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Anselmella miltoni girault molecular detection primer, detection method and application thereof

A technology for Milton's honeybee and molecular detection, which is applied to biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of molecular detection and identification methods without Milton's jellyfish, and achieves the effect of convenient use.

Inactive Publication Date: 2013-03-13
INSPECTION & QUARANTINE TECH CENT OF XIAMEN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, there is no molecular detection method for the identification of A.

Method used

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  • Anselmella miltoni girault molecular detection primer, detection method and application thereof
  • Anselmella miltoni girault molecular detection primer, detection method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] 1) Extraction of total DNA

[0018] Method 1: Extraction of total DNA from multiple insects

[0019] Genomic DNA was extracted using the Universal Genomic DNA Extraction Kit Ver 3.0 produced by TaKaRa, including the following steps:

[0020] (1) Put 30 insects into a mortar, add 500 μL of Solution A (provided by TaKaRa’s kit) and 1.0 μL of RNase A1 (provided by TaKaRa’s kit) for grinding, and collect the homogenate into Collection Tube (TaKaRa's kit provided);

[0021] (2) Rinse the mortar with 200 μL of Solution A and transfer to the Collection Tube, and incubate at 65 °C for 5-10 min;

[0022] (3) Add 400 μL of Solution B (provided by TaKaRa’s kit) to the Collection Tube, shake and mix; then add 1 mL of 4°C pre-cooled Solution C (provided by TaKaRa’s kit), and mix well , centrifuge at 12000 rpm for 2 min;

[0023] (4) Discard the upper organic phase, then add 1 mL of 4 ℃ pre-cooled Solution C, mix thoroughly, and centrifuge at 12,000 rpm for 2 min;

[0024] (5) D...

Embodiment 2

[0043] Specific detection of PCR method in Adenia milton

[0044] Take 30 A. milton, A. eucalyptus and A. eucalyptus, and extract total DNA respectively according to the method in Example 1, then carry out PCR amplification, and detect the results by agarose gel electrophoresis. Results A DNA fragment of 317 bp was detected in A. milton, but no DNA fragment of 317 bp was detected in A. eucalyptus and A. erythrina. Test results such as figure 1 shown. It shows that the established PCR method has good specificity and can be used for rapid detection of A. milton.

Embodiment 3

[0046] PCR detection of a single Adenia milton

[0047] Take a single Adenia milton, extract the total DNA according to the method in Example 1, then perform PCR amplification, and agarose gel electrophoresis to detect the results. Results A DNA fragment of 317 bp was detected in a single A. milton. Test results such as figure 2 shown. It shows that the established PCR method has good sensitivity and can be used for the rapid detection of a single A. milton.

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Abstract

The invention discloses an anselmella miltoni girault molecular detection primer, a detection method and an application thereof, and the DNA (deoxyribonucleic acid) sequence of the anselmella miltoni girault molecular detection primer is as follows: an upstream primer (MITS2F) is 5'-TCGGAAGTGTCAATAGGCG-3'; and a downstream primer (MITS2R) is 5'-TCCATCTCGCATTACCCTC-3'. The method is reliable in result, easy to operate, strong in specificity and high in sensitivity, and can be used for high-sensitivity fast molecular detection of anselmella miltoni girault and provide guarantee for safety of import and export.

Description

technical field [0001] The invention relates to a molecular detection primer of A. milton, a detection method and an application thereof. The invention belongs to the field of crop pest detection, identification and control technology. Background technique [0002] Milton Bee Anselmella Miltoni Girault belongs to the order Insecta, Chalcidoidea, Eulophilidae, Anselmella belongs to. [0003] Miltonia larvae are herbivorous, feeding on the seeds of rose apples for development. This type of fruit is seedless, and after suffering damage, a gall that looks like a walnut is formed, which not only affects the yield of the fruit, but also makes it lose its edible value. The insect reproduces quickly, in large quantities, and is seriously harmful. At present, lotus mist is planted in Hainan, Guangdong, Guangxi, Fujian and other places in my country. Therefore, it is obvious that it is affected by the harm of Adenia milton. [0004] With the development of molecular biology tec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 黄蓬英廖富荣林石明
Owner INSPECTION & QUARANTINE TECH CENT OF XIAMEN ENTRY EXIT INSPECTION & QUARANTINE BUREAU