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Detection method and application of real-time fluorescent PCR for Pythias spp.

A technology of real-time fluorescence and detection methods, applied in the direction of biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of immature importing countries with immature inspection methods, increasing the consumption of quarantine treatment costs, and increasing the risk of prohibiting exports, etc. Achieve the effect of avoiding false positive detection results, high specificity, and high amplification specificity

Inactive Publication Date: 2016-04-20
中华人民共和国库尔勒出入境检验检疫局
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Immature and low-resolution inspection methods still pose a risk of severe sanctions by importing countries when intercepting quarantine pests
Bringing too many technical disputes, increasing the risk of export prohibition, increasing quarantine treatment or returning goods will result in greater cost consumption

Method used

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  • Detection method and application of real-time fluorescent PCR for Pythias spp.
  • Detection method and application of real-time fluorescent PCR for Pythias spp.
  • Detection method and application of real-time fluorescent PCR for Pythias spp.

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Embodiment 1, design and synthesis of primers and probes

[0072] By collecting samples of adult and larvae of Ceratocystis spp. from different areas in Bazhou, Xinjiang, with COI universal primers: forward primer: 5'-GGWGGATTTGGAAATTGAYTAGTWCC-3', reverse primer: 5'-CCHGGTAAAATTAAAATAAACTTC-3', by conventional PCR Amplification and product sequencing obtained the fragment sequence of the COI gene of C. Through the BLAST comparison in NCBI, the COI characteristic sequence of Ceratocystis argentina is compared, and the sequence homology reaches 97% (KC215198.1, KC442311.1, KC442309.1), that is, the COI region is highly conserved, which ensures the primer design accuracy. In addition, we also compared and analyzed the COI sequences of Pythias spp., codling moth, and pear borer to find the difference sites between them, and designed species-specific primers and probes in the difference sequence regions. Needle, and commissioned Takara (Dalian, China) company synthesis an...

Embodiment 2

[0084] The extraction of embodiment 2 total DNA

[0085]Take positive samples of pear borer moth, codling moth, and pear borer, put them into a mortar, add liquid nitrogen and grind them thoroughly, use DNeasybloodkit (Qiagen Company) to extract DNA from the samples, and operate according to the kit instructions. The amount of muscle tissue used in sample extraction: codling moth: single head 2.5-5.0mg, multi-head (4-5) 15-20mg; pear borer: single head 0.8-1.2mg, multi-head (12-13) 12 -16mg; Pear moth: single head 1.8-2.2mg, multi-head (3-5) 8-12mg; cutworm: single head 8-12mg; single head plus 30μLTE buffer for elution, multiple head plus 60μLTE buffer liquid elution. The DNA solution was stored at -20°C for future use.

[0086] The extracted DNA solution was detected by a nucleic acid protein analyzer, and the OD260 / 280 was greater than 1.5, and the OD260 / 230 was greater than 1.0. The extraction effect was good, and the concentration was 30-200ng / μL (without RNase degradat...

Embodiment 3

[0088] Embodiment 3 real-time fluorescent PCR detects

[0089] The 25 μL real-time fluorescent PCR reaction system includes: sterilized distilled water 16.75 μL, 10×PCRBuffer (containing Mg2+) 2.5 μL, dNTP (2.5mM, each) 2 μL, upstream and downstream primers YB-FP, YB-RP (10 μM) each 0.5 μL , 0.5 μL of probe YB-P (10 μM), 0.25 μL of 500 UTaq DNA polymerase and 2 μL of DNA template (DNA solution extracted from Example 2). The reaction mixture was amplified in an ABI7500 real-time quantitative PCR instrument. The reaction conditions were set as pretreatment at 95°C for 15s, denaturation at 95°C for 5s, annealing / extension at 60°C for 34s, and 40 cycles. At the same time, set up repeated experiments and positive control, negative control and blank control experiments. The experimental results are analyzed and processed using ABI7500softwarev2.0.1.

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Abstract

The invention discloses a euzophera pyriella real-time fluorescence PCR detection method. The euzophera pyriella real-time fluorescence PCR detection method sequentially comprises the following steps of 1 extracting genome DNA of a sample to be tested; 2 using the genome DNA as an amplification template, using a specific primer and a Taqman probe to perform real-time fluorescence PCR detection, wherein the primer is F-Primer: 5'-CTGCCTTATTACTACTTCTTTCACTACCT-3', R-Primer: 5'-AATAGGGTCTCCCCCTCCT-3', and the probe is Probe: 5'-(FAM)CGGAATCTTAATACTTCTTTTTTTGACCCTG-(TAMRA)-3'. By means of the euzophera pyriella real-time fluorescence PCR detection method, euzophera pyriella can be distinguished from carposinidae insect pests (including oriental fruit moth, codling moth, euzophera pyriella and the like).

Description

technical field [0001] The invention relates to a molecular detection method for the pollution of the cantaloupe moth, in particular to a fluorescent PCR detection method for the cantaloupe moth, which belongs to the field of biotechnology. Background technique [0002] Euzopherapyriella Yang is an omnivorous pest of the genus Euzophora subfamily Lepidoptera Lepidoptera. It is a new species of pest named by Professor Yang Jikun in 1994. It is a unique fruit tree borer pest in Xinjiang. Since it was first discovered in the late 1980s, it has spread and caused serious damage. Among them, Korla fragrant pear has the highest rate of damaged plants, often as high as 100%. It is one of the main pests of fragrant pears. It mainly harms pears, apples, figs, jujubes, apricots, almonds, peaches and other fruit trees, seriously affecting the yield and quality of fruits. [0003] The larvae harm the branches and trunks of fruit trees and forest trees, and eat between the phloem and xyl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q1/6888C12Q2561/101C12Q2561/113
Inventor 王凤军冯俊丽华鹏张伟张祥林王鹏举郭铁群刘永杰
Owner 中华人民共和国库尔勒出入境检验检疫局
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