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Rapid diagnosis method for plesimonas shigelloides

One-pair technology, applied in the field of rapid diagnosis of P. shigella, can solve the problem of reducing reaction specificity and sensitivity, high requirements for primer specificity, high requirements for ratio optimization solution, etc. problems, to achieve the effect of easy popularization and application, improved detection efficiency, and high sensitivity

Inactive Publication Date: 2014-07-30
ICDC CHINA CDC
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0008] The biggest disadvantage of the LAMP method is that it is prone to false positives. Since LAMP uses multiple primers and is amplified at the same temperature, the primers may complement each other and amplify non-specific bands, resulting in false positives.
Therefore, the LAMP method has high requirements on the specificity of the primers, and the design is more complicated.
Since LAMP omits the 94°C denaturation step, and the annealing and extension steps are carried out at the same temperature (isothermal), the composition of the reaction system is also more complicated than that of traditional PCR, except for the necessary buffer, dNTP primers, enzymes and other components in the reaction system , also need to add betaine (betaine), magnesium sulfate and other essential components, the reaction system is relatively complex, and the requirements for the ratio optimization solution of the main components in the system are relatively high, otherwise the specificity and sensitivity of the reaction will be reduced

Method used

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  • Rapid diagnosis method for plesimonas shigelloides
  • Rapid diagnosis method for plesimonas shigelloides
  • Rapid diagnosis method for plesimonas shigelloides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1: the detection of 4 parts of Shigella-like Plasmomonas

[0045] By comparing the known gene sequences (GeneBank ID number) of P. shigella-like hugA gene in GenBank, we selected a conserved sequence on the hugA gene as the detection target. Using the primer design software PrimerExplorer V4 software ( http: / / primerexplorer.jp ) (Eiken Chemical Co. Ltd., Tokyo, Japan) for LAMP primer design online. Primers were synthesized by Shanghai Sangon Co., Ltd. (see Table 2). The reaction system of LAMP is as follows: the concentration of primers SEQ ID NO 1 and SEQ ID NO 2 is 5pmol, the concentration of primers SEQ ID NO 3 and SEQ ID NO4 is 40pmol, the concentration of primers SEQ ID NO 5 and SEQ ID NO 6 is 20pmol, 10mM Betain, 6mM MgSO 4 , 1 mM dNTP, 2.5 μL of 10×Bst DNA polymerase buffer, 8 U of strand displacement DNA polymerase, 2 μL of template, and add deionized water to 25 μl. The whole reaction was carried out in LA320 real-time turbidimeter (Teramecs, To...

Embodiment 2

[0047] Embodiment 2: Sensitivity evaluation of LAMP detection:

[0048] 1. Construction and preparation of plasmid standards containing hugA gene

[0049] (1) Use hugA-specific primers for PCR amplification to obtain the target fragment;

[0050] (2) Gel cutting recovery, purification of PCR products;

[0051] (3) connected to the T carrier;

[0052] (4) transform JM109 competent cells;

[0053] (5) Screen positive clones, verify by PCR, and finally confirm by sequencing;

[0054] (6) Extract the plasmid, and convert the concentration of the plasmid sample to the copy number concentration according to the molecular weight of the plasmid: the copy number of the detected gene in each μL sample=concentration (ng / μL)×Avogadro’s constant×10 -9 / (660×recombinant plasmid base number);

[0055] (7) Use Easy Dilution to dilute the above plasmids to 1.0×10 0 Copy / μL~1.0×10 6 copies / μL, a total of 7 concentration gradients.

[0056] 2. Sensitivity of LAMP detection system

[005...

Embodiment 3

[0063] Example 3: Evaluation of the specificity of LAMP detection: the specificity of the LAMP system was evaluated using DNA of common pathogenic bacteria and conditional pathogenic bacteria (see Table 2) as templates.

[0064]The specificity of LAMP detection was evaluated using the DNA of 52 strains of common pathogenic bacteria and conditional pathogenic bacteria (see Table 2). The 20 strains of P. shigella-like bacteria all showed positive amplification within 1 hour, and the 32 strains of non-P. shigella-like bacteria showed no amplification, which indicated that the specificity of the LAMP detection technique was good.

[0065] Bacterial strains used in the experiment of table 1

[0066]

[0067]

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Abstract

The invention provides a method for detecting plesimonas shigelloides. The method comprises the following steps of: adopting a loop-mediated isothermal amplification technology; using a hug A gene as a target gene; putting designed three pairs of DNA (Deoxyribonucleic Acid) primers and a DNA template to be detected to an amplification buffer solution including MgSO4 and glycine betaine; amplifying in a constant-temperature environment of 65 DEG C, and terminating the reaction in a constant-temperature environment of 80 DEG C; and detecting the amplified product. The method disclosed by the invention is simple and easy to operate, and has good specificity, sensitivity and repeatability.

Description

technical field [0001] The invention relates to a rapid diagnosis method for Pseudomonas shigella, belonging to the field of rapid diagnosis of microorganisms. Background technique [0002] Plesiomonas shigelloides is a Gram-negative, motile, facultatively anaerobic, oxidase-positive, nonspore-producing bacterium that has been associated with human pathogenic Vibrios and Aeromonas The genus belonged to Vibriaceae together, and later studies found that it was closer to Pseudomonas Shigella and Proteus at the molecular level. In the second edition of "Bergey's Handbook of Systematic Bacteriology", Shigella-like Pseudomonas is assigned to Enterobacteriaceae, Pseudomonas genus. [0003] Freshwater sources and river ecological environments are the main storage places for P. shigella-like bacteria, and fish are common hosts for P. shigella-like bacteria. In recent years, the cases of gastrointestinal infection caused by this bacteria in infants, the elderly and patients with imm...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 孟双徐建国叶长芸
Owner ICDC CHINA CDC
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