Method for identifying bentgrass nematode by real time fluorescent PCR technology

A technical identification and real-time fluorescence technology, which is applied in the field of biological identification, can solve the problems of long identification time, complicated procedures, and difficulties in identifying the nematodes bentgrass, so as to speed up the quarantine, improve the detection level, and promote the quarantine of nematodes. Effect

Inactive Publication Date: 2009-04-29
SOUTH CHINA AGRI UNIV +1
View PDF0 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The identification of bentgrain nematodes is an important task in my country's entry-exit inspection and quarantine. However, there is no standard method for the identification of bentgrain nematodes in my country. The traditional morphological and morphometric identification methods are mainly based on the morphology of adult nematodes. It takes a long time to identify the biological characteristics and shape measurement data; and only when adults are found, combined with factors such as hos

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for identifying bentgrass nematode by real time fluorescent PCR technology
  • Method for identifying bentgrass nematode by real time fluorescent PCR technology
  • Method for identifying bentgrass nematode by real time fluorescent PCR technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Take the worms in a sample to be tested, use a picking needle to pick several live larvae from the sample one by one, wash them in deionized water for 3 times, and set aside. Add 2 μl deionized water to a clean glass slide, pick a washed nematode, cut the nematode into 3 sections with a scalpel, suck it into a 200 μl Eppendorf tube, and wash the scalpel and slide with 6 μl deionized water Finally, suck water into the same Eppendorf tube; add 1.0 μl of 10-fold PCR buffer and 1.0 μl of proteinase K, and put it into an ultra-low temperature refrigerator—70°C for 20 minutes. Take out the frozen nematodes, quickly put them into a PCR instrument or a water bath, and incubate at 65°C for 1 to 2 hours, then place the Eppendorf tube in a PCR instrument or a water bath, and incubate at 95°C for 10 minutes to lose proteinase K. active. Make 10 μl of DNA template.

[0078] Perform fluorescent PCR detection on the sample DNA template, and the composition ratio of each component in...

Embodiment 2

[0111] The worm body in a certain sample to be tested is taken, and the preparation method of the worm body DNA template is the same as in Example 1.

[0112] The DNA template of the sample is detected by fluorescent PCR, and the composition ratio of each component in the PCR reaction system and the fluorescent PCR amplification procedure are the same as those in Example 1. Similarly, 1 μL of the reporter plasmid was also tested by fluorescent PCR to prove the validity of the test. The constituent ratios of the components in the PCR reaction system used to detect the plasmid, the PCR amplification program and the sample detection program in this implementation are the same as in Example 1.

[0113] The detection result is the sample amplification result, which is automatically analyzed by the instrument, and an obvious amplification curve can be found, such as Figure 4 Shown in curve 2. The test results showed that the sample contained Nematode bentgrass. The amplification...

Embodiment 3

[0116] The worm body in a certain sample to be tested is taken, and the preparation method of the worm body DNA template is the same as in Example 1.

[0117] The DNA template of the sample is detected by fluorescent PCR, and the composition ratio of each component in the PCR reaction system and the fluorescent PCR amplification procedure are the same as those in Example 1. Similarly, 1 μL of the reporter plasmid was also tested by fluorescent PCR to prove the validity of the test. The constituent ratios of the components in the PCR reaction system used to detect the plasmid, the PCR amplification program and the sample detection program in this implementation are the same as in Example 1.

[0118] The test result is the sample amplification result, which is automatically analyzed by the instrument, and no amplification curve is found. The test results showed that the sample did not contain Nematode bentgrass. The amplification results of the reporter plasmid are automatical...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a method for identifying Anguina agrostis by applying a real-time fluorescent PCR technology. The specific DNA sequence of the Anguina agrostis is utilized; a bioinformatics method is used to design a specific primer and a specific probe; a 5' end of the probe is labeled with a FAM; a 3' end of the probe is labeled with TAMRA; a target gene of the Anguina agrostis is amplified by applying a fluorescent PCR method; and whether the Anguina agrostis is contained in a sample is identified through the change of a fluorescent signal of the probe during the reaction. The method is not affected by larva instar and the quantity of polypides, is quicker and more accurate compared with morphology and morphometry identification methods, has simple operation, can be standardized, solves the problem of a standard method for quickly identifying the Anguina agrostis, greatly saves detection time, accelerates the acceptance of grass seeds at ports, promotes the import and export trade, and saves the cost generated by the long-term storage of goods in a goods yard.

Description

technical field [0001] The invention relates to a biological identification technology, in particular to a nematode molecular biological identification technology, that is, the technology of using real-time fluorescent PCR reaction to identify the nematode nematodes, especially the nematode nematodes bentgrass. Background technique [0002] Anguinaagrostis is an important species in the order Tylenchida, the suborder Tylenchina, the superfamily Tylenchoidea, the family Anguinidae, and the genus Anguina. Plant nematodes are important pests of pastures. Its host plants mainly include Agrostisspp., Calamagrostis spp., Carex spp., Dactylis spp., and Eragrostis spp. , Festuca spp., Hordeum spp., Lolium spp., Phleum spp., Poa spp., Puccinelliaspp .), Sporobolus spp., Trisetum spp., etc., mainly damage the inflorescence of the host plant, resulting in a decrease in the yield of seeds and shoots of the diseased plant. Especially in areas where forage such as bentgrass is widely p...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): G01N21/64C12Q1/68
Inventor 谢辉马以桂王金成黄国明
Owner SOUTH CHINA AGRI UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap