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A primer for rapid identification of two cryptic species of Bemisia tabaci, meam1 and med, and its identification method

A Bemisia tabaci and rapid technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial determination/inspection, etc., can solve the problem of long cycle, high cost, and the inability to adapt to a large number of single Bemisia tabaci identification methods. Sample identification and other problems, to achieve the effect of low cost, high reliability, and improved DNA extraction efficiency

Active Publication Date: 2017-11-17
INST OF PLANT PROTECTION FAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide primers for rapid identification of two cryptic species of Bemisia tabaci, MEAM1 and MED, in view of the problems that the identification method of whitefly cryptic species in the prior art cannot adapt to the identification of a large number of single samples, and the cycle is long and the cost is high. A double-primer PCR amplification system for the identification of whitefly MEAM1 and MED cryptic species was developed, and the results are reliable

Method used

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  • A primer for rapid identification of two cryptic species of Bemisia tabaci, meam1 and med, and its identification method
  • A primer for rapid identification of two cryptic species of Bemisia tabaci, meam1 and med, and its identification method
  • A primer for rapid identification of two cryptic species of Bemisia tabaci, meam1 and med, and its identification method

Examples

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Effect test

Embodiment 1

[0042] Example 1: Primers specific amplification of different cryptic species of Bemisia tabaci

[0043] Materials: 50 samples of different Bemisia tabaci collected from eight cities in Fujian in 2014, each sample was compared with the mtDNA COI gene sequencing results.

[0044] 1. Single DNA extraction

[0045] Carefully put the single Bemisia tabaci sample into the bottom of the PCR tube, use a self-made electric grinder (self-made grinding rod + small electroporation), stick a little DNA extraction solution (1% SDS, 10mmol / L Tris-HCl, pH 8.0 , 25mmol / LNaCl, 5mmol / L EDTA), placed at the bottom of the PCR tube in contact with the sample, after grinding for 15 seconds, add 40ul extract and 2ul proteinase K solution to each tube, mix well for later use; put the ground and mixed sample in Water bath at 58°C for 20min, then add 80u1 of pre-cooled absolute ethanol, mix gently, and precipitate at -20°C for 10min; centrifuge the precipitated sample at 12000r / min for 3min, discard t...

Embodiment 2

[0051] Embodiment 2: Sensitivity detection of primers

[0052] 1. DNA concentration dilution: the extracted Bemisia tabaci (cryptic species MED, provided by the Plant Protection Institute of the Chinese Academy of Agricultural Sciences) genomic DNA, after measuring the concentration with a Thermo ultraviolet spectrophotometer, six samples were used in a series of concentrations (10, 1, 0.1 , 0.01, 0.001, 0.0001ng / μl) dilution.

[0053] 2. Test results: such as image 3 As shown, in the 20μl reaction system, the genomic DNA of six samples of Bemisia tabaci amplified the obvious target band except the sixth sample, and the other samples all amplified the obvious target band, and the detection sensitivity could reach 1pg.

Embodiment 3

[0054] Example 3: Specific amplification of two cryptic species of Bemisia tabaci with double primers

[0055] 1. Sample DNA Extraction

[0056] Put the mixed sample of the two cryptic species of Bemisia tabaci into the bottom of the centrifuge tube, use a self-made electric grinder (self-made grinding rod + small electric transfer), stick a little DNA extraction solution (1% SDS, 10mmol / L Tris-HCl, pH 8.0, 25mmol / LNaCl, 5mmol / L EDTA), placed at the bottom of the PCR tube in contact with the sample, after grinding for 15 seconds, add 100ul extract solution and 5ul protease solution to each tube, mix well for later use; 58℃ water bath for 20min, then add pre- Cold absolute ethanol 80u1, mix gently, precipitate at -20°C for 10min; centrifuge at 12000r / min for 3min, discard the supernatant, dry naturally or ventilated, add TE solution (100ul), save for later use.

[0057] 2. Double-primer detection of Bemisia tabaci MEAM1 and MED cryptic species

[0058] Double-primer PCR ampli...

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Abstract

The present invention discloses primers and identification method for rapid identification of two bemisia tabaci cryptic species of MEAM1 and MED. A pair of primers are designed respectively mainly aiming at mitochondrial COI gene sequences of two bemisia tabaci cryptic species of MEAM1 and MED: primers for the MEAM1 cryptic species are: 5'-CACTATAATTATTGCTGTTCCC-3', and 5'-ACCTAAGATTAGTGGAAATC-3'; and primers for the MED cryptic species are 5'TTAATTAGCAGCGAGGCTGG-3', and 5'-AGGAAC GGCAATAATCATAGTAGC-3'. The sample DNA extraction were improved, and a method for rapid extraction of a large number of single bemisia tabaci sample DNA is provided, so as to stably obtain a large number of single bemisia tabaci sample DNA applied to identification of both cryptic species in shortened time. The invention constructs a double primer PCR amplification system for molecular identification of bemisia tabaci cryptic species of MEAM1 and MED. The present invention can be applied in rapid identification, field distribution and risk assessment of a large amount of alien invasive bemisia tabaci cryptic species of MEAM1 and MED in the field, and provides a scientific basis to develop strategies for the prevention and treatment of bemisia tabaci.

Description

technical field [0001] The present invention relates to the field of crop pest detection, identification and control technology, in particular, it relates to a primer for rapid identification of Bemisia tabaci MEAM1 and MED two cryptic species and its identification method, which is specially used for alien invasive organisms Bemisia tabaci MEAM1 and MED The rapid, sensitive and accurate molecular identification of cryptic species can also be used for monitoring and forecasting of invasive cryptic species in a large population of Bemisia tabaci in the field. Background technique [0002] Bemisia tabaci (Gennadius) is widely distributed in all continents except Antarctica, and is an important pest in tropical, subtropical and adjacent temperate regions (Brown et al., 1995). Bemisia tabaci is a compound group in a continuous evolution process. According to the biological differences of different geographical populations in host range, hazard habit, virus transmission ability a...

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 郑宇何玉仙姚凤銮赵建伟丁雪玲翁启勇
Owner INST OF PLANT PROTECTION FAAS