Primers, kits and identification methods for assisting the identification of carnivorous mite and common carnivorous mite in Malacca

A technology for auxiliary identification and kits, applied in biochemical equipment and methods, measurement/inspection of microorganisms, DNA/RNA fragments, etc., can solve the problems of time-consuming, large-scale, time-consuming and money-consuming sequences, and achieve short time-consuming , easy operation and low cost

Active Publication Date: 2017-10-20
ACAD OF NAT FOOD & STRATEGIC RESERVES ADMINISTRATION +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This process requires personnel with a certain background in molecular technology to operate. At the same time, sequencing requires a large amount of funds, and the subsequent processing and analysis of the sequence also takes time, which is a time-consuming and expensive process.

Method used

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  • Primers, kits and identification methods for assisting the identification of carnivorous mite and common carnivorous mite in Malacca
  • Primers, kits and identification methods for assisting the identification of carnivorous mite and common carnivorous mite in Malacca
  • Primers, kits and identification methods for assisting the identification of carnivorous mite and common carnivorous mite in Malacca

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1, the design of specific primer pair

[0034] 1. Implementation process

[0035] Firstly, samples of common mites on grain depots and warehouses were collected, including morphologically similar carnivorous mite and common carnivorous mite. The mitochondrial cytochrome oxidase subunit I barcode (mtDNA COI barcode) sequence was obtained by genomic DNA extraction and PCR amplification, and the mtDNA COI barcode gene sequence of mites in the BOLD database and GenBank were collected. On this basis, the DNAMAN software was used to compare and analyze the mtDNA COI barcodes of different species, and to find the specific sequence segments of the Malacca carnivorous mite and the common carnivorous mite. The specific primer pairs of Melaka carnivorous mite and common carnivorous mite were artificially designed, and the primers were evaluated and checked by Oligo software and Blast program. Under the standard PCR reaction system and reaction conditions, the specific...

Embodiment 2

[0056] Composition and use of embodiment 2 kit

[0057] The kit includes the primer pair (Cma-F / Cma-R) designed in Example 1 for identifying carnivorous mite in Malacca and the primer pair (Cer-F / Cer-R) for identifying common carnivorous mite, 10×PCR Buffer (with Mg 2+ ), dNTPs, Taq DNA polymerase and DEPC water, the above reagents were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0058] Use of the kit:

[0059] (1) Extract the genomic DNA of the mite to be tested, which can be extracted using commercial kits or methods known in the prior art;

[0060] (2) Using the genomic DNA extracted in step (1) as a template, use the above two pairs of primers to carry out PCR amplification reaction respectively; reaction system (25μL): Taq enzyme (5U / μL) 0.2μL, 10×PCR Buffer (containing Mg 2+ ) 2.5 μL, dNTPs (2.5 μM) 2 μL, upstream and downstream primers 0.5 μL each (one PCR with primer pair Cma-F / Cma-R, another PCR with primer pair Cer-F / Cer-R), template 2 μL, ...

Embodiment 3

[0063] Embodiment 3, specificity experiment

[0064] Cheyletus eruditus, Schrank, Cheyletus malaccensis Oudemans, Acarus siro L., Tyrophagus putrescentiae (Schrank), Tyroborus lini Oudemans, Aleuroglyphus ovatus (Troupeau), Lepidoglyphus destructor (Schrank), Glycyphagus domesticus (De Geer), Chortoglyphus arcuatus (Troupeau), and Carpoglyphus lactis (L.): preserved in the Scientific Research Institute of the National Grain Administration.

[0065] 1. Experimental samples

[0066] The samples of 10 kinds of storage mites were collected from my country and the Czech grain depots, among which the Malacca carnivorous mites were from the Haikou grain depot in Hainan, my country, and the rest of the mite samples were from the Czech Republic. The samples were collected in 2012 and stored at -80°C in In absolute ethanol, the common carnivorous mite and the malacca carnivorous mite were live insects raised in the laboratory, and the specific information is shown in Table 2.

[0067] ...

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Abstract

The invention discloses a primer for auxiliary authentication on Malacca cheyletus and common cheyletus, a kit and an authentication method. The primer is shown in the sequence table SEQ ID NO:1 to SEQ ID NO:4 and can be used for auxiliary authentication of the Malacca cheyletus and the common cheyletus. According to the primer for auxiliary authentication on the Malacca cheyletus and the common cheyletus, the kit and the authentication method, fast authentication of the Malacca cheyletus and the common cheyletus is achieved, authentication of non-adult-form samples is achieved, and a new approach of authenticating two kinds of predatory mites (the Malacca cheyletus and the common cheyletus) similar in form is achieved; operation is simple and convenient, an economical property and easy implementation are achieved, a slide preparation skill is needed generally in a traditional form authentication method, the requirement for sample integrity and the like is high, a specific primer molecular authentication method can be standardized, operation is easy and convenient, needed cost is low, workers without the professional taxonomy knowledge background can conveniently conduct operation and application, and easy application and popularization can be achieved in practical work; the detection sensitivity is high, consumed time is short, and high-flux detection can be achieved.

Description

technical field [0001] The invention relates to the fields of mite identification and mite monitoring, in particular to a method for assisting the identification of two predatory mites, the malacca carnivorous mite and the common carnivorous mite, and a pair of special primers. Background technique [0002] Acarids are a large group of tiny arthropods belonging to the class Arachinida and the subclass Acari. Most of them live in warehouses, grain storage and processing plants, Chinese herbal medicines, litter and other places. The stored-grain mites are widely distributed and there are many kinds. Acarina, Radiata, Gamaina and Oribati are important components of stored-grain mites. Among the stored grain mites, there are both harmful mites that endanger the quality of stored grains and predatory mites that prey on harmful mites and pests. Common carnivorous mites and Malacca carnivorous mites are the most common species of predatory mites that are widely distributed and ab...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888
Inventor 伍祎曹阳李志红张涛汪中明贺培欢
Owner ACAD OF NAT FOOD & STRATEGIC RESERVES ADMINISTRATION
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