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Primers and method of identifying cryptic specie of bemisia tabaci and trialeurodes vaporariorum

A technology of whitefly and whitefly, applied in biochemical equipment and methods, measurement/inspection of microorganisms, DNA/RNA fragments, etc., can solve the problems of difficulty in successful amplification

Inactive Publication Date: 2014-07-02
BIOTECH RES CENT SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Khansdan et al. (2005) used mtCOI and nuclear sodium ion channel genes to develop a method for identifying the MEAM1 cryptic species and MED cryptic species Bemisia tabaci. Since the amplified DNA fragments are all larger than 800bp, it is difficult to amplify successfully

Method used

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  • Primers and method of identifying cryptic specie of bemisia tabaci and trialeurodes vaporariorum
  • Primers and method of identifying cryptic specie of bemisia tabaci and trialeurodes vaporariorum
  • Primers and method of identifying cryptic specie of bemisia tabaci and trialeurodes vaporariorum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Bemisia tabaci MEAM1 cryptic species and Bemisia tabaci MED cryptic species described in Example 1 and Example 2 are populations raised in this laboratory;

Embodiment 3

[0036] Bemisia tabaci and whitefly described in Example 3 were collected from various places in Shandong Province in 2013 (Table 1).

[0037] Table 1

[0038]

[0039] The Sac II endonuclease described in the examples was purchased from Fermentas Company, Tris-HCl, ethylenediaminetetraacetic acid, and sodium lauryl sulfate were all purchased from Shanghai Bioengineering Company, and other reagents were all commercially available products.

[0040] Example 1

[0041] (1) Extraction of Genomic DNA of Bemisia tabaci MEAM1 and Bemisia tabaci MED

[0042] Place a single whitefly in a 0.2ml centrifuge tube containing 60 μl of alkaline lysate: 50 mmol L - 1 Tris-HCl (pH8.0), 20mmol·L -1 NaCl, 1mmol L -1 EDTA (ethylenediaminetetraacetic acid) and 1% SDS (sodium dodecyl sulfate) are fully ground and homogenized with a sealed gun head, placed in a water bath at 65°C for 15 minutes, and then placed in a water bath at 95°C for 10 minutes to obtain B. tabaci MEAM1 cryptic and B. t...

Embodiment 2

[0054] A method for distinguishing whitefly MEAM1 cryptic species and whitefly MED cryptic species, the steps are as follows:

[0055] (1) Put the laboratory-raised B. tabaci MEAM1 cryptic species and B. tabaci MED cryptic species as identification samples into 0.2 ml centrifuge tubes containing 60 μl of alkaline lysate: 50 mmol L -1 Tris-HCl (pH8.0), 20mmol·L - 1 NaCl, 1mmol L -1 EDTA, 1% SDS, fully grind the homogenate with a sealed pipette tip, put it in a water bath at 65°C for 15 minutes, and then put it in a water bath at 95°C for 10 minutes to obtain a genomic DNA solution;

[0056] (2) Using the genomic DNA prepared in step (1) as a template, performing PCR amplification on the mitochondrial COI gene in the genomic DNA to obtain a PCR amplification product;

[0057] The PCR amplification system is:

[0058] Genomic DNA solution 2.5μl, 20μM primer 0.5μl, 5U / μl Taq enzyme 0.25μl, 10×Taq Buffer 2.5μl, 10mM dNTP 0.5μl, ddH 2 O supplemented to 25 μl;

[0059] The prim...

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Abstract

The invention relates to a pair of primers and method of identifying a cryptic specie of bemisia tabaci and trialeurodes vaporariorum. The pair of primers of identifying the cryptic specie of bemisia tabaci MEAM1 (Middle East-Asia Minor one), the cryptic specie of bemisia tabaci MED (Mediterranean) and trialeurodes vaporariorum is respectively nucleotide sequences shown as SEQ ID NO. 1 and SEQ ID NO. 2. The invention further relates to a method of identifying the cryptic specie of bemisia tabaci and trialeurodes vaporariorum by the primers. The primers of identifying the cryptic specie of bemisia tabaci MEAM1, the cryptic specie of bemisia tabaci MED and trialeurodes vaporariorum can amplify BtabCSP2 genes in genomic DNAs of cryptic species of bemisia tabaci MEAM1 and MED but have no amplification effect on genomic DNA of trialeurodes vaporariorum. The primers provide simple, convenient and stable molecular markers for identifying the cryptic specie of bemisia tabaci MEAM1, the cryptic specie of bemisia tabaci MED and trialeurodes vaporariorum and solve the problem that an existing method of identifying the cryptic specie of bemisia tabaci MEAM1, the cryptic specie of bemisia tabaci MED and trialeurodes vaporariorum is tedious.

Description

technical field [0001] The present invention relates to a primer and identification method for identifying whitefly cryptic species and whitefly, in particular to a primer and identification method for identifying whitefly cryptic species Bemisia tabaci MEAM1, whitefly MED and whitefly, belonging to biotechnology field. Background technique [0002] Whitefly (Bemisa tabaci) and whitefly (Trialeurodes vaporariorum) are two worldwide omnivorous pests. Whitefly has always been an important agricultural pest in northern my country. Since the late 1990s, Bemisia tabaci has suddenly become a disaster in large areas in my country. In addition to directly harming crops, the two whiteflies also transmit a variety of important plant virus diseases. In northern China, they have overlapping hosts, the same hazard habits, and very similar life histories. They are also small in size and similar in appearance, and often occur in the same domain. It is difficult to distinguish them only ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/683C12Q1/686C12Q2531/113C12Q2521/301
Inventor 刘国霞让·弗朗西斯·皮西姆邦宣宁步迅谢红艳岳寿松杨连群张全芳范仲学
Owner BIOTECH RES CENT SHANDONG ACADEMY OF AGRI SCI
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