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Multi-PCR primer and method for quickly identifying blattaria insect variety

A technology of Blattata and multiplex, applied in the field of identification of Blattata insects, can solve the problems of losing morphological characteristics, etc., and achieve the effects of saving reagents, low cost, and simple operation

Active Publication Date: 2018-01-19
SICHUAN GOODDOCTOR PANXI PHARMA
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for medicinal cockroaches such as Periplaneta americana and soft-shelled turtle, after rough processing in the place of origin and processing of decoction pieces, they lose their morphological characteristics, and traditional morphological identification cannot be used indiscriminately. It is necessary to scientifically and accurately identify such medicinal materials to ensure Safe and effective application of medicinal materials

Method used

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  • Multi-PCR primer and method for quickly identifying blattaria insect variety
  • Multi-PCR primer and method for quickly identifying blattaria insect variety

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Embodiment 1: the monomer of six kinds of cockroach insects and the extraction of different mixed population genomic DNA

[0037] 1) Single genome DNA extraction of six species of Blattoides: before the genome preparation of the test samples of six known species of Blattoides, the surface of the samples was first wiped with 75% ethanol to eliminate exogenous contamination, and after the ethanol volatilized, Abdominal muscle tissue was selected and thoroughly ground as a genome extraction sample. DNA extraction kits (TIANamp Genomic DNA Kit (Beijing Tiangen Biochemical Technology Co., Ltd.)) were used to extract the monomeric genomic DNAs of six known species of cockroaches. Dilute the DNA concentration of the sample to 50ng / ul with deionized water, and store the DNA at -20°C.

[0038] 2) Genomic DNA extraction of different mixed populations of six cockroaches: take about 10 mg of abdominal muscle tissue of each known species of cockroaches, and mix them according to th...

Embodiment 2

[0039] Embodiment 2: PCR system and procedure: Carry out the amplification of two groups of PCR, wherein one group uses six kinds of monomeric cockroaches insect genome DNA as template, and another group uses 13 kinds of mixed cockroaches insect group genome DNA as template template.

[0040] 1) The multiplex PCR system for genomic DNA of six monomeric cockroaches is: 2×TSINGKE TM Master MixBuffer (Qingke Biotechnology Co., Ltd.) 30ul; the concentration of 2umol / L primers P ame-F and P ame-R each 0.4ul; the concentration of 2umol / L primers Pful-F and Pful-R each 0.75 ul; concentration is 0.5ul each of primers Paus-F and Paus-R of 2umol / L; concentration is each 0.5ul of primers Pbru-F and Pbru-R of 2umol / L; concentration is the primer Bger-F of 2umol / L and 0.5ul each of B ger-R; 0.9ul each of primers E sin-F and E sin-R with a concentration of 2umol / L; 1ul of DNA template; make up to 50ul with sterile ultrapure water.

[0041] 2) The multiplex PCR system for genomic DNA of di...

Embodiment 3

[0043] Example 3: The PCR product is detected by 2% agarose gel electrophoresis, and the 50bp DAN Marker is used as a reference to determine the size of the PCR product to determine the species of the Blattata insect monomer and the mixed population contained in the Blattata insect: Periplaneta americana is 452bp, Periplaneta melanogaster is 125bp, Periplaneta australis is 160bp, Periplaneta brown spot is 284bp, Blattella germanica is 253bp and ground turtle is 380bp (see figure 1 and figure 2 ). At the same time, in order to further confirm the reliability of the identification method, the corresponding PCR target bands of each cockroach insect were sent to Qingke Biotechnology Co., Ltd. (Chengdu) for sequencing, and the sequences of the DNA fragments obtained by sequencing were compared with those in the NCBI database. The relative sequences of the existing cockroach species all have very high homology (more than 97%), thus further confirming the accuracy of the species id...

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Abstract

The invention relates to a multi-PCR primer and a method for quickly identifying a blattaria insect variety, belonging to the technical field of applied biology. The multi-PCR primer for quickly identifying the blattaria insect variety comprise a periplaneta americana primer, a periplaneta fuliginosa primer, a periplaneta australasiae primer, a periplaneta brunnea primer, a blattella germanica primer and a ground beetle primer. The identifying method comprises the following steps: extracting blattaria insect genome total DNA, performing multi-PCR amplification, and judging a variety of a blattaria insect to be detected according to PCR product size obtained by sepharose gel electrophoresis detection. PCR product strip sizes of periplaneta americana, periplaneta fuliginosa, periplaneta australasiae, periplaneta brunnea, blattella germanica and ground beetle are respectively 452bp, 125bp, 160bp, 284bp, 253bp and 380bp. The identifying primer and the multi-PCR method can be used for sensitively and quickly determining the variety of the blattaria insect sample to be detected, and the method has the advantages of simple operation, high repeatability, low cost and the like in comparisonto a traditional detection method.

Description

technical field [0001] The invention relates to a method for identifying insects of the order Blatella, in particular to a multiplex PCR primer and a method for quickly identifying six species of insects of the order Blatella. Background technique [0002] The order Blattata is an order of the subclass Insecta. There are more than 5,000 known species in the world, and more than 250 species are known in my country, which belong to three families: Blattidae, Blattidae and Geocharidae. Some species of insects in the order Blattata are important disease vectors and are one of the main objects of sanitation control; however, some species of insects in the order Blattata can be used as medicinal materials to extract biologically active substances and treat various difficult and miscellaneous diseases of humans (such as : Periplaneta americana and Weetweet). Periplaneta americana, commonly known as "cockroach", is an important traditional medicinal material in my country. Its use...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12N15/11
Inventor 姜顺日耿福能刘彬吴桃清
Owner SICHUAN GOODDOCTOR PANXI PHARMA
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