阅读说明：本技术 一种快速检测大肠杆菌的方法 (Method for rapidly detecting escherichia coli ) 是由 刘玲 吴娓娓 董绍俊 于 2019-12-23 设计创作，主要内容包括：本发明涉及一种快速检测大肠杆菌的方法,属于大肠杆菌检测技术领域。解决现有技术中采用366nm激发和450nm发射组合,由于MUG本底经366nm激发后,在450nm也有一定荧光强度,当大肠杆菌浓度很低时4-MU产量少,产物信号与本底信号分不开,导致对于水样中大肠杆菌浓度极低的样品现有方法无法检测的技术问题。本发明的快速检测大肠杆菌的方法,采用比率荧光方法,可以将底物MUG的吸收峰的降低与产物4-MU的吸收峰的增加同时利用起来,通过以二者吸收峰峰值比率与大肠杆菌的浓度做进行线性拟合,与单纯利用366nm激发,450nm发射的方法相比,所拟合的线性更好,提高了检测的准确性；可以缩短大肠杆菌检测的温育时间,在4-MU的产生量达不到区分度的时候,通过比率荧光可以达到区分。(The invention relates to a method for rapidly detecting escherichia coli, and belongs to the technical field of escherichia coli detection. The technical problem that a 366nm excitation and 450nm emission combination is adopted in the prior art, and the existing method cannot detect a sample with extremely low escherichia coli concentration in a water sample due to the fact that a certain fluorescence intensity exists at 450nm after the MUG background is excited by 366nm, the 4-MU yield is low when the escherichia coli concentration is very low, and a product signal and a background signal cannot be separated is solved. According to the method for rapidly detecting escherichia coli, a ratio fluorescence method is adopted, the reduction of the absorption peak of a substrate MUG and the increase of the absorption peak of a product 4-MU can be simultaneously utilized, linear fitting is carried out by using the ratio of the two absorption peak values to the concentration of the escherichia coli, compared with a method which only uses 366nm excitation and 450nm emission, the fitted linearity is better, and the detection accuracy is improved; can shorten the incubation time of Escherichia coli detection, and can achieve differentiation by ratiometric fluorescence when the yield of 4-MU cannot reach differentiation degree.)

1. A method for rapidly detecting escherichia coli is characterized in that a ratio fluorescence method is adopted to detect the escherichia coli.

2. The method for rapidly detecting Escherichia coli according to claim 1, wherein the method comprises the steps of linearly fitting the ratio of the peak values of two emission peaks to the concentration of Escherichia coli by using the absorption peak of background 4-methylumbelliferone- β -D-glucuronide (MUG) at 380nm and the absorption peak of product 4-methylumbelliferone (4-MU) at 450nm when the sample is excited at 320nm, drawing a standard curve, measuring the fluorescence intensity of the sample to be detected, exciting at 320nm, reading the peak values of the emission peaks at 380nm and 450nm, substituting the ratio of the peak values of the two emission peaks into the standard curve, and calculating the concentration of Escherichia coli in the sample to be detected.

3. The method for rapidly detecting Escherichia coli according to claim 1, wherein one embodiment is:

step 1, preparing a culture solution containing a MUG component;

step 2, pure culture of escherichia coli, centrifugal cleaning of the pure culture of escherichia coli, dilution and preparation of a series of concentration gradients as samples to prepare a standard curve;

step 3, quantifying the bacterial liquid sample obtained in the step 2 by using a standard flat plate counting method (CFU) to obtain the true concentration of the pure cultured escherichia coli;

step 4, inoculating each bacterial liquid diluted to a series of concentrations in the step 2 into the culture solution containing the MUG component prepared in the step 1, and calculating the number of escherichia coli contained in each bacterial liquid according to the CFU quantitative result in the step 3;

step 5, inoculating an actual sample to be detected into the culture solution containing the MUG component prepared in the step 1;

step 6, respectively incubating the culture mediums of the step 4 and the step 5, adjusting the pH value to acidity, measuring the fluorescence intensity, exciting at 320nm, and respectively reading the emission peak values at 380nm and 450 nm;

step 7, drawing a standard curve according to the concentration of the escherichia coli calculated in the step 4 and the ratio of the emission peak value of the culture medium in the step 4 at 380nm to the emission peak value of the culture medium in the step 6;

and 8, substituting the ratio of the emission peak values of the culture medium in the step 5 at 380nm and 450nm measured in the step 6 into a standard curve, and calculating to obtain the concentration of the escherichia coli in the sample to be measured.

4. The method for rapidly detecting Escherichia coli according to claim 3, wherein step 6 isUsing HCl or H₂SO₄Adjusting the pH value to be acidic.

Technical Field

The invention relates to the technical field of escherichia coli detection, in particular to a method for rapidly detecting escherichia coli.

Background

Coliform is an internationally recognized indicator for detecting the epidemiological safety of various foods, medicines and environmental water quality, and the coliform is the best indicator for fecal pollution. How to detect the escherichia coli, especially how to detect the escherichia coli with low concentration is significant.

The traditional E.coli detection methods mainly comprise multitubular fermentation, a filter membrane method and a plate counting method (GB4789.3-2016 replaces GB/T4789.32-2002), and the methods have the defects of complex operation, long time consumption and low sensitivity. In recent years, scientists have developed many rapid detection methods, which are mainly classified into three categories: molecular biological methods, immunoassay techniques, and metabolic techniques. The molecular biology method is based on the detection of genetic materials, and the accuracy is guaranteed. However, the method needs professional instruments and technologies and cannot realize online monitoring; and the detection signal is based on genetic material amplification (PCR, etc.), and the detection limit is generally 10³The immunoassay technology can quickly detect escherichia coli, but only aims at the detection of known bacteria of surface antigen antibodies, such as an escherichia coli O157: H7 kit, and cannot complete the broad-spectrum detection of all escherichia coli in a water body, in the technology based on the metabolic detection, the 4-methylumbelliferone- β -D-glucuronide (MUG) is decomposed to generate fluorescent 4-methylumbelliferone (4-MU) by utilizing the characteristic of β -glucuronidase generated by E.coli to carry out detection, and 94-97 percent of escherichia coli with the metabolic pathway can be detected.

Disclosure of Invention

The invention aims to solve the technical problem that the existing method cannot detect samples with extremely low escherichia coli concentration in a water sample due to the fact that a 366nm excitation and 450nm emission combination is adopted in the prior art, and the MUG background has certain fluorescence intensity at 450nm after being excited by 366nm, when the escherichia coli concentration is very low, the 4-MU yield is low, and a product signal and a background signal are not separated, and provides a method for quickly detecting escherichia coli.

In order to solve the technical problems, the technical scheme of the invention is as follows:

the invention provides a method for rapidly detecting escherichia coli, which adopts a ratio fluorescence method to detect the escherichia coli.

In the technical scheme, the method for rapidly detecting the escherichia coli preferably comprises the steps of utilizing that when 320nm is excited, the absorption peak of background 4-methylumbelliferone- β -D-glucuronide (MUG) is 380nm, the absorption peak of a product 4-methylumbelliferone (4-MU) is 450nm, carrying out linear fitting according to the ratio of the two emission peak values and the concentration of the escherichia coli, drawing a standard curve, measuring the fluorescence intensity of a sample to be detected, exciting at 320nm, respectively reading the emission peak values at 380nm and 450nm, substituting the ratio of the two emission peak values into the standard curve, and calculating the concentration of the escherichia coli in the sample to be detected.

In the above technical solution, it is further preferable that: one specific embodiment of the method for rapidly detecting escherichia coli is as follows:

step 1, preparing a culture solution containing a MUG component;

step 2, pure culture of escherichia coli, centrifugal cleaning of the pure culture of escherichia coli, dilution and preparation of a series of concentration gradients as samples to prepare a standard curve;

step 3, quantifying the bacterial liquid sample obtained in the step 2 by using a standard flat plate counting method (CFU) to obtain the true concentration of the pure cultured escherichia coli;

step 4, inoculating each bacterial liquid diluted to a series of concentrations in the step 2 into the culture solution containing the MUG component prepared in the step 1, and calculating the number of escherichia coli contained in each bacterial liquid according to the CFU quantitative result in the step 3;

step 5, inoculating an actual sample to be detected into the culture solution containing the MUG component prepared in the step 1;

step 6, respectively incubating the culture mediums of the step 4 and the step 5, adjusting the pH value to acidity, measuring the fluorescence intensity, exciting at 320nm, and respectively reading the emission peak values at 380nm and 450 nm;

step 7, drawing a standard curve according to the concentration of the escherichia coli calculated in the step 4 and the ratio of the emission peak value of the culture medium in the step 4 at 380nm to the emission peak value of the culture medium in the step 6;

and 8, substituting the ratio of the emission peak values of the culture medium in the step 5 at 380nm and 450nm measured in the step 6 into a standard curve, and calculating to obtain the concentration of the escherichia coli in the sample to be measured.

In the above technical solution, it is preferable that: step 6 using HCl or H₂SO₄The pH is adjusted to acidity, such as pH 5.

The invention has the beneficial effects that:

according to the method for rapidly detecting escherichia coli, the ratio fluorescence method is adopted, the reduction of the absorption peak of the substrate MUG and the increase of the absorption peak of the product 4-MU can be simultaneously utilized, linear fitting is carried out by using the ratio of the two absorption peaks to the peak value and the concentration of escherichia coli, compared with a method which only uses 366nm excitation and 450nm emission, the fitted linearity is better, and the detection accuracy is improved.

According to the method for rapidly detecting escherichia coli, provided by the invention, when 320nm is used for excitation, the absorption peak of background MUG is 380nm, the absorption peak of product 4-MU is 450nm, and the linear relation can be corrected through ratio calculation, so that the product detection signal and the background signal of low-concentration escherichia coli can be distinguished; the method can shorten incubation time for detecting Escherichia coli, and can distinguish by ratio fluorescence when 4-MU production amount is not distinguishable.

Drawings

The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

FIG. 1 is a graph showing the peak-to-peak ratio of absorption peaks of products and substrates of Escherichia coli measured by the rapid Escherichia coli detection method of the present invention, as compared with the concentration of Escherichia coli in example 1.

FIG. 2 is a graph showing the peak-to-peak ratio of absorption peaks of products and substrates of Escherichia coli measured by the rapid Escherichia coli detection method of the present invention, as compared with the concentration of Escherichia coli in example 2.

Detailed Description

The present invention will be described in detail with reference to the accompanying drawings.