A multiple PCR detection kit for wheat aphids and their parasitoids

A kit and parasitic wasp technology, applied in the field of multiplex PCR detection, can solve the problems of difficulty in identifying species, time-consuming and labor-intensive, etc.

Inactive Publication Date: 2020-11-10
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

Traditional research methods include dissection, bee breeding, and morphological identification, which are time-consuming and laborious, and sometimes it is difficult to identify specific species based on morphological identification characteristics due to the small size of aphids and parasitoids
In order to accurately evaluate the control effect of parasitoids on aphids in wheat fields, a rapid and accurate molecular detection method needs to be established, but there is currently no systematic standard detection method for aphids and parasitoids in wheat fields

Method used

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  • A multiple PCR detection kit for wheat aphids and their parasitoids
  • A multiple PCR detection kit for wheat aphids and their parasitoids
  • A multiple PCR detection kit for wheat aphids and their parasitoids

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Example 1 The specific amplification effect of the multiplex PCR system MP1 on the target species

[0092] 1. Preparation of the target aphid species genome

[0093] Put the single-headed wheat without nets, Aphids aphids, Aphids aphids, and Aphis granola into 1.5mL centrifuge tubes for tissue grinding and DNA extraction, and mark the centrifuge tubes storing different aphid DNA solutions Species name, spare at -20°C.

[0094] 2. Synthesize the primers required in the multiplex PCR system MP1, see the 4 pairs of primers corresponding to the MP1 system in Table 1. 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 shown in Table 1.

[0095] 3. PCR amplification reaction system is calculated in 10 μL, including ddH 2 O, 2.0 μL; 2×Multiplex PCR MasterMix, 5.0 μL; Primer Mix, 1.0 μL; 10 μg / μL BSA, 0.5 μL and DNA template, 1.5 μL.

[0096] The PCR reaction conditions were: pre-...

Embodiment 2

[0099] Example 2 Multiplex PCR System MP2 Specific Amplification Effect on Target Species

[0100] 1. Preparation of the genome of the target parasitoid species

[0101] The single-headed winged aphids, Aphididae, Uzbekia, Aphidia, and Aphidia were placed in 1.5mL centrifuge tubes for tissue grinding and DNA extraction, and the centrifuge tubes storing DNA solutions of different parasitoids were marked with Species name, spare at -20°C.

[0102] 2. For the primers required in the synthesis of the multiplex PCR system MP2, see the corresponding primers for the MP2 system in Table 1. Each pair of primers was prepared in proportion to a mixed primer solution, and the final concentration of each primer in the reaction system is shown in Table 1.

[0103] 3. PCR amplification reaction system is calculated in 10 μL, including ddH 2 O, 1.8 μL; 2×Multiplex PCR Master Mix, 5.0 μL; Primer Mix, 1.0 μL; 10 μg / μL BSA, 0.5 μL; 5M TMAC, 0.2 μL and DNA Template, 1.5 μL.

[0104] The PCR r...

Embodiment 3

[0107] Example 3 Multiplex PCR System MP3 Specific Amplification Effect on Target Species

[0108] 1. Preparation of the genome of the target parasitoid species

[0109] Refer to Example 2 for the extraction methods of Chrysopus spp., Aphids aphids Phaenoglyphis villosa and Phaenoglyphis villosa.

[0110] 2. Synthesize the primers required in the multiplex PCR system MP3. The primer sequences in the system and the final concentration of each primer in the reaction system are shown in Table 1.

[0111] 3. PCR amplification reaction system is calculated in 10 μL, including ddH 2 O, 2.0 μL; 2×Multiplex PCR MasterMix, 5.0 μL; Primer Mix, 1.0 μL; 10 μg / μL BSA, 0.5 μL and DNA template, 1.5 μL.

[0112] The PCR reaction conditions were: pre-denaturation at 95°C for 15 minutes; denaturation at 94°C for 30 seconds, annealing at 58°C for 1 minute and 30 seconds, and extension at 72°C for 1 minute, a total of 35 cycles.

[0113] 4. Identification of PCR products Take 5.0 μL of PCR amp...

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Abstract

The invention provides a wheat aphid and a multiplex PCR (polymerase chain reaction) detection kit of parasitic wasp thereof and belongs to the technical field of multiplex PCR detection. The kit comprises three multiplex PCR detecting systems and five single PCR detecting systems; specific primer sequences applied to three multiplex PCR detecting systems are shown as SEQ ID NO. 1-8, SEQ ID N 0.9-16, SEQ ID NO. 17-22; the specific primer sequences of five single PCR detecting systems are shown as SEQ ID NO. 23-32. The kit can accurately detect 4 wheat aphid and its parasitic wasp, and has advantages of high sensitivity, strong specificity, short time waste, simple and fast operation; moreover, the kit has good application prospect.

Description

technical field [0001] The invention relates to the technical field of multiplex PCR detection, in particular to a kit for detecting wheat aphids and their parasitoids in North China and applications thereof. Background technique [0002] Wheat aphids are worldwide agricultural pests and are widely distributed in my country's wheat producing areas. They suck wheat leaf juice, affect photosynthesis, and spread wheat virus diseases, resulting in a serious decline in wheat yield and quality. The types of aphids in wheat fields in North China include Sitobion avenae (Fabricius), Rhopalosiphum padi (Linnaeus), Schizaphisgraminum (Rondani), and Metopolophium dirhodum (Walker). The population of wheat aphids occurs in a large number, which causes a large amount of production reduction in wheat fields every year. The number of aphids per hundred plants exceeds 2000, and the thousand-grain weight of wheat decreases by 45%. [0003] The wheat aphid parasitoid colony is an important p...

Claims

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Application Information

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Patent Type & Authority Patents(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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