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Enterobacter cloacae 16SrDNA and primer group Yg2 and application thereof in aspect of enterobacter cloacae molecule detection

A technology of Enterobacter cloacae and primer set, which is applied in the direction of recombinant DNA technology, DNA/RNA fragments, microbial determination/inspection, etc., can solve the problems of insufficient research technology, achieve high sensitivity, reliable detection results, and good sensitivity Effect

Inactive Publication Date: 2018-04-10
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no relevant technical report on the application of LAMP technology for the detection of mulberry wilt. It can be expected that the research technology based on relevant primers and key detection conditions is insufficient, and LAMP technology has not yet been applied to the detection of mulberry wilt.

Method used

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  • Enterobacter cloacae 16SrDNA and primer group Yg2 and application thereof in aspect of enterobacter cloacae molecule detection
  • Enterobacter cloacae 16SrDNA and primer group Yg2 and application thereof in aspect of enterobacter cloacae molecule detection
  • Enterobacter cloacae 16SrDNA and primer group Yg2 and application thereof in aspect of enterobacter cloacae molecule detection

Examples

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Effect test

Embodiment 1

[0052] Example 1 Detection primer design and establishment of LAMP amplification method

[0053] 1. Primer design

[0054] In the present invention, a high-throughput sequencing method is used to successfully obtain a new Enterobacter cloacae 16SrDNA complete gene sequence SEQ ID NO.1 with the bacterial universal primer 27F / 1492R (Jiang and Wu et al., 2011), and then a large number of Primers, after analysis and screening, finally obtained a set of primers with excellent specificity and sensitivity, named Yg2. The sequence of the primer set is as follows:

[0055]

[0056]

[0057] 2. Establishment of LAMP amplification method

[0058] Using the isolated Enterobacter DNA as a template, using the positive and negative (water) of the kit (Guangzhou Double Helix Company) as a control, use the primer set Yg2 to refer to the following reaction system, and carry out LAMP amplification sequence at 63°C, such as SEQ ID Shown in NO.10. The amplified products were detected by ...

Embodiment 2

[0066] Specificity detection of embodiment 2 Yg2 primer set

[0067] 1. Use a variety of bacteria such as Pseudomonas DNA, Bacillus DNA, and Klebsiella DNA isolated from the roots and stems of diseased plants, and Phyllactinia moricola, the pathogenic fungus of mulberry trees. ), mulberry bacterial wilt pathogen Laueria DNA, mulberry virus disease mosaic wilt pathogen DNA, mulberry leaf stain pathogen DNA and Beauveria bassiana DNA, Aspergillus nomius DNA, Penicillium DNA, Cladosporium DNA DNA and other fungi were used as a control group, and the yg2 primer set was used to perform fluorescence amplification, color reaction, and agarose gel electrophoresis detection by the method of Example 1.

[0068] 2. The amplification results of the primers are as attached Figure 4 , Figure 5 , Figure 6 shown. The results showed that only the DNA of the mulberry wilt pathogen (Enterobacter cloacae) showed an S-shaped curve at the target position, and the display results turned green...

Embodiment 3

[0069] Sensitivity detection of embodiment 3 Yg2 primer set

[0070] 1. Extract the DNA of Enterobacter cloacae, the original concentration is 50ng / μL.

[0071] Dilute the above DNA with 1×TE, dilute 10, 10 2 、10 3 、10 4 、10 5 、10 6 times. That is to say, the concentration gradient is 5.0, 5.0×10 -1 , 5.0×10 -2 , 5.0×10 -3 , 5.0×10 -4 , 5.0×10 -5 ng / μL.

[0072] 2. Using the above concentrations of DNA as a template and the Yg2 primer set, LAMP fluorescence amplification, color reaction, and agarose gel electrophoresis detection were carried out by the method of Example 1.

[0073] 3. The result is as follows Figure 7 , Figure 8 , Figure 9 As shown, the Yg2 primer set can detect the DNA concentration of pathogenic bacteria at 5.0×10 -4 , with good detection sensitivity.

[0074] 4. Culture to 1×10 8 CFU / mL bacterial liquid gradient dilution is 1×10 7 CFU / mL, 1×10 6 CFU / mL, 1×10 5 CFU / mL, 1×10 4 CFU / mL, 1×10 3 CFU / mL, 1×10 2 CFU / mL, 1×10 1 CFU / mL, 1×1...

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Abstract

The invention discloses enterobacter cloacae 16SrDNA and a primer group Yg2 and an application thereof in an aspect of enterobacter cloacae molecule detection. A designed sequence of the enterobactercloacae rDNA is shown as SEQ ID NO.1; the primer group includes primers Yg2-FIP (F1 and F2), Yg2-BIP (B1 and B2), Yg2-F3, Yg2-B3, Yg2-LoopF and Yg2-LoopB; and nucleotide sequences of the primers are shown as SEQ ID NO.2-9. On the basis of the excellent specificity of the primer group, a kit for detecting enterobacter cloacae molecules is designed, and a detection method is scientifically determined. With the application of the primer group and the detection method provided by the invention, specific detection can be achieved in the early stage of pathogen infection, and the primer group and the detection method are strong in specificity, high in sensitivity and being visible; moreover, the primer group and the detection method are reliable and accurate in detection result and easy to operate; and a broad application value and an important significance can be achieved in practical detection of the enterobacter cloacae molecules and in the preparation of related products for detecting the enterobacter cloacae molecules.

Description

technical field [0001] The invention relates to the field of biotechnology, more specifically, relates to Enterobacter cloacae 16SrDNA, a primer set Yg2 and their application in molecular detection of Enterobacter cloacae. Background technique [0002] Enterobacter mulberry wilt, also known as mulberry wilt, is an important disease of mulberry trees. In recent years, it has commonly occurred in mulberry gardens in sericulture areas in South China. Following the first discovery of the pathogenic bacteria of mulberry bacterial disease in my country as the Enterobacter cloacae complex, E. cloacae asburiae, E. cloacae and an unnamed species (Enterobacter sp. .) and other 3 pathogenic bacteria. Among them, Enterobacter cloacae [0003] (Enterobactercloacae) is the most reported plant pathogenic bacteria in the Enterobacter genus, which was first reported by JordanEO in 1890. However, Enterobacter is a pathogenic bacterium with a variety of hosts and complex sources, so the dete...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/689C12Q2531/119C12Q2563/107Y02A50/30
Inventor 刘吉平杨宏宇
Owner SOUTH CHINA AGRI UNIV
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