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Primer group for multi-fold PCR detection of regiella insecticola and detection method

A technology of symbiotic bacteria and primer sets, which is applied in the field of microbial detection, can solve the problems of time-consuming and labor-consuming detection of aphid symbiotic bacteria, and achieve the effects of saving detection costs, high sensitivity, and improving work efficiency

Active Publication Date: 2015-12-09
INST OF PLANT PROTECTION HENAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aiming at the problems existing in the prior art, the present invention provides a primer set and a detection method for multiplex PCR detection of aphid symbiotic bacteria. It overcomes the shortcomings of time-consuming and labor-consuming detection of aphid symbiotic bacteria in the prior art, and saves a certain amount of manpower and material resources for downstream related research

Method used

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  • Primer group for multi-fold PCR detection of regiella insecticola and detection method
  • Primer group for multi-fold PCR detection of regiella insecticola and detection method
  • Primer group for multi-fold PCR detection of regiella insecticola and detection method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Design and synthesis of primers:

[0046] (1) Screen the target gene or DNA fragment for primer design:

[0047] In the present invention, firstly, the target genes detected by multiplex PCR of each aphid symbiotic bacteria are obtained through literature analysis (see Table 1).

[0048] Table 1 Target genes detected by multiplex PCR of three common aphid symbiotic bacteria

[0049] target microorganism

target gene

Wolbachia

wsp gene

andricidal

yaeT gene

Aphid U-shaped symbiont

gltA gene

[0050] (2) Primer design of wsp gene:

[0051] Download the wsp gene sequences of multiple different Wolbachia strains in Genbank (the gene sequence numbers are: KJ596656.1, KC137225.1, AB096235.1, AY860419.1, KP208723.1, AB512756.1, AB231470 .1, AY157684.1, AB094386.1, AB094385.1, AB109567.1, AB109611.1, GQ385974.1, AB109591.1, KF898395.1), and then import the downloaded wsp gene sequence into MEGA6.0 software for Mu...

Embodiment 2

[0067] Embodiment 2: the preparation of template

[0068] 1. Extraction of total DNA from aphid samples

[0069] (1) Aphid material for testing: the aphid material used in the present invention is collected from Zhengzhou, Henan, Yuanyang wheat field in April 2013 (Sitobionmiscanthi) and grain constricted tube aphid (Rhopalosiphumpadi), then in the laboratory Single-female feeding was carried out to detect the infection of symbiotic bacteria, and to establish indoor populations of different symbiotic bacteria infections. Specifically used in the present invention are Aphids graminis infected with Wolbachia alone, Aphids graminis infected with andrcidal genus alone, Aphis spp. Four kinds of aphid samples of Aphis sativa were used as experimental materials for experimental research.

[0070] (2) Extraction steps of the total DNA of aphid samples: take out the raised single aphids, rinse them several times in 75% alcohol to remove bacterial contamination on the surface of the a...

Embodiment 3

[0103] Example 3: Concentration Gradient Screening of Primers and Detection of Amplification Sensitivity

[0104] 1. Primer concentration gradient screening

[0105] (1) Primer concentration screening for single primer PCR amplification experiments

[0106] Respectively with the three plasmids obtained above (the concentration of each plasmid is 10 4 copies / μl) as a template, compared the expansion of the PCR reaction when the concentrations of the single primer pairs Wol-F / R, Ars-F / R, and Reg-F / R in the PCR reaction system were 0.2 μm / L and 0.4 μm / L, respectively. increase the effect.

[0107] The reaction system of single-primer PCR amplification reaction (in the PCR reaction system, only one plasmid is added as a template each time, and then the primer pair corresponding to the template is added) is 50 μl: 10×PCRBuffer (100mM / LTris-HCl, 500mM / LKCl) 5μl, 2.5mM / L dNTPs4μl, 5U / μl TaqDNA polymerase 0.5μl, 10μm / L upstream primer 1-2.0μl, 10μm / L downstream primer 1-2.0μl, DNA ...

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Abstract

The invention discloses a primer group for multi-fold PCR detection of regiella insecticola. The primer group consists of primer pairs of Wolbachia pipientis, Arsenophonus and regiella insecticola. The invention further discloses a method for performing multi-fold PCR detection on regiella insecticola. According to the method, the primer group and the general DNA of an aphid sample to be detected are subjected to multi-fold PCR reaction; after the reaction, a PCR reaction product is subjected to electrophoretic analysis so as to determine whether the aphid sample contains regiella insecticola or not. The detection method is quick, accurate and high in sensitivity, and can be used for simultaneously authenticating the infection situations of three types of regiella insecticola in the aphid sample in one PCR reaction, so that the workload is greatly reduced, the working efficiency is improved, and the detection cost is reduced to a certain extent.

Description

technical field [0001] The invention belongs to the field of microbial detection, and in particular relates to a primer set and a detection method for multiple PCR detection of aphid symbiotic bacteria. Background technique [0002] Aphids, belonging to the order Hemiptera and the suborder Sternorrhyncha, are a class of important economic pests. Aphids and bacteria are closely related, and symbiotic bacteria play a variety of functions in aphids, such as supplementing nutrition, enhancing aphids' tolerance to high temperature, enhancing aphids' resistance to parasitic wasps and fungal infections, and expanding the host range of aphids. However, aphid symbionts have strict requirements on the living environment, and basically cannot be purely cultured in vitro of aphids. Therefore, the infection detection of aphid symbionts is identified by molecular means. Aphid symbionts are the third heritable substance besides aphid ribosomes and mitochondrial DNA. The analysis of the ge...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/01
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2537/143C12Q2565/125
Inventor 李彤蒋月丽段云苗进巩中军赵明茜武予清
Owner INST OF PLANT PROTECTION HENAN ACAD OF AGRI SCI