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A kind of multiple PCR primers and method for rapid identification of cockroach species

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, simple operation, and low cost

Active Publication Date: 2019-03-08
SICHUAN GOODDOCTOR PANXI PHARMA
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  • Summary
  • 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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  • A kind of multiple PCR primers and method for rapid identification of cockroach species
  • A kind of multiple PCR primers and method for rapid identification of cockroach species

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Extraction of genomic DNA from single and different mixed populations of six species of Blattaria

[0037] 1) Extraction of single genomic DNA of six species of Blattaria: Before the genome preparation of the six known species of Blattaria, the test samples of the six known species of Blattaria were first wiped with 75% ethanol on the sample surface to eliminate exogenous pollution. After the ethanol evaporates, The abdominal muscle tissue is selected and fully ground as a genome extraction sample. A DNA extraction kit (TIANamp Genomic DNAKit (Beijing Tiangen Biochemical Technology Co., Ltd.)) was used to extract the monomeric genomic DNA of six known species of Blattaria. The concentration of the extracted template DNA was detected with a microspectrophotometer, and the DNA Bacteria's deionized water will dilute the DNA concentration of the sample to 50ng / ul, and store the DNA at -20°C.

[0038] 2) Genomic DNA extraction from different mixed populations of six s...

Embodiment 2

[0039] Example 2: PCR system and procedures: two sets of PCR amplifications were performed, one of which used six monomeric Blattaria genomic DNA as a template, and the other group used 13 mixed Blattaria genomic DNA as a template template.

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

[0041] 2) The genomic DNA multiplex PCR system of different mixed populations of six species of Blattaria: 2×TSINGKE TM Master Mix Buffe...

Embodiment 3

[0043] Example 3: The PCR product was detected by 2% agarose gel electrophoresis, and the 50bp DAN Marker was used as a reference to determine the size of the PCR product to determine the species of Blattaria in the individual and mixed population of Blattaria: The American cockroach is 452bp, the black-breasted cockroach is 125bp, the Australian cockroach is 160bp, the brown-spotted cockroach is 284bp, the German cockroach is 253bp and the ground turtle is 380bp (see figure 1 with 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 Kinco Biotechnology Co., Ltd. (Chengdu) for sequencing, and the sequence of the DNA fragment obtained by sequencing was in the NCBI database. The existing sequences of Blattaria species have very high homology (over 97%), which further confirms the accuracy of the species identified by this method.

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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 Blattaria insects, in particular to a multiple PCR primer and method for quickly identifying six Blattaria insect species. Background technique [0002] Blattaria is the first order of the Insecta Pterygota. More than 5,000 species are known in the world, and more than 250 species are known in my country, which belong to 3 families of Cockroach family, Cockroach family and Geochelone family. Some species of insects in the order Blattella are important disease vectors and one of the main targets for sanitary control; but some species of cockroaches can be used as medicinal materials to extract biologically active substances and treat a variety of intractable human diseases (such as : American cockroach and ground borer). Periplaneta americana, commonly known as "cockroach", is an important traditional medicinal material in my country. Its medicinal material was first published in the "Shen Nong's Materia Me...

Claims

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Application Information

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Patent Type & Authority Patents(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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