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TaqMan probe quantitative detection method for detecting pseudomonas putida and corresponding kit

A Pseudomonas putida quantitative detection method technology, applied in the field of molecular biology, can solve the problems of inability to quantify, fail to achieve accurate detection, long detection time, etc., achieve high sensitivity and reproducibility, rigorous and accurate results , The effect of stable test results

Inactive Publication Date: 2020-08-11
广东美格基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the first three types of methods are complicated to operate, time-consuming and low in detection sensitivity, and can only diagnose fish and shrimps with obvious diseases; although the results of PCR detection method are accurate, but the detection time is long, the operation is complicated, and cannot be quantified, so it is difficult to carry out in production. Popularization and application; although the detection time of the LAMP method is fast, the false positives are high, which cannot meet the requirements of accurate detection

Method used

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  • TaqMan probe quantitative detection method for detecting pseudomonas putida and corresponding kit
  • TaqMan probe quantitative detection method for detecting pseudomonas putida and corresponding kit
  • TaqMan probe quantitative detection method for detecting pseudomonas putida and corresponding kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1 detection primer amplification standard curve and plasmid sensitivity experiment

[0045] 1. Microbial culture

[0046] The LB medium was used as the medium for the Pseudomonas putida plasmid, and it grew well on a plate with a pH of 7.0 to 7.4. The diameter of the colony on the plate is 2mm, round, smooth and transparent. The vigorously growing microbial samples were selected for subsequent experiments.

[0047] 2. Genomic DNA Extraction

[0048] Genomic DNA was extracted according to the instructions of Qiagen's QIAamp DNA Mini Kit.

[0049] 3. Production of standard plasmids for amplified products

[0050] Use the DNA extracted from Pseudomonas putida to amplify with primers of 16sRNA gene. After the amplified product is treated with A, it is connected into the T vector using T4 ligase, and after transducing competent cells, the plasmid is obtained massive expansion. The primer sequences and TaqMan probe sequences of the 16sRNA gene are shown in SEQ ...

Embodiment 2

[0065] Example 2 Plasmid Repeatability Experiment

[0066] 1. Microbial culture

[0067] With embodiment 1.

[0068] 2. Genomic DNA Extraction

[0069] With embodiment 1.

[0070] 3. q-PCR detection of target genes

[0071] Perform q-PCR amplification of the sample according to the instructions of TaKaRa Taq PCR Mix, and obtain the Ct value reading corresponding to the primers. The q-PCR reaction in this experiment was set at two concentrations, which were 1×10 6 fg / μL and 1×10 5 fg / μL, 4 biological replicates and 5 technical replicates were set up for each concentration, and the q-PCR reaction system and amplification reaction conditions are shown in Table 5.

[0072] table 5

[0073]

[0074] 4. Result analysis

[0075] In this example, the correlation coefficient and reliability of the amplification curve of the 16sRNA gene and the amplification efficiency at two concentrations were analyzed, all of which met the requirements of the optimal state, and the primers...

Embodiment 3

[0078] Embodiment 3 Positive and negative sample detection situation

[0079] 1. Microbial culture

[0080] With embodiment 1.

[0081] 2. Genomic DNA Extraction

[0082] With embodiment 1.

[0083] 3. q-PCR detection of target genes

[0084] Perform q-PCR amplification of the sample according to the instructions of TaKaRa Taq PCR Mix, and obtain the Ct value reading corresponding to the primers. The q-PCR reaction was set up with 3 biological repetitions and 3 technical repetitions, and the q-PCR reaction system and amplification reaction conditions were the same as in Example 2.

[0085] 4. Result Analysis

[0086] as in Table 7 and Figure 4 As shown, the q-PCR amplification results of the primers showed that Pseudomonas putida showed positive results for the 16sRNA gene, while other insect species or bacteria genera showed negative results. Therefore, for Pseudomonas putida and other species or genera, the detection of 16sRNA gene can better distinguish Pseudomonas ...

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Abstract

The invention discloses a TaqMan probe quantitative detection method for detecting pseudomonas putida and a corresponding kit. Specific gene detection is ingeniously applied to distinguish pseudomonasputida from strains or insect species of other species, and accurate bacterial genus information is obtained through comprehensive judgment. Compared with the existing mainstream detection kit, the kit provided by the invention for detecting the pseudomonas putida has the advantages of high sensitivity, rapidness, convenience, high specificity, rigorous and accurate judgment and the like, and hashigh application prospect and market value.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method for quantitatively detecting Pseudomonas putida with a TaqMan probe through a specific gene and a corresponding detection kit. Background technique [0002] Pseudomonas putida belongs to the family Pseudomonadaceae and the genus Pseudomonas. It is already a common pathogenic bacteria of aquaculture animal diseases, and can cause diseases of sea and freshwater fish. [0003] The bacterium is a Gram-negative short bacillus with round ends, polar single or multiple flagella, motility, no spores, and the size of the bacteria is 0.6-1.0μm×1.5-3.0μm. On the ordinary nutrient agar plate, there are neat, round, flat, white and transparent moist colonies, and they grow slowly on the TCBS medium, showing green and transparent colonies. It does not grow in salt-free peptone water, but can grow in peptone water with 6% NaCl concentration, grows faster at 15-30°C and grow...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/04
CPCC12Q1/689C12Q1/6851C12Q2531/113C12Q2561/101
Inventor 唐伟杨荣束浩然黄斌蒋华束文圣
Owner 广东美格基因科技有限公司
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