CN101498666A

CN101498666A - Fast alive bacteria amount measurement by fluorescence method

Info

Publication number: CN101498666A
Application number: CNA2008101400951A
Authority: CN
Inventors: 王静雪; 林洪; 王晶
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2008-09-22
Filing date: 2008-09-22
Publication date: 2009-08-05

Abstract

The invention relates to a method for detecting the total number of bacteria based on the fluorescent method of NADH, in particular to a novel method for rapidly detecting the total number of colon bacteria based on the fluorescent characteristic of the NADH in a colon bacteria cell and the stable-content characteristic of the NADH in the colon bacteria cell, which comprises the processes of extracting NADH in the colon bacteria cell, detecting the content of the NADH in the colon bacteria cell, detecting the fluorescent intensity of the NADH in the colon bacteria cell with a fluorescent spectrophotometer and rapidly detecting the total number of bacteria. The fluorescent detection limit of the NADH is 1 nM, and the content of the NADH ranges from 10 nM to 80 microns and keeps a favorable liner relationship (R<2> = 0.9905) with the fluorescent intensity. The colon bacterial cell is obtained in a decentering way, the NADH in the colon bacteria cell is extracted in a Tris-HCl method, the fluorescent intensity of extracting solution of the NADH is extracted with the excitation wavelength of 342 nm and the emission wavelength of 461 nm, and the colon bacteria of 10<4> cfu/mL can be detected in one hour. The method adopts novel detection theory, has rapid detection speed and good repeatability, is sensitive and easy, is suitable for quantitatively detecting the number of colon bacteria in the fields of food sanitation and safety, environment detection, and the like, and can replace the traditional method for detecting the number of living cells.

Description

Fast alive bacteria amount measurement by fluorescence method

Technical field

The present invention relates to a kind of method of fast measuring alive bacteria amount, particularly relate to a kind of method of the fast measuring alive bacteria amount based on NADH fluorescence principle.

Background technology

According to the inventor by known to the inspection information, literature search, the method of the mensuration total number of bacteria that generally adopts is mainly the method for plate culture count and turbidimetry at present, the time that the method for plate culture count needs bacterium colony to cultivate, therefore minute is more than 1 day, and complex steps, easily cause pollution, artificial counting can be owing to subjective error reduces accuracy of measurement.The turbidimetry method is easy, and is consuming time few, but this method can't be differentiated cell anyway, and its application is restricted.In recent years, both at home and abroad shortening detection time, simplifying and studied the detection method that makes new advances aspect the trace routine, mainly contain the enzyme linked immunosorbent assay (ELISA) and immunofluorescence (IFA) method that have in the molecular biology in polymerase chain reaction (PCR) method and in situ hybridization (ISH) method and the immunology, and gene chips, biology sensor.Above method differs from one another, but many detection methods or relatively more expensive or comparatively complicated or comparatively strict because of experiment condition because of operating process because of equipment and reagent are not to be applicable to all mechanisms and unit.

Reduced coenzyme nicotinamide adenine dinucleotide (NADH) extensively is present in the living cells of animal and plant and microorganism; it is the coenzyme of at present known more than 300 kind of dehydrogenasa; simultaneously also be the important substance in many biological oxidation electron transport chains, play an important role at aspects such as growth and proliferation of cell, signal transmission, gene regulation, chondriosome protectives.Can be used as the indicant of cell with metabolic activity.Because NADH is a kind of hyperfluorescence material, as far back as the sixties, the variation with regard at first having proposed to measure cell metabolism such as B.Chano by oxidation state of coming coenzyme I in the observation of cell with the microscopic fluorescence art and the variation of going back ortho states.The eighties, the method for usefulness laser induced chemical vapor deposition NADH fluorescence such as G.Renault is studied the metabolism of the biologic-organ that exsomatizes.Fluorescence intensity signals is directly proportional with the amount of the NADH of existence, quantitative amounts relation such as various biochemistry metabolism parameters and NADH fluorescence intensity, kind and the state thereof of differentiating cell according to the fluorescent effect of NADH in the cell (are living cells, dead cell, normal cell and pathology, mutant), thereby being applied to the early diagnosis and the food freshness of disease, malignant tumour more, the NADH fluorescence method judges.

Because NADH extensively is present in the viable bacteria body cell.After the bacterium death, under the desmoenzyme effect, NADH will be decomposed very soon.And the amount of NADH is proportional with the bacterial number that is present in the nutrient culture media, thereby bacterium bacterium number and fluorescence intensity are interrelated.Therefore, by the NADH concentration in the working sample, can extrapolate viable count.The quantity that therefore can directly reflect bacterium by the signal of fluorescence intensity.Consider that in addition fluorescence method is few in whole extraction testing process step, only need to use fluorospectrophotometer that do not need injector easy to operate, this method meets the requirement of fast detecting.

The report that directly utilizes NADH fluorescence spectrometry Escherichia coli sum is not arranged at present as yet.

Summary of the invention

The objective of the invention is to set up a kind of new method of fast detecting Escherichia coli sum based on the NADH fluorescent characteristic in the bacterium born of the same parents and in born of the same parents' intensive amount stable properties.

The present invention measures extraction and the NADH Determination on content that colibacillary process comprises NADH in the bacterium born of the same parents.By the fluorescence intensity of NADH in the fluorescent spectrophotometer assay bacterium born of the same parents, the efficient somatic sum of fast measuring.

The present invention has at first set up the method for fluorescent spectrophotometer assay NADH.Select by NADH being carried out the condition of scanning, determine that maximum excitation wavelength and emission wavelength are respectively 342nm and 461nm.And the final optimal detection condition parameter that obtains: sweep velocity: 3000nm/min; Postpone: 0; EX slit: 10.0nm; EM slit: 20.0nm; Photomultiplier transit tube voltage: 400V; Response: 0.08s.The detection of the NADH of this fluorescence method is limited to 1nM, and NADH content is good linear relation (R with fluorescence intensity between 10nM～80 μ M ²=0.9905).

Next comparison by the extracting method of NADH in the different bacterium born of the same parents of the present invention, determine optimum extraction condition: (0.02M pH8.0), handles 30min down for 80 ℃ to Tris-HCl.Sample is cooled to the room temperature back 4 ℃ of centrifugal 10min of 10000rpm that fully vibrate, and gets the detection that supernatant 1mL carries out NADH immediately.This method detects the principle novelty, and detection method is easy fast, and good reproducibility can detect 1 * 10 in the 1h ⁴The cfu/mL Escherichia coli.

The present invention is applicable to the detection by quantitative of field Escherichia coli living cells quantity such as food hygiene and safety, environment measuring, and can develop into the total number of bacteria method for measuring, is used for substituting the method for traditional mensuration active somatic cell sum.

Description of drawings

Accompanying drawing 1 NADH emission wavelength fluorogram.

Accompanying drawing 2 NADH excitation wavelength fluorograms.

The typical curve of accompanying drawing 3 fluorescence spectrometry NADH.

The bacteria suspension of accompanying drawing 4 different OD values and the graph of relation of fluorescence intensity.

Embodiment

Embodiment 1

To concentration is that the NADH of 0.1mM carries out the condition of scanning and selects, and determines to excite and emission wavelength.

As shown in Figure 1 under the different excitation wavelengths of 310nm～390nm scope, scanning 220～800nm scope, find all to have single emission peak to occur, change along with excitation wavelength, peak position in the emission collection of illustrative plates does not almost have displacement, its wavelength coverage wherein when excitation wavelength 340nm, reaches the fluorescence intensity peak-peak at 460～462nm.

Rescan to obtain with 3 emission wavelength 460nm, 461nm obtaining and 462nm and excite crest, the excitation wavelength in scanning 220～550nm scope, respectively at 232nm, fluorescence peak (Fig. 2) appears in 273nm and 342nm place.

Make excitation wavelength with 232nm, at the no fluorescence peak in 460～462nm place.Wherein the NADH excitation wavelength that obtains for us of 273nm and 342nm is different from NAD and 342nm is NADH ⁺Exclusive fluorescence peak.Determined that finally excitation wavelength is 342nm, emission wavelength is 461nm.

The parameter setting of the condition of scanning: sweep velocity: 3000nm/min; Postpone: 0; EX slit: 10.0nm; EM slit: 20.0nm; Photomultiplier transit tube voltage: 400V; Response: 0.08s.

Embodiment 2

Accurately take by weighing the pure product 0.0355g of NADH, be settled to 50mL, be mixed with the NADH storing solution of 1mM with aseptic PBS (pH is 8.0).The dilution storing solution between 0.1nM～10mM, according to fluorescence spectrometry, is a horizontal ordinate with NADH concentration with the NADH dilution, and fluorescence intensity is an ordinate, draws the typical curve (Fig. 3) of NADH.

Embodiment 3

Add the 0.02M of 3mL in the somatic cells of collecting, the aseptic Tris-HCl of pH8.0 handles 30min down for 80 ℃.Sample is cooled to the room temperature back 4 ℃ of centrifugal 10min of 10000rpm that fully vibrate, and gets the detection that supernatant 1mL carries out NADH immediately.

Embodiment 4

Bacteria suspension to the E.coli of incubated overnight dilutes, and obtains the OD value and is respectively 0.1,0.2, the bacteria suspension of 0.4,0.8 four gradient.Fig. 6 is for being blank with hot Tris-HCl, the graph of relation of OD value and fluorescence intensity.Fluorescence intensity and bacteria suspension OD value are good linear relationship, both coefficient R ²Reached 0.9975.Because the bacterium number of OD value and bacterium is also linear, can obtains fluorescence intensity and bacterium number in view of the above and be good linear relation (Fig. 4).

Claims

The method of 1 one kinds of fast measuring totals number of bacteria is characterized in that:

(1) based on NADH fluorescence principle;

(2) based on the characteristics of NADH stable content in the bacterium born of the same parents that live;

(3) utilize fluorospectrophotometer directly to detect;

(4) excitation wavelength range 330-350nm;

(5) emission wavelength ranges 450-470nm.
Utilize high temperature Tris-HCl to carry out the extracting method of viable bacteria body cell NADH among 2 the present invention, it is characterized in that:

(1) lysis buffer is Tris-HCl;

(2) cracking temperature〉60 ℃.