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

本发明涉及了一种基于NADH荧光法原理的细菌总数快速检测方法。本发明测定细菌总数的过程包括细菌胞内NADH的提取以及NADH含量的测定。通过荧光分光光度计测定细菌胞内NADH的荧光强度，快速测定出活体细胞的总数。本发明基于细菌胞内的NADH荧光特性及其在胞内含量稳定的特性，建立一种快速检测大肠杆菌数量的新方法。该荧光法的NADH的检测限为1nM，NADH含量在10nM～80μM间与荧光强度呈良好线性关系(R²＝0.9905)。经离心获得菌体细胞，热Tris－HCl法提取胞内NADH，以342nm为激发波长，461nm为发射波长测定提取液荧光强度，1h内可检测到1×10⁴cfu/mL大肠杆菌。该方法检测原理新颖，检测方法快速，灵敏，简便，重复行好，可适用于食品卫生与安全、环境检测等领域大肠杆菌活细胞数量的定量检测，用于替代传统的测定活体细胞总数的方法。The invention relates to a rapid detection method for total number of bacteria based on the principle of NADH fluorescence method. The process of measuring the total number of bacteria in the present invention includes the extraction of NADH in the bacteria cells and the determination of the NADH content. The fluorescence intensity of NADH in bacterial cells is measured by a fluorescence spectrophotometer, and the total number of living cells can be quickly determined. The invention establishes a new method for rapidly detecting the number of coliform bacteria based on the NADH fluorescence characteristic in the bacterial cell and the characteristic of its stable content in the cell. The detection limit of NADH in the fluorescence method is 1 nM, and the NADH content has a good linear relationship with the fluorescence intensity between 10 nM and 80 μM (R ² =0.9905). The bacterial cells were obtained by centrifugation, and the intracellular NADH was extracted by hot Tris-HCl method. The fluorescence intensity of the extract was measured with 342nm as the excitation wavelength and 461nm as the emission wavelength. 1×10 ⁴ cfu/mL E. coli could be detected within 1 hour. The detection principle of this method is novel, the detection method is fast, sensitive, simple, and repeatable, and it can be applied to the quantitative detection of the number of E. coli living cells in the fields of food hygiene and safety, environmental testing, etc., and is used to replace the traditional method of measuring the total 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

1 A method for rapidly determining the total number of bacteria, characterized in that:

(1) Based on the principle of NADH fluorescence;

(2) Based on the stable NADH content in living bacteria;

(3) Utilize the direct detection of fluorescence spectrophotometer;

(4) The excitation wavelength range is 330-350nm;

(5) The emission wavelength range is 450-470nm.

2 Utilize high-temperature Tris-HCl in the present invention to carry out the extraction method of living bacterial cell NADH, it is characterized in that:

(1) The lysis buffer is Tris-HCl;

(2) Cracking temperature > 60°C.