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Composition for identifying or assisting identification of American white moth and detection method thereof

A technology for assisting the identification of white moth, which is applied in the field of identification or auxiliary identification of white moth, can solve the problems of long identification time and inaccurate results, and achieve the effect of high sensitivity and strong specificity

Inactive Publication Date: 2016-01-20
NANJING FORESTRY UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Adults, larvae, and pupae of the American white moth can spread and spread along with the fruit and packaging materials. After being intercepted at the port, if the morphological identification method is used, the identification time is too long and the result is not accurate enough.

Method used

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  • Composition for identifying or assisting identification of American white moth and detection method thereof
  • Composition for identifying or assisting identification of American white moth and detection method thereof
  • Composition for identifying or assisting identification of American white moth and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 The present invention identifies or assists in identifying the composition of American white moth

[0031] The COI gene fragment of the sample was amplified and sequenced using the general primer LepF1 / LepR1 of Lepidoptera (Hebert, 2003). The sequence was compared and analyzed using MEGA5.02, and the American white moth specificity was designed according to the difference site Primers HC-F / HC-R and MGB probe / HC-TZ.

[0032] extract total DNA

[0033] GenMagBio Animal Cell Tissue / Cell Genomic DNA Magnetic Beads Extraction Kit was used to extract the total DNA of insect samples as follows: Cut 30-90 mg of the feet and chest of the test specimen into a test tube, add small steel balls, and use MM400 ball mill to Grind at a frequency of 15r / s for 2min; add 540μL LysisBuffer and 60μL proteinase K, incubate at 24℃ for 3h, incubate at 37℃ for 3h, centrifuge at 13000r / min for 5min, take 300μL of the supernatant into a 1.5mL centrifuge tube; add 300μL BindingBuffe...

Embodiment 2

[0041] Example 2 The composition of the present invention is used to identify the establishment of a real-time fluorescent PCR method for the white moth

[0042] 1. Extraction of total DNA from American white moth

[0043] The DNA of each tested insect sample in Table 1 was extracted according to the method in Example Title 1.

[0044] Establishment and optimization of real-time fluorescent PCR reaction system

[0045] Using primers HC-F, HC-R and MGB probe to detect white moth by real-time fluorescent PCR.

[0046] PCR reaction system: the total volume is 20 μl, and the composition is as follows: 0.1 μl of 5U / μl TaqDNA polymerase, 1 μl of 6.75 ng / μl template DNA, 10 μl of TaqManmix mixed reaction solution, and the concentration of upstream primer HC-F is from 100 nmol / L to 1000 nmol / L ( Increments of 50nmol / L), the concentration of downstream primer HC-R is from 100nmol / L to 1000nmol / L (increments of 50nmol / L), the concentration of MGB probe is from 50nmol / L to 500nmo...

Embodiment 3

[0051] Embodiment 3 The present invention is used for identifying the specificity of the real-time fluorescent PCR method of American white moth

[0052] 1. Extract total DNA from worm samples

[0053] The DNA of each tested insect sample in Table 1 was extracted according to the method in Example Title 1.

[0054] real-time fluorescent PCR reaction

[0055] Real-time fluorescent PCR reaction was carried out using the DNA of each tested insect sample as a template. The reaction system is: TaqManmix mixed reaction solution 10μl, 500nmol / L upstream primer HC-F 0.5μl, 500nmol / L downstream primer HC-R 0.5μl, 250nmol / L probe HC-TZ 0.25μl, 6.75ng / μl Template 1μl, 5U / μl TaqDNA polymerase 0.1μl, add ddH 2 O to make up to 20 μl. The reaction program is: 50°C for 2min, 95°C for 10min, and then cycle, 95°C for 15s, 60°C for 60s, a total of 40 cycles.

[0056] result

[0057] see results Figure 4 , the four samples of the American white moth all showed fluorescent signals,...

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Abstract

The invention provides a composition for identifying or assisting in identifying the white moth and a detection method thereof, and relates to the field of biotechnology. A composition for identifying or assisting in identifying American white moth, consisting of a primer pair and a probe; the primer pair consists of SEQ? ID? NO:1 and SEQ? ID? The composition of two single-stranded DNA molecules shown in NO:2; the nucleotide sequence of the probe is as SEQ? ID? NO:3 shown. The composition of the invention for identifying or assisting the identification of white moth can quickly, accurately, efficiently and reliably identify adults, pupae, larvae and eggs, and determine whether they are white moths. The method of the invention has strong specificity and can distinguish the American white moth from other morphologically similar species. The method of the invention has high sensitivity, and the minimum detection limit can reach 0.25pg / μl total DNA.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a composition for identifying or assisting in identifying the white moth and a detection method thereof. Background technique [0002] American White Moth Hyphantria cunea (Drury) belongs to the family Lepidoptera and is native to North America. It is widely distributed in the northern United States, southern Canada and Mexico, and then spread to Europe, Japan, and Korea along with vehicles. In 1979, the American white moth was first discovered in Dandong, Liaoning, my country, and has now spread to Liaoning, Shaanxi, Hebei, Shandong, Tianjin, Beijing and other provinces and cities. American white moth has miscellaneous feeding habits, large food intake, strong fecundity, and a wide range of hosts. Its hosts are more than 600 species in the United States, Europe, Japan, and North Korea. In my country, the host plants have reached 49 families, 108 genera, and 175 species. It mainly h...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q2563/107
Inventor 钱路安榆林叶兼菱吴晶宋俊贤徐梅郑斯竹吕飞吴翠萍李彬汪潇琳
Owner NANJING FORESTRY UNIV