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Fluorescent PCR detection method and application of pear borer

A technology of pear worm and detection method, which is applied in the directions of biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of increasing the risk of prohibition of export, immature inspection methods of importing countries, and increasing the consumption of quarantine treatment costs. , to avoid false positive test results, high specificity, and high amplification specificity

Inactive Publication Date: 2017-01-11
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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  • Fluorescent PCR detection method and application of pear borer
  • Fluorescent PCR detection method and application of pear borer
  • Fluorescent PCR detection method and application of pear borer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Embodiment 1, design and synthesis of primers and probes

[0070] By collecting samples of adults and larvae of the pear borer moth from different areas in Bazhou, Xinjiang, the COI universal primer: forward primer: 5'-GGWGGATTTGGAAATTGAYTAGTWCC-3', reverse primer: 5'-CCHGGTAAAATTAAAATAAACTTC-3' was amplified by conventional PCR and The sequence of the COI gene fragment of the pear borer was obtained by sequencing the product. Through the BLAST comparison in NCBI, the COI characteristic sequence of Pear borer was compared, and the sequence homology reached 99% (AB603521.1, HQ538466.1, JF733841.1), that is, the conservation of the COI region was high, which ensured the accuracy of primer design sex. In addition, we also compared and analyzed the COI sequences of the pear borer moth, codling moth, and P. Needle, and commissioned Takara (Dalian, China) company synthesis and marking.

[0071] Upstream primer LX-FP: 5'-CAGCTCTTTTTATTACTACTTTCACTACCA-3'

[0072] Downstrea...

Embodiment 2

[0081] The extraction of embodiment 2 total DNA

[0082]Negative control samples such as pear borer moth, codling moth, and pear borer were taken, put into a mortar, and liquid nitrogen was added to grind thoroughly, and the sample DNA was extracted with DNeasy blood kit (Qiagen Company), and operated 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; Leptospirosis: single head 1.8-2.2mg, multi-head (3-5) 8-12mg; cutworm: single head 8-12mg; single head add 30μL TE buffer for elution, multi-head add 60μL TE buffer elution. The DNA solution was stored at -20°C for future use.

[0083] 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 (witho...

Embodiment 3

[0085] Embodiment 3 real-time fluorescent PCR detects

[0086] The 25 μL real-time fluorescent PCR reaction system includes: 16.75 μL of sterilized distilled water, 10×PCR Buffer (containing Mg 2+ ) 2.5 μL, dNTP (2.5mM, each) 2 μL, upstream and downstream primers (10 μM) each 0.5 μL, probe (10 μM) 0.5 μL, 500 U TaqDNA polymerase 0.25 μL and DNA template (DNA solution extracted from Example 2) 2 μL. The reaction mixture was amplified in ABI 7500 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 ABI 7500software v2.0.1.

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Abstract

The invention discloses an oriental fruit moth fluorescence PCR (Polymerase Chain Reaction) detection method. The oriental fruit moth fluorescence PCR detection method comprises the following steps of (1) extracting DNA (Deoxyribose Nucleic Acid) of a to-be-authenticated sample genome; (2) arranging a real-time fluorescence quantification PCR amplification system and performing real-time fluorescence PCR detection through a specific primer and a probe with the DNA of the genome serving as an amplification template; (3) analyzing and judging an amplification result. The probe is 5'-(FAM) CGAAATCTTAATACTTCATTTTTTGACCCTG (TAMRA)-3'. THE oriental fruit moth fluorescence PCR detection method can effectively district an oriental fruit moth from fruit boring pests (comprising the oriental fruit moth, codling moth, euzophera pyriella yang and the like).

Description

technical field [0001] The invention relates to a molecular detection method for contamination by the small pear borer, in particular to a fluorescent PCR detection method for the small pear borer, which belongs to the field of biotechnology. Background technique [0002] Pear molesta (Grapholitha molesta (Busck)) belongs to Lepidoptera Toriidae. Plaster, widely distributed in all parts of the world, all over the country, is an important pest of pear trees, especially in orchards where pears and peach trees are mixed. Most of the larvae eat the fruit from the calyx depression, directly to the core of the fruit, there is insect excrement from the borehole, and there is a fruit removal hole where the larvae come out of the damaged fruit. When larvae attack young shoots, they usually enter from the base of the petiole of the third leaf at the top of the young shoots, until the pith, and eat downwards. There is a small amount of insect droppings discharged from the borehole, a...

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祥林张伟郭铁群刘永杰王鹏举
Owner 中华人民共和国库尔勒出入境检验检疫局
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