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Quantitative detection method of polyphosphate in a kind of microbial cell

A quantitative detection method, polyphosphate technology, applied in the biological field, can solve problems such as incomprehension, and achieve the effect of small sample demand, sample pretreatment and simple reaction process

Active Publication Date: 2017-05-31
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In view of the differences between disciplines, although scholars engaged in spectroscopic analysis or chemical analysis, from their own point of view, various factors that affect the accuracy and reproducibility of the experiment (such as storage methods of standards and samples, pretreatment methods of samples, The impact of various ions and their concentrations in the detection system, the settings of various controls during detection, etc.) have been analyzed in detail, but for ordinary scientific researchers who are not professional, when faced with complicated samples When applying this method, it seems a little obscure and difficult to understand, and I don’t know where to start. Therefore, it is necessary to develop a simple and efficient method for the direct quantification of poly P in microbial cells based on this principle, which is suitable for general biological experiments.

Method used

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  • Quantitative detection method of polyphosphate in a kind of microbial cell
  • Quantitative detection method of polyphosphate in a kind of microbial cell
  • Quantitative detection method of polyphosphate in a kind of microbial cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 Construction of poly P standard curve and detection of poly P concentration in samples

[0071] Take a sampling in a daily experiment as an example, such as figure 1 As shown, it contains all the parameters of DAPI-poly P complex detection, data A1-B6 is the original data of this test bracket construction, data C1-H11 is the original data of this test sample and its control, all data The units are relative fluorescence values ​​(Relative fluorescence unit, Rfu).

[0072] In view of the fact that 1U=3 μM orthophosphate, for the convenience of graphical description, we refer to the different concentration gradients of the standard as the relative concentration of poly P (Relative Concentration). After processing the raw data of A1-B6 standard products, the data listed in Table 3 can be obtained. For example, the data corresponding to the first column "0U" is the average value of A2B2 minus the average value of A1B1, and so on.

[0073] Table 3 Rfu correspondin...

Embodiment 2

[0079] The construction of embodiment 2 BSA standard curve and the detection of sample protein concentration

[0080] After obtaining the sample Y P After the value, you also need to get the corresponding Y A value. Such as image 3 As shown, it contains all the parameters of protein concentration detection, data A1-B6 is the original data of this detection standard curve construction, data C1-H11 is the original data of this detection sample (wherein G1-H11 is the sample S1-S11 The corresponding data, the rest of the data is the data of other samples of the same batch), and the unit of all data is the absorbance of the sample at 750nm.

[0081] After processing the raw data of A1-B6 standard products, the data in Table 5 can be obtained. For example, the data corresponding to "0.2" in the first column is the average value of A2B2 minus the average value of A1B1, and so on.

[0082] Table 5 Absorbance at 750nm of different concentrations of BSA standard

[0083]

[008...

Embodiment 3

[0088] Example 3 The final quantification of microbial intracellular poly P

[0089] After getting all samples of Y P and Y A After the value, according to the final quantitative formula of poly P: T PP =30.9738Y P / Y A The intracellular poly P content of each microbial sample can be calculated, and the detailed data are shown in Table 7.

[0090] Table 7 Polyp content in samples S1-S11

[0091]

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Abstract

The invention discloses a method for quantitatively detecting polyphosphates in microbial cells. The method comprises the steps of preprocessing and preparing a sample, determining the content of polyphosphates in the microbial cells, determining bacterial proteins, and quantifying the polyphosphates poly P in the microbial cells. Compared with the prior art, the sample preprocessing and reaction process is simple, the demand of the sample is small, and a plurality of samples can be simultaneously detected. Meanwhile, the minimum detection limit of the poly P in the method can reach an ng scale, thus having significance for the basic scientific research.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a quantitative detection method for polyphosphate in microbial cells. Background technique [0002] Polyphosphate (poly P) is a linear polymer composed of several to thousands of phosphate residues polymerized through phosphoric anhydride bonds, and widely exists in bacteria, fungi and higher mammalian cells. Studies have shown that the biological functions of poly P mainly include: (1) a way of cellular energy storage; (2) storage of phosphorus; (3) chelating agent for metal ions; (4) buffering agent for alkaline ions; 5) A regulator that resists external environmental pressure; (6) One of the auxiliary factors for gene expression regulation. In microorganisms, poly P is also directly related to the following physiological processes: (1) Bacterial motility; (2) Biofilm formation; (3) Quorum sensing of bacteria; (4) Stringent response (Strigent response) ; (5) spore for...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68
CPCG01N21/6402G01N33/68
Inventor 杨柳燕王鑫陈旭张文李丽王爱丽吴丹
Owner NANJING UNIV
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