阅读说明：本技术 一种基于生物膜干涉技术的沙门氏菌快速检测方法 (Rapid salmonella detection method based on biofilm interference technology ) 是由 张晓光 藏程琳 孙春燕 张鸣镝 刘佰潼 刘凯 刘泽同 刘志伟 于 2019-09-25 设计创作，主要内容包括：本发明公开了一种基于生物膜干涉技术的沙门氏菌快速检测方法,其方法为：步骤一、传感器活化；步骤二、抗体固定化；步骤三、封闭；步骤四、样品检测及结果判断；本发明的有益效果：本发明提供的基于生物膜干涉技术的沙门氏菌快速检测方法步骤少、操作简单易学、无需任何标记；该方法检测时间短,由于该方法的其他环节可以提前准备妥当,所以可以做到样品的随到随检。根据样品中所含目标菌浓度的不同,单纯的样品检测环节只需要数十秒到几分钟的时间。本发明所述方法可应用于粗样品的检测,检测前样品无需经过离心和脱气。本发明所述方法配合可商购的基于BLI技术的设备和系统,可实现多个样品(例如96个样品)的同时批量检测。(The invention discloses a method for quickly detecting salmonella based on a biofilm interference technology, which comprises the following steps: step one, activating a sensor; step two, immobilizing the antibody; step three, sealing; step four, sample detection and result judgment; the invention has the beneficial effects that: the method for rapidly detecting the salmonella based on the biomembrane interference technology has the advantages of few steps, simple and easy operation, no need of any mark; the method has short detection time, and other links of the method can be prepared properly in advance, so that the samples can be detected at any time. According to the difference of the concentration of target bacteria contained in the sample, the simple sample detection process only needs tens of seconds to several minutes. The method can be applied to the detection of a crude sample, and the sample does not need to be centrifuged or degassed before detection. The method of the present invention, in conjunction with commercially available equipment and systems based on BLI technology, enables simultaneous batch testing of multiple samples (e.g., 96 samples).)

1. A salmonella rapid detection method based on a biofilm interference technology is characterized by comprising the following steps: the method comprises the steps of sensor activation, antibody immobilization, sealing, sample detection and result judgment, and comprises the following specific steps:

step one, sensor activation: immersing the tail end of the second generation amino coupling sensor into pure water for prewetting for at least 10 minutes, and then immersing the second generation amino coupling sensor into a mixed activation reagent of 15-25mM 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 8-12mM N-hydroxysuccinimide for activation for 150 seconds and 450 seconds;

step two, antibody immobilization: immersing the activated second generation amino coupled sensor into an antibody solution with the concentration of 12.5-100 mu g/mL and diluted by acetic acid-sodium acetate buffer solution with the pH value of 4-6 for immobilization for 180 seconds;

step three, sealing: after the antibody is immobilized, the second generation amino coupling sensor is sequentially immersed in 0.8-1.5M ethanolamine hydrochloride solution and 0.8-1.5% bovine serum albumin solution for sealing for 150-450 seconds;

step four, sample detection and result judgment: soaking the sealed second-generation amino coupled sensor into phosphate buffer solution containing 0.01-0.03% of Tween-20 for balancing for 60-180 seconds, then soaking the sensor into a sample to be detected for salmonella detection, reading a binding signal in real time in the detection process, and judging that the sample contains salmonella when the binding signal is greater than or equal to 0.0075nm of an effective signal value for qualitative analysis; for the quantitative analysis, the binding signals y at the binding times of 60 seconds, 120 seconds, 180 seconds, and 300 seconds were substituted into the following mathematical formulas (1), (2), (3), and (4) between the bacterial concentration and the binding signal, respectively:

C₆₀＝10^{ln(41349.7y+277.0)^0.98499} (1)

C₁₂₀＝10^{ln(11293.1y+160.5)^1.11054} (2)

C₁₈₀＝10^{ln(16331.9y+179.7)^1.04064} (3)

C₃₀₀＝10^{ln(13112.1y+146.1)^1.04585} (4)

solving to obtain C which is the specific concentration of the salmonella in the sample, wherein C₆₀、C₁₂₀、C₁₈₀And C₃₀₀The minimum detection limits of Salmonella at the binding times of 60 seconds, 120 seconds, 180 seconds and 300 seconds are 1.9X 10⁶CFU/mL、1.3×10⁶CFU/mL、8.8×10⁵CFU/mL and 5.6X 10⁵CFU/mL。

Technical Field

The invention relates to a method for rapidly detecting salmonella, in particular to a method for rapidly detecting salmonella based on a biomembrane interference technology.

Background

At present, food poisoning is a public health problem worldwide, and food-borne pathogenic bacteria are the leading cause of food poisoning. Salmonella is a common food-borne pathogenic bacterium, and the salmonella often pollutes animal food such as meat, fish, milk, eggs and the like and related products to cause food poisoning. According to related statistics at home and abroad, the food poisoning caused by salmonella accounts for 42.6-60% of bacterial food poisoning. Salmonella can cause severe digestive tract infections in addition to food poisoning. According to the European food safety agency, the economic loss of the European Union due to salmonella pollution is estimated to be up to 30 hundred million euros each year.

The rapid and accurate detection of salmonella is a key to the prevention and control of salmonella food poisoning and related diseases. The traditional detection method for salmonella has the disadvantages of multiple steps, complex operation and long time consumption, and can not meet the requirement of rapid development of modern society. The existing rapid detection methods widely applied comprise an immunological detection technology (such as ELISA) based on antigen-antibody reaction and a molecular biology detection technology represented by PCR, and compared with the traditional method, the method shortens the detection time and has higher sensitivity and specificity; however, the method has the defects of easy contamination to cause false positive results (PCR), high selectivity to reagents, insufficient accuracy (ELISA), long detection time and the like. There is an urgent need to develop a more rapid and accurate salmonella detection method.

The biofilm interference (BLI) technology is a new generation of real-time label-free rapid detection technology that has just recently emerged. The technology can analyze the interaction among various biological molecules, and a plurality of scholars at home and abroad apply the technology to the fields of antibody screening, antibody-antigen/virus affinity determination, protein-protein/DNA/RNA/nanoparticle affinity determination, quantitative determination of antibodies and other proteins and the like, thereby obtaining favorable scientific achievements. The technical advantages displayed by the method, such as rapidness, high efficiency, high accuracy, simplicity and easy use, are well recognized by a plurality of experts and scholars at home and abroad.

Disclosure of Invention

The invention aims to provide a salmonella rapid detection method based on a biofilm interference technology, which aims to solve the problems of multiple steps, complex operation and long time consumption in the traditional salmonella detection method.

The invention provides a salmonella rapid detection method based on a biomembrane interference technology, which comprises the steps of sensor activation, antibody immobilization, sealing, sample detection and result judgment, and comprises the following specific steps:

step one, sensor activation: immersing the tail end of the second generation amino coupling sensor into pure water for prewetting for at least 10 minutes, and then immersing the second generation amino coupling sensor into a mixed activation reagent of 15-25mM 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 8-12mM N-hydroxysuccinimide for activation for 150-450 seconds;

step two, antibody immobilization: immersing the activated second generation amino coupled sensor into an antibody solution with the concentration of 12.5-100 mu g/mL and diluted by acetic acid-sodium acetate buffer solution with the pH value of 4-6 for immobilization for 180 seconds;

step three, sealing: after the antibody is immobilized, the second generation amino coupling sensor is sequentially immersed in 0.8-1.5M ethanolamine hydrochloride solution and 0.8-1.5% bovine serum albumin solution for sealing for 150-450 seconds;

step four, sample detection and result judgment: soaking the sealed second-generation amino coupled sensor into phosphate buffer solution containing 0.01-0.03% of Tween-20 for balancing for 60-180 seconds, then soaking the sensor into a sample to be detected for salmonella detection, reading a binding signal in real time in the detection process, and judging that the sample contains salmonella when the binding signal is greater than or equal to 0.0075nm of an effective signal value for qualitative analysis; for quantitative analysis, the binding signals y at binding times of 60 seconds, 120 seconds, 180 seconds, and 300 seconds were substituted into the following mathematical formulae (1), (2), (3), and (4) between the bacterial concentration and the binding signal, respectively:

C₆₀＝10^{ln(41349.7y+277.0)^0.98499} (1)

C₁₂₀＝10^{ln(11293.1y+160.5)^1.11054} (2)

C₁₈₀＝10^{ln(16331.9y+179.7)^1.04064} (3)

C₃₀₀＝10^{ln(13112.1y+146.1)^1.04585} (4)

solving to obtain C which is the specific concentration of the salmonella in the sample, wherein C₆₀、C₁₂₀、C₁₈₀And C₃₀₀Respectively represent the cell concentrations determined at the binding times of 60 seconds, 120 seconds, 180 seconds and 300 seconds, and the minimum detection limits of Salmonella at the binding times of 60 seconds, 120 seconds, 180 seconds and 300 seconds are 1.9X 10⁶CFU/mL、1.3×10⁶CFU/mL、8.8×10⁵CFU/mL and 5.6X 10⁵CFU/mL。

The invention has the beneficial effects that:

the method for rapidly detecting the salmonella based on the biomembrane interference technology has the advantages of few steps, simple and easy operation, no need of any mark; the method has short detection time, and other links of the method can be prepared properly in advance, so that the samples can be detected at any time. According to different target bacteria concentrations in the sample, a simple sample detection process only needs tens of seconds to several minutes. For example, when the concentration of the target bacteria in the actual sample is 8.8X 10⁶CFU/mL, only 60 seconds are required to determine the presence of the target bacteria in the sample. This time is shorter if the bacteria concentration is higher. The method of the invention can obtain the detection signal in real time, so the detection signal can be read at any time according to the actual requirement to analyze so as to judge whether the detection process is necessary to be continuously carried out, and the detection can be stopped if the detection signal meets the requirement, thereby the detection time can be saved. The method can be applied to the detection of a crude sample, and the sample does not need to be centrifuged or degassed before detection. The process of the invention is coupled with a commercially available B-basedThe LI technology equipment and system can realize the simultaneous batch detection of a plurality of samples (for example, 96 samples).

Drawings

FIG. 1 is a schematic diagram of the specific detection result of the detection method of the present invention.

FIG. 2 is a standard graph of bacterial concentration and binding signal for a 60 second binding time for the assay of the invention.

FIG. 3 is a standard graph of the binding time of the assay of the invention between the bacterial concentration and the binding signal at 120 seconds.

FIG. 4 is a standard graph of bacterial concentration versus binding signal for a 180 second binding time assay according to the present invention.

FIG. 5 is a standard graph of bacterial concentration versus binding signal for a detection method of the present invention at a binding time of 300 seconds.

Detailed Description

Please refer to fig. 1 to 5:

the following examples are carried out on the premise of the technical scheme of the invention, and give detailed implementation modes and processes, and experimental methods without specific conditions noted in the examples are generally carried out according to conventional conditions or according to conditions recommended by reagent manufacturers.

The information on the main reagents and consumables used in the following examples is shown in Table 1; the main instrument information is shown in table 2.

TABLE 1 Main reagent consumables information table

TABLE 2 Main Instrument information Table