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Hyphantria cunea constant temperature nucleic acid detection kit and detection method thereof

A detection kit and the technology of American white moth, which are applied in the direction of biochemical equipment and methods, and the determination/inspection of microorganisms, can solve the problems of false positives and expensive detection, and achieve high specificity, fast response speed, and high sensitivity. Effect

Inactive Publication Date: 2014-10-15
郑斯竹
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although FQ-PCR has the advantages of simplicity, speed and sensitivity, its detection requires expensive instruments, and it is easy to cause false positives and other problems

Method used

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  • Hyphantria cunea constant temperature nucleic acid detection kit and detection method thereof
  • Hyphantria cunea constant temperature nucleic acid detection kit and detection method thereof
  • Hyphantria cunea constant temperature nucleic acid detection kit and detection method thereof

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Experimental program
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Effect test

Embodiment 1

[0034] Embodiment 1 Composition and preparation of detection kit

[0035] (1) DNA detection device: a test strip for the rapid detection method of nucleic acid thin film chromatography disclosed in CN1811447A, and a fully enclosed rapid detection device for target nucleic acid amplification products disclosed in CN1888902A.

[0036] (2) Reaction solution for isothermal amplification of white moth nucleic acid: two peripheral primers (0.05 μmol), two probes (0.5 μmol), and amplification cross primer (0.5 μmol), 10×Thermol buffer, MgSO 4(6mmol), dNTPs solution (0.4mmol), Bst DNA polymerase (10U) and sterile double distilled water, the total reaction volume is 18μL. Among them: the forward peripheral primer sequence of the two peripheral primers is 5'- AGCAGGAACTGGATGAACA-3', the reverse peripheral primer sequence is 5'-GTATGGTAATAGCTCCAGC-3'; the sequences of the two probes are: forward 5' end Biotin Labeled probe 5'-biotin- ATTACTACAATCATTAACATACGATT-3', reverse 5' end fluores...

Embodiment 2

[0041] Embodiment 2 The method for detecting the American white moth nucleic acid with the kit of embodiment 1

[0042] Using 16 species of Luphidae insects, including the American white moth, as test specimens, the adult larvae of these 16 species of Luphididae insects are indistinguishable from the American white moth in terms of external morphology. The test specimens are shown in Table 1.

[0043] Table 1 Test specimens

[0044]

[0045] 1) DNA preparation: DNA was extracted using GenMagBio Animal Cell Tissue / Cell Genomic DNA Magnetic Beads Extraction Kit. The specific process is as follows:

[0046] Cut directly 30 mg of the feet and chest of the test specimen into a 2 mL test tube, soak in absolute ethanol and save the specimen, rinse and soak in sterile distilled water for 4 to 5 times, and discard the water; soak the dry specimen in sterile distilled water for 3 hours and blot dry. Place in a 2mL centrifuge tube, shake and grind in MM400 ball mill (30 times / s) fo...

Embodiment 3

[0051] The DNA of the cultured white moth was extracted, quantified, and diluted to a concentration of 10 4 copies / μL, 10 3 copies / μL, 10 2 copies / μL, 10 1 copies / μL, the method described in Example 2 was used to determine the sensitivity of the kit of the present invention for the detection of H. americana nucleic acid.

[0052] The result is as figure 2 As shown, 1-4 in the figure represent 10 4 copies / μL, 10 3 copies / μL, 10 2 copies / μL, 10 1 Copy / μL, 5 is the negative control, it can be found that the kit can detect 10 copies in each reaction system, which has high sensitivity and can meet the requirements of rapid detection of white moth (copy is the clonal unit).

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Abstract

The invention discloses a constant temperature detection kit for Hyphantria cunea nucleic acid and a detection method thereof. The kit includes: 1) a DNA detection device; 2) a Hyphantria cunea nucleic acid constant temperature amplification reaction solution; 3) a positive control, which is a DNA fragment containing a Hyphantria cunea mitochondrion COI gene; and 4) a negative control, which is sterile double distilled water. The detection kit provided by the invention has the characteristics of high specificity, high sensitivity and fast reaction speed. From sample treatment to the end of detection on a single sample, only 1-1.5 hours are needed. The kit can meet middle flux and low flux sample detection, is especially suitable for field detection, and the whole reaction process only needs a constant temperature instrument.

Description

technical field [0001] The invention belongs to the technical field of detection of white moth, and in particular relates to a detection kit for constant temperature nucleic acid of white moth and a detection method thereof. Background technique [0002] The American white moth (Hyphantria cunea) (Drury), also known as the American lamp moth (Drury), is a world-renowned quarantine pest. It mainly harms fruit trees, street trees and ornamental trees, and causes serious harm to garden trees, economic forests, farmland protection forests, etc. It has been included in the first batch of invasive alien species in my country. The main expansion route of American white moth can be spread from various stages with goods by means of transportation. The larvae and pupae above the 4th instar have the most chances of transmission. In recent years, it has been frequently seen that this insect is imported into China with packing boxes imported from abroad, and attention should be drawn....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6844C12Q2545/101
Inventor 詹国辉郑斯竹陈景云葛华林
Owner 郑斯竹
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