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Specific SS-COI (species-specific-COI) primer based PCR rapid detection method for Frankliniella intonsa

A detection method and a specific technology, which are applied in the field of molecular biology, can solve the problems that the SS-COI method has not yet been applied in the detection of thrips, and achieve the effects of simple and rapid operation, strong specificity and high accuracy.

Inactive Publication Date: 2018-02-06
INST OF PLANT PROTECTION OF XINJIANG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Flower thrips is a major pest on cotton and a variety of agricultural and forestry crops in Xinjiang. At present, there are few related research reports in Xinjiang, and the SS-COI method has not been applied in the detection of flower thrips.

Method used

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  • Specific SS-COI (species-specific-COI) primer based PCR rapid detection method for Frankliniella intonsa
  • Specific SS-COI (species-specific-COI) primer based PCR rapid detection method for Frankliniella intonsa
  • Specific SS-COI (species-specific-COI) primer based PCR rapid detection method for Frankliniella intonsa

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1, using primers Fi63-4F / Fi63-4R to amplify flower thrips

[0042] (1) Preparation of the genome of Thrips florensis

[0043] Collect flower thrips in the cotton field, put single-head flower thrips into a 1.5mL centrifuge tube and grind it, and use OMEGA InsectGenomic DNA Kit extraction kit (OMEGA Company, USA) to extract single-head thrips according to the operating procedures attached to the kit. Genomic DNA of Thrips analis. Measure the concentration of the DNA solution (10.1 ng / μL) on a DNA concentration analyzer (Nanodrop, ND2000C, American Thermoelectric), store it at -20°C for later use, and draw 1 μL of the solution as a DNA template during PCR amplification.

[0044] (2) synthetically detect the specific SS-COI primer of Thrips floridae, the sequence is

[0045] Specific Forward Primer Fi63-4F for Thrips analis: 5'-CGCTAAGTATTTTAATTCGG-3'(SEQ ID No.1)

[0046] Specific reverse primer Fi63-4R for Thrips analis: 5'-AGTGGCACTAGTCAGTTTCC-3' (SEQ ID No.2...

Embodiment 2

[0052] Example 2, the specific amplification effect of primers Fi63-4F / Fi63-4R on Thrips florida

[0053] Utilize primers Fi63-4F / Fi63-4R, use Thrips floridae DNA as a template, and 32 other insects in the same domain as Thrips floridae (see Table 1) as controls for SS-COI PCR amplification, the results are as follows figure 1 shown. Only the 150bp target band was amplified in the 1 and 2 lanes corresponding to Thrips florida, and there were no corresponding bands in the other lanes, indicating that the SS-COI primers designed according to the mitochondrial DNA COI gene of Thrips florida had strong specificity.

[0054] Table 1. Insect species involved in primer-specific assays

[0055]

[0056]

Embodiment 3

[0057] Example 3, Primer Fi63-4F / Fi63-4R Determination of Minimum Detection Amount of Thrips floridae

[0058] According to the method in Example 1, the genomic DNA of Thrips unicephalus was extracted, and then the original template concentration of 10.1 ng / μL was diluted by 4 times until no band was detected, and 1 μL was used as a template for PCR detection, and PCR reaction was carried out. Its reaction system is the same as above.

[0059] Using primers Fi63-4F / Fi63-4R to measure the minimum detection amount, the minimum detection concentration of DNA of Thrips floridus is 0.158ng / μL, the results are as follows figure 2 shown.

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Abstract

The invention provides specific SS-COI (species-specific-COI) primers for Frankliniella intonsa, a kit containing the primers and used for specifically detecting Frankliniella intonsa, an applicationof the primers or the kit containing the primers to specific detection of the Frankliniella intonsa and a specific SS-COI primer based PCR rapid detection method for the Frankliniella intonsa. The SS-COI primers comprise Fi63-4F:5'-CGCTAAGTATTTTAATTCGG-3'(SEQID No.1) and Fi63-4R:5'-AGTGGCACTAGTCAGTTTCC-3'(SEQ ID No.2). The specific SS-COI primer based PCR rapid detection method comprises the following steps: 1) extracting DNA of a sample; 2) performing a PCR amplification reaction with DNA extracted in the step 1) as a template as well as the primers; 3) analyzing a PCR product.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a flower thrips specific SS-COI primer, a kit containing the primer and a PCR detection method based on the flower thrips specific SS-COI primer. Background technique [0002] Flower thrips (Frankliniella intonsa), also known as Taiwan flower thrips, belongs to Thysanoptera (Thysanoptera), Thripidae (Thripidae), flower thrips (Frankliniella), and is distributed in most parts of my country and the world (Han Yunfa , 1997). Thrips flower is a polyphagous pest with a wide range of hosts, which can harm a variety of crops, vegetables and flower plants. It is one of the important pests of agricultural and forestry crops. [0003] Flower thrips can reduce the yield of crops by eating and transmitting viruses, causing huge economic losses (Lewis, 1997; Nagata and Peters, 2001). There are two ways of damage to host plants by flower thrips: direct damage and indirect damage. ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6895C12Q2565/125
Inventor 李海强王冬梅刘建潘洪生丁瑞丰樊国全徐遥阿克旦·吾外士李号宾
Owner INST OF PLANT PROTECTION OF XINJIANG ACADEMY OF AGRI SCI
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