Kit for detecting Aphis gossypii Glover and parasitic wasp thereof in North China

A technology of cotton aphids and kits, applied in the field of molecular biology, can solve the problems of difficult identification of specific species by morphological characteristics, time-consuming and labor-intensive problems, and achieve the effects of shortening detection time, improving detection efficiency and high accuracy

Pending Publication Date: 2018-08-03
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current research on the control of parasitoids in cotton fields is limited to the investigation of the number and rate of aphids in cotton fields and the collection of samples of aphids
It is not only time-consuming and labor-intensive for parasitoid bees to be identified through breeding, but it is also difficult to identify specific species because of the tiny fuzzy morphological characteristics.
In order to accurately assess the occurrence dynamics of parasitoids in cotton fields and their control effects on aphids, it is necessary to establish a set of rapid and accurate molecular detection methods, but there is currently no systematic standard detection method for aphids and parasitoids in cotton fields

Method used

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  • Kit for detecting Aphis gossypii Glover and parasitic wasp thereof in North China
  • Kit for detecting Aphis gossypii Glover and parasitic wasp thereof in North China
  • Kit for detecting Aphis gossypii Glover and parasitic wasp thereof in North China

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1 The specific amplification effect of the multiplex PCR system SP1 on the target species

[0064] 1. Preparation of the genome of the target aphid species

[0065]Put the single-headed aphid into a 1.5mL centrifuge tube for tissue grinding and DNA extraction. The centrifuge tube containing the DNA solution is marked with the species name and stored at -20°C for later use.

[0066] 2. Specific primers for the synthesis of cotton aphids. The sequence of the specific 16S primers for cotton aphids is as follows:

[0067] Forward primer: 5’-TTGAATGAAAGATTTGATGAGAAATAG-3’

[0068] Reverse primer: 5'-TCACCCCAATAAAATAAATTTTAATT-3'

[0069] 3. PCR amplification reaction system is calculated in 10 μL, including ddH 2 O, 1.5 μL; 2×Multiplex PCR MasterMix, 5.0 μL; Forward primer, 1.0 μL; Reverse primer, 1.0 μL and DNA template, 1.5 μL.

[0070] The PCR reaction conditions were: pre-denaturation at 95°C for 15 minutes; denaturation at 94°C for 30 seconds, annealing at 6...

Embodiment 2

[0073] Example 2 The specific amplification effect of the multiplex PCR system MP1 on the target species

[0074] 1. Preparation of the genome of the target parasitoid species. The single Aphididae, Aphididae, and A. gossypii were placed in 1.5mL centrifuge tubes for tissue grinding and DNA extraction. Label the tube with the species name and store it at -20°C.

[0075] 2. The primer sequences in the primer system required in the synthetic multiplex PCR system MP1 are shown in Table 3 below:

[0076] table 3

[0077]

[0078] Each pair of primers was prepared in proportion to a mixed primer solution, and the final concentration of each primer in the reaction system was 0.4 μM.

[0079] 3. PCR amplification reaction system is calculated in 10 μL, including ddH 2 O, 2.5 μL; 2×Multiplex PCR MasterMix, 5.0 μL; Primer Mix, 1.0 μL and DNA template, 1.5 μL.

[0080] The PCR reaction conditions were: pre-denaturation at 95°C for 15 minutes; denaturation at 94°C for 30 seconds, ...

Embodiment 3

[0083] Example 3 The specific amplification effect of the multiplex PCR system MP2 on the target species

[0084] 1. Preparation of the genome of the target parasitoid species

[0085] The single-headed broad-shouldered A. spp., A. aphids., A. aphids. and Phaenoglyphis villosa were placed in 1.5mL centrifuge tubes for tissue grinding and DNA extraction. Label the tube with the species name and store it at -20°C.

[0086] 2. The primer sequences in the primer system required in the synthetic multiplex PCR system MP2 are shown in Table 4 below:

[0087] Table 4

[0088]

[0089]

[0090] Each pair of primers was prepared in proportion to a mixed primer solution, and the final concentration of each primer in the reaction system was 0.2 μM.

[0091] 3. PCR amplification

[0092] The reaction system is calculated as 10 μL, including ddH 2 O, 2.0 μL; 2×Multiplex PCR Master Mix, 5.0 μL; Primer Mix, 1.0 μL; 10 μg / μL BSA, 0.5 μL and DNA template, 1.5 μL.

[0093] The PCR re...

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PUM

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Abstract

The invention provides a kit for detecting aphis gossypii glover and parasitic wasp thereof in North China. The kit comprises two multiple-PCR detection system and three single-PCR detection system, asequence of a specific primer used by each of the two multiple-PCR detection systems is respectively as shown by SEQ ID NO.1-6 and SEQ ID NO.7-12, and the sequences of the specific primers of the three single-PCR detection systems are as shown by SEQ ID NO. 13-18. The kit can accurately detect the aphis gossypii glover and eight parasitic wasps thereof in North China, a fragment size is within 500bp, the detection limit reaches 1000replica, the sensitivity is high, the specificity is high, the time-consuming is short, simplicity and rapidness can be achieved, by combining a single PRC detection technology, the information of 9 to 10 species can be acquired, while the accuracy and completeness of the detection information are ensured, the aphid-parasitic wasp food network relationship canbe rapidly established, and the application prospect is good.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a kit for detecting wheat aphids and their parasitoids in North China and applications thereof. Background technique [0002] Cotton aphid (Aphis gossypii Glover) is the main aphid species in cotton fields in North China. It feeds on phloem sap and causes serious damage to cotton fields in the early stages of cotton growth. The abuse of insecticides has made cotton aphid resistant to pesticides. Under the environmental conditions suitable for its growth, high density of cotton aphid will affect the quality of cotton and greatly reduce the yield. Therefore, the prevention and control of cotton aphid should be paid enough attention, and natural enemies play a vital role in the control of cotton aphid population. [0003] Cotton aphid parasitoid is an important parasitic natural enemy of cotton aphid, which deserves our in-depth study. However, the current research on the control ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888C12Q2600/16
Inventor 陆宴辉杨帆
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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