Double-target simultaneous rapid detection method for escherichia coli and salmonella enteritidis

Abstract

The invention relates to the field of microbiological detection, in particular to a method for simultaneously and rapidly detecting at least two target objects, which comprises the following steps: S101, obtaining a first mixture; s102, obtaining a second mixture; s103, centrifuging the first mixture, collecting a first supernatant, centrifuging the second mixture, and collecting a second supernatant; s104, obtaining a third mixture; and S105, analyzing the existence and / or concentration of the first target object and / or the second target object in the sample to be detected according to the fluorescence intensity signal. The method has high detection sensitivity, good specificity, reproducibility, stability and anti-interference performance, and realizes enzyme-free, DNA-free, ultra-sensitive and wide-linear-range double-target detection.

Description

[0001] This application claims priority to Chinese invention patent application No. 2024106700186 filed on May 28, 2024, entitled "Establishment and application of a dual-target simultaneous rapid detection method for Escherichia coli and Salmonella enteritidis", which is incorporated by reference in its entirety. Technical Field

[0002] The invention relates to the field of microbial detection, and in particular to a method for rapid dual-target simultaneous detection of Escherichia coli and Salmonella enteritidis. Background Art

[0003] Food contamination by pathogens is a significant problem, and outbreaks of foodborne illness caused by consumption of contaminated food are a serious threat to human health and public safety. Bacteria are the most common cause of foodborne illness and have a variety of forms, types, and characteristics. It is estimated that 21 species of bacteria account for 64% of food-related hospitalizations and deaths. Among them, more than half of bacterial deaths worldwide (64% of hospitalizations and 76% of deaths) are caused by various important bacterial pathogens, including Escherichia coli and Salmonella Enteritidis. Rapid, sensitive, accurate, and reliable detection of these pathogens is essential to effectively prevent outbreaks of foodborne infections and significantly reduce hospitalizations and deaths caused by foodborne diseases.

[0004] At present, the conventional method commonly used in clinical practice to detect foodborne pathogens is mainly bacterial culture method, but this method is time-consuming and laborious, that is, the operation time is usually long, and the technical requirements for equipment and operators are high. These shortcomings determine that it is not suitable for point-of-care detection (POCT) of pathogens. Currently, several new methods, such as enzyme linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), have been developed for rapid detection of foodborne pathogens. Although ELISA and conventional PCR shorten the detection time, both ELISA and PCR involve relatively cumbersome operation methods and expensive and sophisticated instruments, which also increases the difficulty of bacterial POCT.

[0005] Generally speaking, compared with isothermal nucleic acid amplification techniques that do not use enzymes, enzyme-assisted amplification has higher detection sensitivity in DNA-mediated signal amplification. However, enzyme-assisted amplification methods, such as rolling circle amplification (RCA), strand displacement amplification (SDA), loop-mediated isothermal amplification (LAMP), etc., have disadvantages such as high cost, difficult storage, and complex reaction conditions. Chinese patent application CN117551793A discloses a method for detecting E. coli O157:H7 based on cascade strand displacement reaction, which still requires multiple enzymes to participate in the reaction and has a narrow linear range (10 3- 10 6 CFU / mL), high detection limit (10 2 CFU / mL). Chinese patent application CN116445586A discloses a biosensor based on fluorescent hybridization chain reaction, which does not require enzymes to participate in the reaction, but still has a narrow linear range (10 2 - 10 6 That is to say, the above existing technologies can only detect bacteria when the bacterial concentration is high, and can only detect one type of target bacteria, so the application scenarios are relatively limited. Summary of the invention

[0006] In a first aspect, the present invention provides a method for simultaneously and rapidly detecting at least two targets, characterized in that it comprises the following steps: S101 incubating the sample to be tested with a first aptamer-first competitive sequence complex (Apt_C1), so that the first aptamer binds to the first target in the sample to be tested and releases the first competitive sequence (C1), thereby obtaining a first mixture; In some embodiments, the first aptamer specifically binds to the first target.

[0007] In some embodiments, the sample to be tested can be a biological sample. The present invention can be performed in vitro on a sample obtained from or extracted from any organism or microorganism. In some embodiments, the sample to be tested can be a body fluid sample, such as urine, blood, serum, plasma, lymph, cyst fluid, pleural fluid, ascites fluid, peritoneal fluid, amniotic fluid, epididymal fluid, cerebrospinal fluid, bronchoalveolar lavage fluid, breast milk, tears, saliva, sputum or a combination thereof. The sample to be tested can be derived from humans or other mammals. In some embodiments, the sample to be tested can be a non-biological sample, such as food.

[0008] S102 incubating the sample to be tested with the second aptamer-second competitive sequence complex (Apt_C2), so that the second aptamer binds to the second target in the sample to be tested and releases the second competitive sequence (C2), thereby obtaining a second mixture; In some embodiments, the second aptamer specifically binds to the second target.

[0009] In some embodiments, the target may be a pathogen, such as bacteria, virus, fungus, etc.

[0010] In some embodiments, the target is bacteria.

[0011] In some embodiments, the target may also be other biomarkers, such as proteins, nucleic acids, small molecules, etc., as long as the aptamer can specifically bind to them.

[0012] In some embodiments, the first target may be Escherichia coli, and the second target may be Salmonella Enteritidis.

[0013] In some embodiments, the first aptamer-first competitor sequence complex is formed by annealing the first aptamer to the first competitor sequence. In other words, the first aptamer and the first competitor sequence are at least partially complementary.

[0014] In some embodiments, the second aptamer-second competitor sequence complex is formed by annealing the second aptamer to the second competitor sequence. In other words, the second aptamer and the second competitor sequence are at least partially complementary.

[0015] S103 centrifuging the first mixture and collecting a first supernatant, and centrifuging the second mixture and collecting a second supernatant; In some embodiments, the first supernatant comprises a free first competitor sequence (C1), and the second supernatant comprises a free second competitor sequence (C2).

[0016] In some embodiments, the first supernatant further comprises the first aptamer bound to the first target, and the second supernatant further comprises the second aptamer bound to the second target.

[0017] S104: mixing the first probe-first trigger sequence complex (P1T1) and the second probe-second trigger sequence complex (P2T2), adding the first amplification sequence-second amplification sequence complex (A1A2) and the first supernatant and reacting, and then adding the second supernatant and reacting to obtain a third mixture; In some embodiments, the third mixture includes a first probe-first amplification sequence complex (P1A1), a second probe-second amplification sequence complex (P2A2), a free first trigger sequence (T1), and a free second trigger sequence (T2).

[0018] In some embodiments, the first probe (P1) and the second probe (P2) are modified with fluorescent groups; the first amplification sequence (A1) and the second amplification sequence (A2) are modified with quenching groups. In other words, the first probe-first trigger sequence complex (P1T1) and the second probe-second trigger sequence complex (P2T2) are fluorescence-quenching structures and will not generate fluorescence signals. Only when their fluorescence-quenching structures are destroyed, the first probe and the second probe will generate fluorescence signals.

[0019] In some embodiments, the fluorescent group includes HEX, FAM, ROX or CY5.

[0020] In some embodiments, the quenching group comprises BHQ1, BHQ3, or MGB.

[0021] In some embodiments, the first probe-first trigger sequence complex is formed by annealing the first probe to the first trigger sequence. In other words, the first probe is at least partially complementary to the first trigger sequence.

[0022] In some embodiments, the second probe-second trigger sequence complex is formed by annealing the second probe to the second trigger sequence. In other words, the second probe is at least partially complementary to the second trigger sequence.

[0023] In some embodiments, the free first competitive sequence can competitively bind to the first probe to release the first trigger sequence, that is, to obtain a free first trigger sequence.

[0024] In some embodiments, the free second competitive sequence can competitively bind to the second probe to release the second trigger sequence, that is, to obtain a free second trigger sequence.

[0025] It should be understood that the free energy of the sequence released by competitive binding is smaller than the free energy of the sequence bound to the target sequence by competitive binding, for example, the free energy of the first trigger sequence is smaller than that of the first competitor sequence.

[0026] In some embodiments, the free first trigger sequence can competitively bind to the second amplification sequence to release the second amplification sequence, that is, to obtain a free second amplification sequence.

[0027] In some embodiments, the free second trigger sequence can competitively bind to the first amplification sequence to release the first amplification sequence, that is, to obtain a free first amplification sequence.

[0028] In some embodiments, the free first amplification sequence is capable of competitively binding with the first probe to release the first trigger sequence.

[0029] In some embodiments, the free second amplification sequence is capable of competitively binding with the second probe to release the second trigger sequence.

[0030] S105 Analyze the presence and / or concentration of the first target and / or the second target in the sample to be tested according to the fluorescence intensity signal.

[0031] In some embodiments, the concentration ratio of the first probe-first trigger sequence complex (P1T1), the second probe-second trigger sequence complex (P2T2), and the first amplification sequence-second amplification sequence complex (A1A2) comprises 1:1:(1-3).

[0032] In some embodiments, the concentration ratio of the first probe-first trigger sequence complex (P1T1), the second probe-second trigger sequence complex (P2T2), and the first amplification sequence-second amplification sequence complex (A1A2) comprises 1:1:1.5-3, preferably 1:1:2.

[0033] In some embodiments, the third mixture further includes a first probe-first competitor sequence (P1C1) and a second probe-second competitor sequence (P2C2).

[0034] In some embodiments, the third mixture further includes a first trigger sequence-second amplification sequence (T1A2) and a second trigger sequence-first amplification sequence (T2A1).

[0035] In some embodiments, the reaction time of step S104 is 10-40 min.

[0036] In some embodiments, the reaction time of step S104 is 5-10 minutes, preferably 10 minutes.

[0037] In some embodiments, the reaction temperature of step S104 is 30-40°C, preferably 37°C.

[0038] In some embodiments, the concentration of Escherichia coli in the test sample is at least 0.7 CFU / mL.

[0039] In some embodiments, the concentration of Escherichia coli in the sample to be tested can be 10-10 6 CFU / mL.

[0040] In some embodiments, the concentration of Salmonella Enteritidis in the test sample is at least 0.6 CFU / mL.

[0041] In some embodiments, the concentration of Salmonella enteritidis in the sample to be tested can be 10-10 6 CFU / mL.

[0042] In some embodiments, the incubation time of step S101 and step S102 is at least 30 min.

[0043] In some embodiments, the nucleotide sequence of the first aptamer is SEQ ID NO:1, and the nucleotide sequence of the second aptamer is SEQ ID NO:2.

[0044] In some embodiments, the nucleotide sequence of the first competitor sequence is SEQ ID NO:3, and the nucleotide sequence of the second competitor sequence is SEQ ID NO:4.

[0045] In some embodiments, the nucleotide sequence of the first probe is SEQ ID NO:5, and the nucleotide sequence of the second probe is SEQ ID NO:6.

[0046] In some embodiments, the nucleotide sequence of the first trigger sequence is SEQ ID NO:7, and the nucleotide sequence of the second trigger sequence is SEQ ID NO:8.

[0047] In some embodiments, the nucleotide sequence of the first amplified sequence is SEQ ID NO:9, and the nucleotide sequence of the second amplified sequence is SEQ ID NO:10.

[0048] In a second aspect, the present invention provides a kit for rapid simultaneous detection of Escherichia coli and Salmonella enteritidis, characterized in that it comprises: a first aptamer, a second aptamer, a first competitive sequence, a second competitive sequence, a first probe, a second probe, a first trigger sequence, a second trigger sequence, a first amplification sequence, and a second amplification sequence, wherein: The nucleotide sequence of the first adapter is SEQ ID NO:1, and the nucleotide sequence of the second adapter is SEQ ID NO:2; the nucleotide sequence of the first competitive sequence is SEQ ID NO:3, and the nucleotide sequence of the second competitive sequence is SEQ ID NO:4; the nucleotide sequence of the first probe is SEQ ID NO:5, and the nucleotide sequence of the second probe is SEQ ID NO:6; the nucleotide sequence of the first trigger sequence is SEQ ID NO:7, and the nucleotide sequence of the second trigger sequence is SEQ ID NO:8; the nucleotide sequence of the first amplification sequence is SEQ ID NO:9, and the nucleotide sequence of the second amplification sequence is SEQ ID NO:10.

[0049] Compared with the prior art, the beneficial effects of the present invention include at least the following aspects: Although existing technologies have developed biosensors for detecting foodborne pathogens, on the one hand, most existing technologies can only detect foodborne pathogens when the bacterial concentration is high, and detecting foodborne pathogens at very low bacterial concentrations is still a problem. On the other hand, there are no reports of biosensors that can detect two targets (such as two bacteria) at the same time.

[0050] The present invention utilizes aptamer-based target (object) recognition and single-stranded DNA-mediated cyclic amplification and fluorescence amplification processes to develop an enzyme-free dual-target microbial biosensor and a detection method based thereon. The present invention ensures dual-target recognition, signal amplification and efficient circulation through the high affinity of the aptamer to the target, the binding stability of the protection probe and the aptamer sequence, and the introduction of the first amplification sequence-second amplification sequence complex, wherein the first amplification sequence-second amplification sequence complex can simultaneously participate in the reaction of the first target and the second target, and in the reaction it will also promote the mutual enhancement of the detection signals of the above two parts, thereby achieving enzyme-free, DNA-free extraction, ultra-sensitive and wide linear range dual-target detection. The average fluorescence intensity of the target under the method of the present invention is strongly linearly correlated with its concentration, and the linear range of Escherichia coli ATCC25922 is 34-10 5 CFU / mL, the linear range of Enteritidis Salmonella is 17-10 6 CFU / mL; and the detection limit (LOD) of Escherichia coli ATCC25922 is 0.7 CFU / mL, and the detection limit (LOD) of Salmonella Enteritidis is 0.61 CFU / mL. Compared with other aptamer-based biosensors reported for detecting Escherichia coli or Salmonella Enteritidis, the present invention shows higher detection sensitivity. In addition, the present invention also shows good specificity, reproducibility, stability and anti-interference, and is suitable for detecting complex samples to be tested (such as body fluids and food).

[0051] Due to the portable, rapid, cheap, enzyme-free and isothermal characteristics of the biosensor of the present invention, ultra-fast detection is allowed in a POCT environment, and it can be used as a POCT detection kit for clinical use. The present invention is not limited to the detection of bacteria or specific targets, and can also be used to simultaneously detect a variety of different biomarkers, such as proteins, nucleic acids, small molecules, etc., by changing the sequences of the aptamer and the protective probe.

[0052] In addition, the biosensor of the present invention can also be used to distinguish between live and dead Escherichia coli (and Salmonella Enteritidis), which not only reflects that the present invention has good specificity, stability and anti-interference, but also reflects that the present invention can be used to identify the activity of pathogens in the sample to be tested, and then can be used to judge the progression of the subject's disease, trace pathogens in food, and other application scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

[0054] Figure 1 Schematic diagram of the dual-target detection scheme for Escherichia coli and Salmonella Enteritidis; Figure 2 This is a diagram showing the DNA gel electrophoresis verification results of the dual-target detection scheme; Figure 3 The results of adding different targets to verify the applicability of the dual-target detection scheme to dual bacteria; Figure 4 Optimization result graph for the ratio of probes P1T1:P2T2:A1A2; Figure 5 This is the result diagram of reaction time optimization for the cyclic amplification step (the horizontal axes are 0-40 minutes respectively); Figure 6 Standard curve constructed for Escherichia coli ATCC25922; Figure 7 Standard curves constructed for clinically collected Salmonella Enteritidis strains; Figure 8 The figure is a result diagram of the selectivity evaluation of the biosensor; Fig. 9 This is the temperature optimization result diagram of the cycle amplification step (the horizontal axes are 25-40℃ respectively); Fig.10 Results for verifying whether the method of the present invention is applicable to Escherichia coli (upper figure) and Salmonella Enteritidis (lower figure). DETAILED DESCRIPTION

[0055] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0056] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0057] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0058] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0059] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0060] In this specification, some embodiments may be disclosed in a format of being in a range. It should be understood that this description of "being in a range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0061] Detailed description with drawings Figure 2 : The bands from left to right are 100 nM P1T1+100 nM P2T2, 200 nM A1A2, 100 nM P1T1+100 nM P2T2+200 nM A1A2+10 nM C1, 100 nM P1T1+100 nM P2T2+200 nM A1A2+10 nM C2, 100 nM P1T1+100 nM P2T2+200 nM A1A2+10 nM C1+10 nM C2. The gel is labeled with DNA sequences.

[0062] Figure 3:The targets added to the five components in the figure are: a. Negative control group (no target, replaced by PBS solution); b. Staphylococcus aureus strain; c. Escherichia coli ATCC25922 strain; d. Salmonella enteritidis strain; e. Inactivated Escherichia coli ATCC25922 strain. The error bars are the standard deviations of three repeated measurements.

[0063] Figure 4 : The ratios of the five groups on the horizontal axis are 1:1:1, 1:1:1.5, 1:1:2, 1:1:2.5, and 1:1:3; the final concentrations of P1T1 and P2T2 added are 100 nM.

[0064] Figure 8 :A. Fluorescence signal of the corresponding probe of Escherichia coli; B. Fluorescence signal of the corresponding probe of Salmonella enteritidis, used to detect (a) 10 5 CFU / mL of Escherichia coli, (b) 10 5 CFU / mL of Salmonella enteritidis, (c) Staphylococcus aureus, (d) Listeria monocytogenes, (e) Streptococcus enteritidis, (f) Escherichia coli BL21, (g) Escherichia coli DH5alpha and (h) background. The control bacteria concentration was 10 5 CFU / mL. Error bars are standard deviation of 3 replicate measurements.

[0065] Fig.10 : The targets added to the five components in the figure are: a. Negative control group (no target, replaced by PBS solution); b. Inactivated Escherichia coli ATCC25922; c. Escherichia coli ATCC25922; d. Enteritidis Salmonella; e. Inactivated Enteritidis Salmonella. Error bars are standard deviations of three repeated measurements.

[0066] Embodiment 1 Materials and Methods The principle of biosensor (biosensor system) based on chain substitution reaction for detecting Escherichia coli and Salmonella enteritidis is as follows Figure 1 In this biosensing system, the competitive sequences C1 / C2 (also known as protection probes) and the probes for capturing the target (also known as capture probes) are combined to design a complex (Apt_C1, Apt_C2), in which the main part of the capture probe is the aptamer of Escherichia coli / Salmonella enteritidis for recognizing dual-target bacteria, and the rest is used to trigger and protect the binding of the capture probe.

[0067] When there is a target in the system (such as E. coli / Salmonella enteritidis), part of the double-stranded P1T1 / P2T2 will be opened by the competitive sequence C1 / C2, and P1 / P2 with the fluorescent group CY5 / FAM will bind to C1 / C2. Since the fluorescence-quenching structure is destroyed, the system will release some fluorescence. The free single-stranded T1 / T2 that is competed off will participate in the SDR reaction and bind to the toe chains exposed at the 3' and 5' ends of A1A2 in the system. The released A1 / A2 will continue to compete with P1T1 / P2T2 and destroy the fluorescence-quenching structure, and release an enhanced fluorescence signal.

[0068] Chemicals and Materials All high-performance liquid chromatography (HPLC)-purified and polyacrylamide gel electrophoresis (PAGE)-purified oligonucleotides (see Table 1) were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). PBS buffer was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). All reagents were of analytical grade and used without additional purification. Ultrapure water (18.2 MΩ·cm) obtained from a Millipore filtration system (Billerica, MA, USA) was used throughout the experiments. N,N,N ́,N ́-Tetramethylethylenediamine (TEMED), ammonium persulfate (APS), acrylamide (29:1), and TBE buffer were purchased from Sangon Biotech Co., Ltd. (Shanghai, China).

[0069] Table 1 DNA names and sequences used in this example (5'-3') Bacterial sources: Escherichia coli ATCC25922, Salmonella enteritidis ATCC13076, Listeria monocytogenes ATCC19115, and Streptococcus enteritidis are all clinical culture strains. Staphylococcus aureus was purchased from Kuujia Company, and Escherichia coli BL21 and Escherichia coli DH5alpha were purchased from Sangon Company.

[0070] Instrumentation: Fluorescence emission spectra were obtained on a fluorimeter F-7100 (Hitachi, Japan).

[0071] Annealing of DNA primers The aptamers (E. coli aptamer (first aptamer) and Salmonella enteritidis aptamer (second aptamer)) (final concentration 10 nM) and competitor sequences (first competitor sequence (Competition 1) and second competitor sequence (Competition 2), hereinafter referred to as C1 and C2) (final concentration 10 nM) were heated at 95°C for 10 minutes and gradually cooled to room temperature in PBS buffer. The two components after annealing are called Apt_C1 and Apt_C2, respectively.

[0072] The first probe (Probe 1, hereinafter referred to as P1) and the first trigger sequence (Trigger 1, hereinafter referred to as T1), the second probe (Probe 2, hereinafter referred to as P2) and the second trigger sequence (Trigger 2, hereinafter referred to as T2), and the first amplification sequence (Amplification 1, hereinafter referred to as A1) and the second amplification sequence (Amplification 2, hereinafter referred to as A2) were mixed (final concentration 100 nM), heated at 95°C for 10 minutes, and gradually cooled to room temperature in 100 μL of PBS buffer. The three components after annealing are called P1T1, P2T2 and A1A2.

[0073] Bacterial strains and culture conditions Escherichia coli ATCC25922 strain and clinically collected Salmonella Enteritidis ATCC 13076 were spread on blood plates and cultured at 37°C for 24 hours. Monoclonal bacteria were selected from the blood plates, added to 10 mL of lysogeny broth (LB) medium, and placed in a shaker at 37°C for 16 hours. The cultured bacteria were diluted 10 6 100 μL of diluted bacterial solution was evenly spread on the LB solid plate with a coating rod, and incubated at 37°C for 12 hours. The count of the cultured colonies was the number of viable bacteria in the bacterial solution, expressed in CFU / mL. 4 The mixture of E. coli and S. enteritidis with CFU / mL was incubated at 100°C for 15 minutes until inactivated. Subsequently, the heat-inactivated mixed bacterial solution was incubated on LB plates at 37°C for 2 days. If no colonies were observed on the plates at the end, it indicated that the inactivation step was successful.

[0074] Dual Bacteria Detection The two bacteria were mixed with Apt_C1 and Apt_C2, respectively, incubated at room temperature for 30 minutes, and centrifuged (5 minutes, 3000×g, 4°C) to collect the supernatant. The collected supernatants were called the first supernatant (Group 1) and the second supernatant (Group 2).

[0075] After annealing, the mixtures P1T1 and P2T2 (20 μL of 100 nM each) were mixed, and 118 μL of PBS buffer was added and incubated at 37°C for 30 minutes. Then, 20 μL of 100 nM A1A2 was added, and 20 μL of Group 1 was added, and the reaction was carried out at room temperature for 10 minutes. Then, the above system was placed in a fluorescence spectrophotometer for detection, with an excitation wavelength of 640 nm and an emission wavelength range of 660-700 nm, and a standard curve of Escherichia coli ATCC25922 was obtained. Then, 20 μL of Group 2 solution was added, and the reaction was carried out at room temperature for 10 minutes, and the system was placed in a fluorescence spectrophotometer for detection, with an excitation wavelength of 495 nm and an emission wavelength range of 515-550 nm, and a standard curve of Salmonella Enteritidis was obtained.

[0076] Before detecting bacteria, the first aptamer-first competitive sequence complex and the second aptamer-second competitive sequence complex formed after annealing of the aptamer and the competitive sequence were incubated with 1 mL of bacteria at 25°C for 30 minutes. The subsequent process was the same as above.

[0077] Bacterial availability and selectivity In order to analyze the effects of other negative control strains such as non-E. coli and Salmonella Enteritidis on the biosensor system, Staphylococcus aureus, Listeria monocytogenes, Streptococcus Enteritidis, E. coli, Salmonella Enteritidis, inactivated E. coli, and inactivated Salmonella Enteritidis were added to the system. The steps after adding bacteria in this experiment were the same as the bacterial detection steps described previously.

[0078] Clinical stability verification Body fluid samples were added to test the stability and clinical applicability of the method of the present invention. 80 μL of pleural effusion, ascites, and cerebrospinal fluid collected from the clinic were added with 10 μL of Escherichia coli ATCC25922 and Salmonella enteritidis ATCC13076, respectively, to a final concentration of 1×10 5 CFU / mL. Then they are added into the system as targets for reaction, and finally their fluorescence signals are obtained in F-7100.

[0079] Embodiment 2 Feasibility Analysis The core of the method of the present invention lies in the two intermediate cyclic amplification steps, and therefore, the feasibility of this part of the method is verified first.

[0080] In the presence of Escherichia coli ATCC25922 strain or Salmonella enteritidis ATCC13076, multiple double-stranded DNAs can be generated through several strand displacement reactions, among which P1C1, P1A1, P2C2, and P2A2 can release fluorescent signals and can be detected without enzyme action. In order to verify whether these double strands can be generated, that is, whether the reaction system can operate normally, the final reaction solution is added to the electrophoresis lane for intuitive characterization in this example.

[0081] like Figure 2 As shown, when the start sequences C1 and C2 are not added, there are only annealed P1T1, P2T2, and A1A2 double-stranded DNA in the solution; after adding the start sequences C1 and C2, as shown in lanes 3 and 4, P1T1 (P2T2) and A1A2 are reduced to varying degrees (i.e., P1T1, P2T2, and A1A2 are partially consumed), and new bands are obviously generated at other positions of the lanes (i.e., part of the DNA is recombined into new double-stranded DNA).

[0082] The above is the product DNA generated by the reaction, among which P1A1, P1C1, P2A2 and P2C2 can be excited by the fluorescence spectrum to release fluorescence, which is used for qualitative and quantitative determination of the target. In addition, both C1 and C2 can trigger the reaction of the system, which is the basis of dual-target detection. Comparing lane 5 with lanes 3 and 4, it can be seen that the reaction system containing C2 is more efficient than the reaction system containing C1. The above results prove the feasibility of the reaction system. In the presence of Escherichia coli and Salmonella enteritidis, new double-stranded DNA can be generated by SDR cyclic amplification. The above experimental results show that the present invention has successfully developed an enzyme-free dual-target detection method and biosensor suitable for simultaneous detection of different targets, which can not only detect bacterial pathogens, but also detect other biomarkers.

[0083] Validation of the discrimination ability and specificity of biosensors Secondly, in order to prove the applicability of the method of the present invention, different clinically collected and standard strains were added as targets to verify the applicability of the method and control experiments were performed. Fig.10 As shown, a, b, c, d, and e on the x-axis are the blank control group, inactivated Escherichia coli, healthy Escherichia coli, healthy Salmonella enteritidis, and inactivated Salmonella enteritidis, respectively. The concentration of each bacterium added is 1×10 3 CFU / mL. Fig.10 Above and Fig.10 The following figures show the corresponding fluorescence in the presence of healthy E. coli and healthy Salmonella Enteritidis, respectively. Fig.10It shows that in the absence of target bacteria, the two fluorescent probes (P1, P2) of the system are mostly quenched by the corresponding quenching probes (T1, T2), and do not emit fluorescence. The fluorescence displayed is similar to the fluorescence intensity of the blank control. Fig.10 Figure (b) and Fig.10 The fluorescence in the figure below (e) is enhanced compared to the control, which may be due to incomplete inactivation, but it is obvious that the fluorescence produced by the inactivated E. coli and Salmonella enteritidis added to the system is much smaller than that produced by normal healthy bacteria. When healthy E. coli and Salmonella enteritidis are used as targets, Fig.10 Figure (c) and Fig.10 As shown in the figure below (d), the corresponding fluorescence intensities of the two groups increased significantly, proving that the method of the present invention can be used for the detection of these two bacterial microorganisms.

[0084] like Figure 3 As shown, in the three groups of experiments without a target, using Staphylococcus aureus as a target, and using an inactivated Escherichia coli ATCC25922 strain as a target, the fluorescence intensity corresponding to the two fluorescent probes did not increase significantly, while after using Escherichia coli ATCC25922 strain and clinically collected Enteritidis Salmonella strain as targets, the corresponding fluorescence intensity of both groups increased significantly, which proves that this method can be used for the detection of these two bacterial microorganisms. At the same time, by comparing Staphylococcus aureus and inactivated Escherichia coli samples, it can be seen that this method has excellent specificity for Escherichia coli ATCC25922 and Enteritidis Salmonella strains. At the same time, any one target (target) will not cause an increase in the corresponding fluorescence of another target, proving that the method of the present invention has no obvious signal crosstalk, good stability, and can be used for dual-target detection.

[0085] Embodiment 4 This example optimizes the following parameters: (a) the ratio of the three pairs of probe double strands ( Figure 4 ); (b) reaction time ( Figure 5 ); (c) reaction temperature ( Fig. 9 ).

[0086] In short, the biosensor performed better under the following experimental conditions: (a) the ratio of probes P1T1:P2T2:A1A2 was 1:1:1.5-3 (preferably 1:1:2); (b) the reaction time was 5-10 min (preferably 10 min); and (c) the reaction temperature was 30-40 °C (preferably 37 °C).

[0087] Embodiment 5 Detectability Use clinical cultured Escherichia coli and Salmonella enteritidis for incubation, then add 10-10 6Different concentrations of the two strains were used for the experiment, and their two fluorescence intensities were tested at 10 and 20 minutes after the reaction. 5 CFU / mL and Salmonella Enteritidis (17-10 6 CFU / mL), indicating that the generation of fluorescence is highly correlated with the concentration of the target. These results validate the performance of the biosensor of the present invention, in which the dual targets bind to the aptamer and release the trigger sequence, thereby initiating a chain displacement reaction and generating fluorescence.

[0088] like Figure 6 and Figure 7 As shown, as the concentration of target 1 increases from 10-10 5 CFU / mL and the concentration of target 2 ranged from 10-10 6 As the CFU / mL increases, the fluorescence intensity of the system increases, indicating that the generation of fluorescence is highly dependent on the concentration of the target. These results verify the working principle that the dual targets bind to the aptamer and release the trigger sequence, thereby triggering a chain displacement reaction and generating fluorescence.

[0089] Figure 6 and Figure 7 The linear correlation between the fluorescence intensity of the system and the concentration of target 1 (E. coli) and target 2 (Salmonella enteritidis) is also shown. Under the optimized conditions, the average fluorescence intensity of the target is strongly linearly related to the concentration of the target, and the linear range of E. coli ATCC25922 is 34-10 5 CFU / mL, the linear range of Enteritidis Salmonella is 17-10 6 CFU / mL. The limit of detection (LOD) of the developed biosensor was determined to be 0.7 CFU / mL for Escherichia coli (n = 3); and 0.61 CFU / mL for Salmonella Enteritidis (n = 3). This value shows a significantly higher detection sensitivity compared to some other reported aptamer-based biosensors for the detection of Salmonella Enteritidis. This high sensitivity and wide detection range can be attributed to the stability and high amplification efficiency of the DNA chain amplification and fluorescence amplification generated by the target-induced trigger sequence cycle. More importantly, the biosensor developed in the present invention has the advantages of no enzyme, no DNA extraction, and ultrasensitivity. These results indicate that the method of the present invention (especially the developed signal amplification strategy) is sensitive, accurate, and cost-effective for the ultrasensitive detection of Escherichia coli and Salmonella Enteritidis.

[0090] Compared with some other bacteria detection biosensors based on aptamers, the method of the present invention has higher detection sensitivity (Table 2). In particular, the biosensor of the present invention has the advantage of being enzyme-free, that is, it can maintain excellent efficiency without the participation of enzymes in the reaction.

[0091] Table 2: Comparison of reported nanomaterial-based methods for detecting Salmonella Enteritidis Note: 1: Zhang P, Song M, Dou L, et al. Development of a fluorescentDNA nanomachine for ultrasensitive detection of Salmonella enteritidis without labeling and enzymes. Mikrochim Acta . 2020;187(7):376. 2: Chinnappan R, AlAmer S, Eissa S, Rahamn AA, Abu Salah KM, Zourob M. Fluorometric graphene oxide-based detection of Salmonella enteritis using atruncated DNA aptamer. Mikrochim Acta . 2017;185(1):61. 3: Wang Z, Yao X, Wang R, et al. Label-free strip sensor based on surface positively charged nitrogen-rich carbon nanoparticles for rapid detection of Salmonella enteritidis. Biosens Bioelectron . 2019;132:360-367. 4: Zhang P, Liu H, Li X, et al. A label-free fluorescent direct detection of live Salmonella typhimurium using cascade triple triggersequences-regenerated strand displacement amplification and hairpin template-generated-scaffolded silver nanoclusters. Biosensors and Bioelectronics . 2017;87:1044-1049. Selectivity and specificity Seven bacteria, including Escherichia coli, Salmonella enteritidis, Staphylococcus aureus, Listeria monocytogenes, Streptococcus enteritidis, Escherichia coli BL21, Escherichia coli DH5alpha and background control, were used as sensing targets. 5 CFU / mL, the specificity of the biosensor was explored. Figure 8 As shown in the figure, except for Escherichia coli and Salmonella Enteritidis, the fluorescence intensity of the system did not increase significantly after adding the above bacteria compared with the negative background control. Figure 8 A(a) and Figure 8 As shown in B(b), Escherichia coli and Salmonella enteritidis only release higher fluorescence intensity to the probe that can participate in the reaction. This high specificity may be due to the high affinity of the aptamer to Escherichia coli and Salmonella enteritidis. In addition, the design of the protection probe and the precise base complementary chain migration mechanism increase the specificity of the present invention. All these results prove that the biosensor for the dual bacteria developed by the present invention has very outstanding selectivity and specificity.

[0092] Precision and repeatability Precision evaluation is an important evaluation index to illustrate the applicability of a method. This example measures this index through experiments. 3 The CFU / mL of Escherichia coli and Salmonella Enteritidis were measured continuously, and the intra-assay precision was determined to be 4.12% for Escherichia coli and 7.03% for Salmonella Enteritidis (n=5). When the samples were retested after 72 hours under the same reaction conditions, the RSD values ​​between samples were 6.22% for Escherichia coli and 8.95% for Salmonella Enteritidis (n=5). 3 Three parallel experiments were conducted on CFU / mL of Escherichia coli and Salmonella Enteritidis, and the inter-batch precision was analyzed. The results were: 5.42% for Escherichia coli and 8.29% for Salmonella Enteritidis (n=3).

[0093] Detection of Escherichia coli and Salmonella Enteritidis in clinical body fluid samples The feasibility and potential of the designed biosensor in clinical application were evaluated by detecting Escherichia coli and Salmonella enteritidis in body fluid samples. 5 The concentration of CFU / mL was added to cerebrospinal fluid, pleural effusion, and ascites; then, the biosensor was applied to detect bacteria after simple pretreatment of the samples.

[0094] As shown in Table 3, the recovery rates of the two target bacterial standard samples in the three body fluids are very impressive, indicating that the presence of complex components in the body fluid samples will not significantly interfere with the detection of Escherichia coli and Salmonella enteritidis. This result can be attributed to the high affinity between the two bacteria and the aptamer and the high efficiency of the DNA cyclic amplification method. More importantly, these results demonstrate the feasibility and application potential of the biosensor developed by the present invention in clinical sample detection. In addition, the biosensor can be developed into a multifunctional platform for detecting biomarkers of any bacteria or even other species.

[0095] Table 3: Detection of Escherichia coli and Salmonella Enteritidis in different body fluid samples using biosensors Remark: E. coli : Escherichia coli; SE: Salmonella Enteritidis.

[0096] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the enlightenment of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the purpose of the present invention and the claims, which all fall within the protection of the present invention.

Claims

1. A method for rapid simultaneous detection of at least two targets, characterized in that: The following steps are involved: S101 incubating the sample to be tested with a first aptamer-first competitive sequence complex, so that the first aptamer binds to the first target in the sample to be tested and releases the first competitive sequence to obtain a first mixture; S102 incubating the sample to be tested with the second aptamer-second competitive sequence complex, so that the second aptamer binds to the second target in the sample to be tested and releases the second competitive sequence to obtain a second mixture; S103 centrifuging the first mixture and collecting a first supernatant, and centrifuging the second mixture and collecting a second supernatant; S104: after mixing the first probe-first trigger sequence complex and the second probe-second trigger sequence complex, adding the first amplification sequence-second amplification sequence complex and the first supernatant and reacting, then adding the second supernatant and reacting to obtain a third mixture; wherein the first probe and the second probe are modified with a fluorescent group; and the first amplification sequence and the second amplification sequence are modified with a quenching group; S105 Analyze the presence and / or concentration of the first target and / or the second target in the sample to be tested according to the fluorescence intensity signal.

2. The method according to claim 1, characterized in that The concentration ratio of the first probe-first trigger sequence complex, the second probe-second trigger sequence complex and the first amplification sequence-second amplification sequence complex comprises 1:1:(1-3).

3. The method according to claim 1, characterized in that The reaction time of step S104 is 5-10 minutes.

4. The method according to claim 1, characterized in that The reaction temperature of step S104 is 30-40°C.

5. The method according to claim 1, wherein The nucleotide sequence of the first aptamer is SEQ ID NO:1, and the nucleotide sequence of the second aptamer is SEQ ID NO:

2.

6. The method according to claim 1, wherein: The nucleotide sequence of the first competitive sequence is SEQ ID NO:3, and the nucleotide sequence of the second competitive sequence is SEQ ID NO:

4.

7. The method according to claim 1, characterized in that The nucleotide sequence of the first probe is SEQ ID NO:5, and the nucleotide sequence of the second probe is SEQ ID NO:

6.

8. The method according to claim 1, wherein The nucleotide sequence of the first trigger sequence is SEQ ID NO:7, and the nucleotide sequence of the second trigger sequence is SEQ ID NO:

8.

9. The method according to claim 1, wherein: The nucleotide sequence of the first amplified sequence is SEQ ID NO:9, and the nucleotide sequence of the second amplified sequence is SEQ ID NO:

10.

10. A kit for rapid detection of Escherichia coli and Salmonella enteritidis simultaneously, characterized in that: include: A first aptamer, a second aptamer, a first competitive sequence, a second competitive sequence, a first probe, a second probe, a first trigger sequence, a second trigger sequence, a first amplification sequence, and a second amplification sequence, wherein: The nucleotide sequence of the first adapter is SEQ ID NO:1, and the nucleotide sequence of the second adapter is SEQ ID NO:2; the nucleotide sequence of the first competitive sequence is SEQ ID NO:3, and the nucleotide sequence of the second competitive sequence is SEQ ID NO:4; the nucleotide sequence of the first probe is SEQ ID NO:5, and the nucleotide sequence of the second probe is SEQ ID NO:6; the nucleotide sequence of the first trigger sequence is SEQ ID NO:7, and the nucleotide sequence of the second trigger sequence is SEQ ID NO:8; the nucleotide sequence of the first amplification sequence is SEQ ID NO:9, and the nucleotide sequence of the second amplification sequence is SEQ ID NO:10.

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