Method for identifying common warehousing liposcelis quickly based on multiple PCR technology

A technology for storing booklice and booklice, which is applied in the fields of plant protection and biology, and can solve problems such as cumbersome steps, undiscussed identification of individual booklice, and insufficient efficiency

Inactive Publication Date: 2011-09-14
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method is cumbersome and not efficient enough. At the same time, it only extracts genomic DNA from populations of book lice, and does not discuss the identification of individual book lice.

Method used

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  • Method for identifying common warehousing liposcelis quickly based on multiple PCR technology
  • Method for identifying common warehousing liposcelis quickly based on multiple PCR technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Using the STE method to extract the monomeric and population genomic DNA of 6 kinds of booklice

[0026] A. Monomer DNA extraction of 6 species of booklice: Use 25 μL of STE lysate (100 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to fully grind the monomers of 6 known species of booklice, and Add 0.3 μL of proteinase K (20 mg / mL) and incubate at 37°C for 40 min. Immediately after the incubation, place each mixture at 95°C for 5 min to denature proteinase K. After denaturation, the mixture was centrifuged at 1000 rpm for 1 min, and the supernatant was the DNA template of the book lice, which could be stored at -20°C for a long time. The control of the above temperature can be operated in the PCR instrument.

[0027] B. Genomic DNA extraction of 6 kinds of booklice populations: Take about 10 mg of each known species of booklice and grind them thoroughly in liquid nitrogen; add 450 μL of STE extraction solution (100 mM NaCl, 10 mM Tris-HCl, 50 mM EDTA, pH 8....

Embodiment 2

[0028] Example 2: PCR system and procedure: Two sets of PCR amplification were carried out, one of which used the DNA of 6 individual booklices as a template, and the other set used the population DNA of 6 kinds of booklice as a template. One kind of booklice corresponds to one PCR reaction, and the volume of the reaction system is 25 μL. The specific order and dosage of the samples are shown in Table 1:

[0029] 10×PCR buffer

2.5 μL

10×PCR buffer

2.5 μL

Mg 2+ (25mmol / μL)

2 μL

Mg 2+ (25mmol / μL)

2 μL

dNTPs (10mmol / μL)

2 μL

dNTPs (10mmol / μL)

2 μL

A single booklice DNA

1 μL

DNA of a certain booklice population

1 μL

7 primers (10 pmol / μL)

1μL each

7 primers (10 pmol / μL)

1μL each

wxya 2 o

10.2 μL

wxya 2 o

10.2 μL

Taq (5U / μL, TaKaRa)

0.3 μL

Taq (5U / μL, TaKaRa)

0.3 μL

Total

25μL

Total

25...

Embodiment 3

[0032] Example 3: PCR products were detected by electrophoresis on 2% agarose gel, using 50 bp DNA Marker (TaKaRa) as a reference. The criteria for judging the species of booklice are: 300 bp for Booklice volata, 329 bp for Booklice entomophagia, 262 bp for Booklice microphthalmia, 145 bp for Booklice tricolor, 234 bp for Booklice Yunnan, and 450 bp for Booklice achromophora.

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Abstract

The invention relates to a method for identifying common warehousing liposcelis quickly based on a multiple polymerase chain reaction (PCR) technology. The method comprises the following steps of: extracting deoxyribonucleic acid (DNA) of the genome of monomeric liposcelis or colonial liposcelis as a polymerase chain reaction (PCR) template, and designing specific primers to perform a multiple PCR reaction in a system and a program of an ordinary PCR kit, and determining liposcelis varieties by the detection of gel electrophoresis. The method is quick, efficient and high in accuracy and stability, 6 liposcelis varieties can be identified only by one PCR reaction, and regardless of colonial or monomeric liposcelis, female or male individuals, nymphae or imagoes, the reliable conclusion canbe obtained among the same geographical populations or different geographical populations; and compared with the enzyme digestion method, the method is simple, easy and economic, and general technicians can operate without complex technical steps and expensive reagents.

Description

technical field [0001] The invention relates to the fields of plant protection and biotechnology, in particular to a method for quickly identifying six common storage booklices based on multiplex PCR technology. Background technique [0002] Booklice belong to the order Psocoptera, family Liposcelididae, genus Liposcelis, and are the most economically important genus in the order Psocoptera. There are more than 120 known species of the genus Booklice, and 23 species have been reported in my country. Liposcelis bostrychophila ), insectophilic booklice ( L. entomophila ), the small-eyed booklice ( L. paeta ), colorless booklice ( L. decolor ), tricolor booklice ( L. tricolor ) and Yunnan booklice ( L. yunnaniensis ) and other 6 species. Booklice not only directly feed on stored food and cause serious harm, but also can carry pathogenic bacteria and cause food contamination, posing a serious threat to world food and food security. In my country, the resistance of bo...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 王进军魏丹丹豆威王梓英袁明龙
Owner SOUTHWEST UNIVERSITY
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