection cost for two pathogens and the workload are reduced. A duplex PCR detection system which is high in specificity and sensitivity, time-saving, labor-saving and easy to operate is established, and the primer composition can be applied to rapid detection and / or identification of the heterodera avenae and the heterodera filipjevi.

Description

technical field [0001] The invention belongs to the field of biological detection, and relates to a primer composition for double PCR rapid detection of cereal cyst nematodes and philip cyst nematodes and an application thereof. Background technique [0002] Cereal cyst nematodes (CCN) are a group of important pathogenic nematodes that harm wheat, barley, oats and other cereal crops worldwide. [1] Among them, the most widely distributed and the most harmful are mainly cereal cyst nematodes (Heterodera arenaria), Phillip cyst nematodes (H.filipjevi) and wheat cyst nematodes (H.latipons) [2] . [0003] Since the first discovery of cereal cyst nematodes in Tianmen County, Hubei Province in 1989 in my country, [3] So far, the distribution of the pathogen has reached 16 provinces (cities) including Henan, Hebei, Shandong, Jiangsu, and Anhui. [4] , some provinces and regions with serious diseases can reduce production by more than 50%, or even have no harvest [5] . Following ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2537/143C12Q2565/125
Inventor 王暄李红梅牛雯雯陈云芳李师默
Owner NANJING AGRICULTURAL UNIVERSITY
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