CN110632301B - Rapid salmonella detection method based on biofilm interference technology - Google Patents
Rapid salmonella detection method based on biofilm interference technology Download PDFInfo
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Abstract
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).
Description
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＝10ln(41349.7y+277.0)^0.98499 (1)
C120＝10ln(11293.1y+160.5)^1.11054 (2)
C180＝10ln(16331.9y+179.7)^1.04064 (3)
C300＝10ln(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 bacterial concentrations determined at the binding times of 60 seconds, 120 seconds, 180 seconds and 300 seconds, and the binding times of 60 seconds, 120 seconds, 180 seconds and 300 secondsThe minimum detection limit of the bacteria was 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 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).
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
Example 1: investigation of specificity of detection method
Specificity is an important index for judging the quality of a detection method. In order to investigate the specificity of the method, the test is carried out by taking salmonella as a target bacterium and taking listeria monocytogenes, staphylococcus aureus and listeria monocytogenes as non-target bacteria, and the method comprises the following specific steps:
(1) inoculating Salmonella, Listeria Spinosa, Staphylococcus aureus and Listeria monocytogenes into tryptone soybean broth culture medium for overnight culture, centrifuging to obtain thallus precipitate, washing with phosphate buffer solution, and adjusting the concentration of Salmonella suspension to 2.2 × 108CFU/mL、 4.4×107CFU/mL、8.8×106CFU/mL, adjusting the concentration of Listeria monocytogenes, Staphylococcus aureus, Listeria monocytogenes suspension to 1.0 × 107CFU/mL, to be tested.
(2) Pure water, a mixed activation reagent of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide (400mM 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 100. mu.l of 200mM N-hydroxysuccinimide each was added to 1800. mu.l of water) to the corresponding positions in a 96-well plate in sequence, an antibody solution (50. mu.g/ml in 10mM acetic acid-sodium acetate buffer solution at pH 5), ethanolamine hydrochloride at a concentration of 1M (pH 8.5), 1% bovine serum albumin, a phosphate buffer solution containing 0.02% Tween-20, and a sample to be tested.
(3) Putting the 96-well plate after sample application into an Octet Red 96 biomolecule interaction instrument, operating the system, and sequentially immersing the tail end of a second-generation amino coupling sensor into pure water (10 minutes), a 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide mixed activation reagent (300 seconds), an antibody solution (600 seconds), ethanolamine hydrochloride (300 seconds), 1% bovine serum albumin (300 seconds), a phosphate buffer solution containing Tween 20 (120 seconds) and a sample (300 seconds), wherein the related conditions of each step can be observed in real time in the operation process of the system. Where sensor pre-wetting (i.e. 10 minutes immersion in pure water) can also be done in advance before running the system.
(4) The detection results are shown in fig. 1, the signal values of the salmonella samples with three concentrations are far above the effective signal value (0.0075nm), and the signal values of listeria monocytogenes, staphylococcus aureus and listeria monocytogenes are far less than the effective signal value, which indicates that the method has good specificity.
Example 2: detection of salmonella with different concentrations in beef samples
Aseptically weighing 25g beef, placing in aseptic homogenizing bag containing 225mL phosphate buffer solution, beating with beating homogenizer for 1-2 min, sampling appropriate amount of suspension, adding Salmonella to make the bacteria concentration in the suspension 2.2 × 108CFU/mL、4.4×107CFU/mL、8.8×106CFU/mL to obtain the required sample to be tested. The samples were applied to a 96-well plate and the samples were examined by a biomolecular interactor as in (2) and (3) of example 1. The detected binding signal value is substituted into a mathematical formula between the bacterial concentration and the binding signal to calculate the recovered bacterial concentration. Table 3 shows the results of the recovery test of the target bacteria at different binding timesAs can be seen from the table, the average recovery rate is basically over 96%, which shows that the method has high accuracy when used for detecting actual samples. In addition, the recovery rate of the bacteria is increased along with the extension of the binding time and is closer to 100 percent by comparing the recovery rates of the bacteria under different binding times, which shows that the detection result can be more accurate to a certain extent by the extension of the binding time.
TABLE 3 test results of recovery rates of target bacteria added to beef at binding times of 60 seconds, 120 seconds, 180 seconds, and 300 seconds
Example 3: detection of salmonella with different concentrations in whole egg powder sample
Weighing 25g of whole egg powder in aseptic operation, placing in an aseptic homogenizing bag containing 225mL of phosphate buffer solution, beating with a beating homogenizer for 1-2 min, taking appropriate amount of sample suspension, adding Salmonella to make the bacteria concentration in the sample suspension 2.2 × 108CFU/mL、4.4×107CFU/mL、8.8×106CFU/mL to obtain the required sample to be tested. The samples were applied to a 96-well plate and the samples were examined by a biomolecular interactor as in (2) and (3) of example 1. The detected binding signal value is substituted into a mathematical formula between the bacterial concentration and the binding signal to calculate the recovered bacterial concentration. Table 4 shows the results of the recovery rate tests of the target bacteria at different binding times, and it can be seen from the table that the average recovery rate is basically over 95%, further indicating that the method has high accuracy when used for detecting actual samples. In addition, the recovery rate of the bacteria under different binding time can be seen by comparing the recovery rate of the bacteria under different binding time, and the recovery rate of the bacteria is closer to 100% along with the extension of the binding time, which shows that the detection result can be more accurate to a certain extent due to the extension of the binding time.
TABLE 4 results of the recovery rate test of the target bacteria added to the whole egg powder at the binding time of 60 seconds, 120 seconds, 180 seconds, 300 seconds
Claims (1)
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＝10ln(41349.7y+277.0)^0.98499 (1)
C120＝10ln(11293.1y+160.5)^1.11054 (2)
C180＝10ln(16331.9y+179.7)^1.04064 (3)
C300＝10ln(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。
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| CN102928590A (en) * | 2012-11-22 | 2013-02-13 | 上海师范大学 | Kit adopting fluorescent quantum dots to quickly screen, separate and detect salmonella |
| CN104089997A (en) * | 2014-05-29 | 2014-10-08 | 北京盈盛恒泰科技有限责任公司 | Electrochemical immunosensor, and preparation method and application thereof |
| CN109971683A (en) * | 2019-04-03 | 2019-07-05 | 吉林大学 | Escherichia coli, salmonella and the compound enriched medium of Listeria monocytogenes |
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| CN102928590A (en) * | 2012-11-22 | 2013-02-13 | 上海师范大学 | Kit adopting fluorescent quantum dots to quickly screen, separate and detect salmonella |
| CN104089997A (en) * | 2014-05-29 | 2014-10-08 | 北京盈盛恒泰科技有限责任公司 | Electrochemical immunosensor, and preparation method and application thereof |
| CN109971683A (en) * | 2019-04-03 | 2019-07-05 | 吉林大学 | Escherichia coli, salmonella and the compound enriched medium of Listeria monocytogenes |
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