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Real-time fluorescence quantitative PCR detection kit for deformed pseudomonas plecoglossicida TaqMan and preparation method thereof

A Pseudomonas mutans, real-time fluorescence quantitative technology, applied in biochemical equipment and methods, microbe-based methods, microbiological determination/inspection, etc., can solve the problems of low accuracy, cumbersome operation, and low sensitivity. Achieve the effect of high practical value, simple operation and complete reagents

Pending Publication Date: 2020-07-24
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, some of these methods are time-consuming, cumbersome to operate, and some have low accuracy and low sensitivity.

Method used

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  • Real-time fluorescence quantitative PCR detection kit for deformed pseudomonas plecoglossicida TaqMan and preparation method thereof
  • Real-time fluorescence quantitative PCR detection kit for deformed pseudomonas plecoglossicida TaqMan and preparation method thereof
  • Real-time fluorescence quantitative PCR detection kit for deformed pseudomonas plecoglossicida TaqMan and preparation method thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0059] 4. Preparation of positive control

[0060] Extract the constructed pMD18-gyrB plasmid, then use a spectrophotometer to detect the quality and concentration of the plasmid, calculate the copy number of the target gene according to the following formula, and then dilute it with sterilized ultrapure water to a concentration of 1×10 8 Copies / μL of positive gyrB plasmid as a positive standard control

[0061] 5. Design TaqMan probes

[0062] According to the sequencing sequence results of positive clones of recombinant plasmids, use Primer Premier 5 to design probes that meet the following requirements: ①The first letter cannot be "G"; ②Try not to appear consecutive "G"s ≥ 3; ③Generally try to use "C" bases Base content > "G" base, A, T, C, G content is uniform, GC content is 40% to 60%; ④The length of the probe should be ≤27bp, preferably not more than 30bp, and the Tm value should be ≥10ºC than the primer. The needle was synthesized by Shanghai Sangon Co., and its 5' en...

Embodiment 1

[0090] The specificity experiment of embodiment 1 primer

[0091] In order to determine the primer specificity of the TaqMan probe real-time fluorescent quantitative PCR detection kit, Vibrio cannellii, Streptococcus agalactiae, Pseudomonas aeruginosa, Aeromonas hydrophila, Vibrio parahaemolyticus, Harvey Vibrio, Pseudomonas mutans, Vibrio alginolyticus, Shewanella, Vibrio nocardia, Streptococcus dysgalactiae, Pseudomonas putida, Pseudomonas fluorescens DNA as templates, sterile ultra- Pure water was used as a blank control without a template, and the established routine PCR was used for detection. The experimental results showed that only Pseudomonas mutans was detected positive, and all others were negative (such as figure 2 ), which indicated that the designed primers had excellent specificity and could be used for rapid detection of Pseudomonas mutans.

Embodiment 2

[0092] Embodiment 2 Sensitivity detection experiment

[0093] Dilute the positive control plasmid (pMD18-gyrB) by 1 × 10 8 , 1×10 7 , 1×10 6 , 1×10 4 , 1×10 3 , 1×10 2 , 5×10 1 , 1×10 1 Copy number / μL was serially diluted, and at the same time, the concentration of the original bacterial solution of Pseudomonas mutans was measured by plate counting, and then diluted according to 10-fold gradients, and then 1 mL of bacterial DNA of each gradient was extracted, and 0.2 μL was taken as a template for each, and the samples were tested in a fluorescent PCR instrument. The results showed that the number of cycles (Ct) required for the fluorescent signal in each reaction tube to reach the threshold value and the logarithm of the initial template copy number had an obvious linear relationship (R 2 =0.996) (such as image 3 ), the lower limit of detection for plasmids is 5×10 1 Copy number / μL (eg Figure 4 ), the detection limit for pure bacterial cultures is 50 cfu / mL (such ...

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Abstract

The invention provides a real-time fluorescence quantitative PCR detection kit for a deformed pseudomonas plecoglossicida TaqMan and a preparation method thereof. The kit comprises a primer and a probe for specifically detecting the deformed pseudomonas plecoglossicida, wherein the sequence is as follows: a formula as shown in the specification. The kit provided by the invention is complete in reagents and simple to operate, and provides a detection tool for the scale detection and control of the disease.

Description

technical field [0001] The invention relates to the field of detection of fish pathogenic bacteria, in particular to a Pseudomonas mutans ( Pseudomonas plecoglossicida ) TaqMan probe real-time fluorescent quantitative PCR detection kit and preparation method thereof. Background technique [0002] Pseudomonas denatus ( Pseudomonas plecoglossicida ) is a straight or slightly curved Gram-negative bacillus with polar flagella, and its size is 0.5~1 μm × 2.5~4.5 μm. It was first obtained in 1991 from sweetfish suffering from bacterial hemorrhagic ascites ( Plecoglossus altivelis ) were separated. It has been reported that Pseudomonas mutans can infect sweetfish ( P. altivelis ), large yellow croaker ( Larimichthys crocea ), slanted grouper ( Epinephelus coioides ) and rainbow trout ( Oncorhynchus mykiss )Wait. Visceral white spot disease caused by Pseudomonas mutans is the most serious seasonal infectious disease of large yellow croaker cultured in cages, and it ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/06C12N15/11C12R1/38
CPCC12Q1/689C12Q1/6851
Inventor 陈新华李承伟何天良
Owner FUJIAN AGRI & FORESTRY UNIV
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