CN117947143A - Method for quantitatively detecting viable bacteria with hands-free taking, amplification-free and high sensitivity and application thereof - Google Patents
Method for quantitatively detecting viable bacteria with hands-free taking, amplification-free and high sensitivity and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of microorganism detection, and particularly relates to a high-sensitivity quantitative detection method for living bacteria without hands-free taking, amplification and application thereof, which is used for high-sensitivity quantitative detection of living bacteria without nucleic acid extraction and amplification. The hand-free extraction means that protease treatment is not needed, and target strain nucleic acid is directly released from living cells by utilizing phage, wherein the detection accuracy of the related living bacteria is improved, and the related hand-free extraction simplifies the test operation and reduces the cost; the invention adopts DNA and ATP to cooperatively trigger PfAgo enzyme to cut the fluorescent probe to realize signal enhancement, and can realize high-sensitivity detection without amplifying target strain nucleic acid, thereby avoiding the risk of cross contamination; the design of PfAgo enzyme guide chain and the target sequence selection are not limited by PAM, so that the complexity of primer design is reduced; the method has the linear range of 10 2-107 CFU/mL, the detection limit of 20 CFU/mL, high detection sensitivity and accurate detection result, and is suitable for quantitative detection of live bacteria of food-borne pathogenic bacteria.
Description
Technical Field
The invention belongs to the technical field of microorganism detection, and particularly relates to an extraction-free, amplification-free and high-sensitivity quantitative detection method for viable bacteria and application thereof.
Background
According to the report of World Health Organization (WHO), unsafe foods cause 6 hundred million food-borne diseases and cause 42 ten thousand deaths each year, the incidence rate of the food-borne diseases is the first total incidence rate of various diseases, and food-borne pathogenic bacteria are the main causes of the food-borne diseases. Therefore, accurate detection of food-borne pathogenic bacteria is an important means for ensuring food safety.
Traditional methods of detection of pathogenic bacteria culture are very time consuming, typically require 2-5 days, and cannot detect strains in a non-culturable state. In recent years, some gene level detection methods, such as real-time fluorescent amplification techniques such as PCR, RT-PCR, immunoenrichment PCR, immunomagnetic separation PCR, and the like, and other isothermal nucleic acid amplification techniques, have shown excellent performances of rapidness and high sensitivity in pathogen detection. Although these nucleic acid amplification methods can achieve exponential amplification of nucleic acids in a short period of time, they still suffer from problems such as complex primer design and susceptibility to non-specific amplification. The CRISPR effector protein is a novel tool for detecting food-borne pathogenic bacteria because of being capable of accurately identifying nucleic acid sequences and having side-cutting activity, and is widely applied to nucleic acid-based detection. However, CRISPR/Cas needs to rely on PAM recognition sites, which are not present in many biological motifs. In addition, CRISPR/Cas enzymes require cleavage under crRNA guidance, and RNA is extremely easily degraded, and thus, the use of CRISPR has been greatly limited. Argonaute (Ago) protein is a nucleic acid leader enzyme widely used in detection, and can accurately identify a target nucleic acid sequence through base complementary pairing. Wherein the Ago protein isolated from the species Pyrococcus furiosus (Pyrococcus furiosus) cleaves the phosphodiester bond between bases 10 and 11 of the complementary target strand under high temperature conditions, guided by 5' -phosphorylated single-stranded DNA (gDNA). Pyrococcus furiosusArgonaute (PfAgo) the guide strand design and target sequence selection are not PAM limited and remain highly cleavage active for single-stranded DNA and most double-stranded DNA substrates. Compared with CRISPR/Cas enzyme, the detection based on PfAgo enzyme has the advantages of no PAM dependence and no RNA participation. However, none of the above methods can distinguish whether the detected food-borne pathogenic bacteria are live or dead bacteria, and often require detection in combination with nucleic acid extraction and amplification steps, which are prone to cross-contamination, leading to false positives. In the aspect of food detection, the existence of dead bacteria DNA enables the detection method of gene level to overestimate the level of living bacteria in a sample, so that the detection result is inaccurate. Therefore, it is important to quantitatively detect living bacteria in foods based on existing detection tools.
At present, the living bacteria/dead bacteria detection is generally carried out by combining azide propidium bromide dye or calcein-ETH 3 dye with real-time fluorescence quantitative PCR, and compared with the traditional dye method, the living bacteria quantitative detection has the characteristics of high sensitivity, simple and convenient operation, easy resolution of results and the like, but the detection sensitivity still needs to be improved by means of an amplification step, so that the risk of cross contamination cannot be avoided. At present, some researches are carried out on RNA as a live bacteria indicator, and quantitative detection of live bacteria is realized by detecting RNA, but RNA is easily degraded by various environmental factors to cause false positive, and the stability and accuracy of detection results are affected. On the other hand, some scholars consider that the RNA refers to mRNA, and the mRNA is a vector for expressing protein, and the mRNA can be detected only to prove that the target strain is in replication and expression, the dead activity of the target strain cannot be distinguished, and the expression quantity of the mRNA is low and is not easy to detect. In view of the drawbacks of the above living bacteria detection technology, it is necessary to develop an accurate and highly sensitive living bacteria quantitative detection method.
The content of cell endogenous Adenosine Triphosphate (ATP) can reflect the number of living cells, and the cell endogenous Adenosine Triphosphate (ATP) and the ATP have good linear relation, so that a new idea can be provided for an accurate living bacteria quantitative detection method. Based on this, the detection technology of measuring the number of living cells by using ATP bioluminescence has been widely used in the food industry, for example, for measuring bacterial contamination in meat products, the content of lactic acid bacteria in dairy products, bacteriological measurement of dehydrated vegetables, and the like. However, ATP is present in a variety of cells, and this type of detection technique lacks the ability to identify bacterial species, and non-target strains in the sample matrix can affect the accuracy of the detection results. Therefore, there is also a need to improve the specific recognition ability of target strains in order to achieve accurate live bacteria quantification.
Phages are viruses that infect bacteria, the oldest and most abundant biological entities on earth. Phage will only attack a specific host and will not destroy the normal flora. Therefore, the phage has the characteristics of high specificity, safety and low research and development cost. Phages can specifically recognize and lyse living host bacteria and release cell lysates such as DNA and ATP. Therefore, the DNA and ATP in the pathogenic bacteria can be detected by utilizing the specificity of the phage to identify and lyse the pathogenic bacteria, thereby solving the false positive problem caused by the fact that the bacterial species cannot be identified in the existing ATP bioluminescence detection technology. The accurate and high-sensitivity quantitative detection of the live bacteria of the food-borne pathogenic bacteria can be further realized by combining the existing detection tool.
Disclosure of Invention
The invention provides an extraction-free, amplification-free and high-sensitivity quantitative detection method for viable bacteria and application thereof, which enable the detection process to be simple to operate, accurate in result interpretation, improve the detection sensitivity and provide a rapid, accurate, high-sensitivity and high-specificity technical means for detecting viable bacteria of food-borne pathogenic bacteria.
In order to achieve the aim of the invention, phage is utilized to carry out nucleic acid extraction, pfAgo enzyme is utilized as nucleic acid guiding enzyme, target strain nucleic acid sequence is accurately identified, divalent aptamer is utilized to capture ATP molecules, and a PfAgo-mediated biosensing system and an enhancement system based on DNA and ATP synergistic triggering are constructed and are used for high-sensitively and quantitatively detecting food-borne pathogenic bacteria living bacteria without nucleic acid extraction and amplification.
The technical scheme of the invention is as follows:
A live bacteria detection kit comprising:
The PfAgo enzyme is guided to target the gDNA of bacteria, specifically gDNA-1 shown as a sequence SEQ ID NO.1, gDNA-2 shown as a sequence SEQ ID NO.2 and gDNA-3 shown as a sequence SEQ ID NO. 3;
Guiding PfAgo enzyme to cut gDNA of the fluorescent probe 1, wherein the gDNA is partial DNA fragments cut by bacteria, specifically gDNA-4 shown as sequence SEQ ID NO.4, of gDNA-1, gDNA-2 and gDNA-3 guiding PfAgo enzyme;
Guiding PfAgo enzyme to cut gDNA of the fluorescent probe 2, wherein the gDNA is an oligonucleotide sequence hybridized with an ATP aptamer to form a bivalent aptamer, and specifically gDNA-5 shown as a sequence SEQ ID NO. 5;
the method comprises the steps of constructing a bivalent aptamer for ATP, wherein the bivalent aptamer is obtained by hybridization of two ATP aptamer sequences and one oligonucleotide sequence, specifically an ATP aptamer shown as a sequence SEQ ID NO.6 and specifically an oligonucleotide sequence shown as a sequence SEQ ID NO. 5;
the fluorescent probe with a fluorescent group and a quenching group is specifically a fluorescent probe 1 shown as SEQ ID NO.7 and a fluorescent probe 2 shown as SEQ ID NO. 8;
the detection kit is used for quantitative detection or auxiliary quantitative detection of bacteria, and the quantitative detection or auxiliary quantitative detection aims at quantitative detection or auxiliary quantitative detection of live bacteria of food-borne pathogenic bacteria.
Preferably, the bacterium is salmonella typhimurium ATCC14028.
A method for quantitative detection of viable bacteria, the method comprising the steps of:
s1: design gDNA:
S2: preparation of magnetic nanoparticle-Phage complex (MNP-phase): washing the carboxyl magnetic nano particles with the size of 1000nm by using MEST buffer solution; removing the supernatant by magnetic separation, re-suspending by MES buffer solution, adding 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and placing on a rotary mixer for activation; washing the activated cells with MEST buffer solution, removing the supernatant by magnetic separation, and adding phage solution for incubation; at the end of incubation, the supernatant was removed by magnetic separation and blocked by addition of PBS (1% BSA) buffer; washing the closed end with PBST buffer solution, removing the supernatant by magnetic separation, and adding PBS buffer solution for resuspension to obtain a magnetic nanoparticle-Phage complex, namely MNP-Phage;
S3: preparation of magnetic nanoparticle-bivalent aptamer complex (MNP-BAPT): washing the 150 nm-sized carboxyl magnetic nanoparticles with MEST buffer; removing the supernatant by magnetic separation, re-suspending by MES buffer solution, adding 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and placing on a rotary mixer for activation; washing the activated cells with PBST buffer, removing the supernatant by magnetic separation, re-suspending the cells with PBS buffer, and adding streptavidin for incubation; at the end of incubation, the supernatant was removed by magnetic separation and blocked by addition of PBS (1% BSA) buffer; washing the end of the sealing by using PBST buffer solution, removing the supernatant by magnetic separation, and adding ultrapure water for resuspension to obtain streptavidin-modified magnetic nano particles, namely MNP-SA;
Adding the biotinylated ATP aptamer, gDNA-5, a binding buffer solution and ultrapure water into a centrifuge tube, heating, immediately performing ice bath, and placing on a rotary mixer for hybridization; adding MNP-SA solution for incubation after hybridization; washing with PBST buffer solution after incubation, removing supernatant by magnetic separation, and adding ultrapure water for resuspension to obtain a magnetic nanoparticle-bivalent aptamer complex, namely MNP-BAPT;
S4: pfAgo enzyme cleavage: incubating the MNP-phase solution with the bacterial liquid; washing with PBST buffer solution after incubation, removing supernatant by magnetic separation, and adding ultrapure water and MNP-BAPT solution for incubation; after the incubation is finished, collecting supernatant by magnetic separation, sequentially adding PfAgo enzymes, gDNA-1, gDNA-2, gDNA-3, a reaction buffer, a fluorescent probe 1 and a fluorescent probe 2, and reacting;
S5: fluorescence reading;
and (5) finishing quantitative detection of viable bacteria.
Preferably, in the step S1, three complementary 15-22 nt gDNA are respectively designed according to the bacterial nucleic acid sequence to be detected, and are respectively named gDNA-1, gDNA-2 and gDNA-3; under the guidance of gDNA-1, gDNA-2 and gDNA-3, activating PfAgo enzyme to cut, and then releasing a 15-22 nt new DNA fragment serving as secondary gDNA, which is named gDNA-4; an oligonucleotide sequence 15-22 nt which hybridizes to the ATP aptamer to form a bivalent aptamer designated gDNA-5; wherein, the base at the 5' end of the gDNA is modified by a phosphate group; the gDNA-4 is complementary to the sequence of the fluorescent probe 1 (FAM-ssDNA 1 -BHQ 1) of 16-25 nt; the gDNA-5 is complementary to the 16-25 nt fluorescent probe 2 (FAM-ssDNA 2 -BHQ 1) sequence.
Preferably, in the step S2, the carboxyl-modified magnetic nanoparticles are washed with 500-1000 uL MEST buffer; after magnetic separation, re-suspending with 500-1000 uL MES buffer, adding 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), placing on a rotary mixer, rotating and mixing at 300-500 rpm/min, and activating at 25-37deg.C for 15-30 min; washing with MEST buffer solution after activation, removing supernatant after magnetic separation, adding 50-200 uL phage solution (phage culturing step: adding 20 uL phage solution (10 8 PFU/mL) into 200 uL salmonella solution (10 8 CFU/mL) to logarithmic phase, inoculating into 5mL 2× Tryptic Soy Broth (TSB) culture medium, incubating at 37deg.C for 12 h with constant temperature shaker, centrifuging at 4deg.C 8000 g for 15 min to absorb supernatant, filtering supernatant with 0.22 um filter membrane, preserving at 4deg.C in refrigerator for use), incubating at 37deg.C for 1-3 h; after the incubation is finished, removing the supernatant after magnetic separation, adding 500-1000 uL of PBS buffer solution containing 1% bovine serum albumin, and incubating at 37 ℃ for 30-60 min; after magnetic separation, the supernatant is removed, washed by PBST buffer solution and resuspended by 500-1000 uL PBS buffer solution, and the magnetic nanoparticle-Phage complex, namely MNP-Phage, is obtained.
Further preferably, in the step S2, 5 mg/mL of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) 10-30 uL and 5 mg/mL of N-hydroxysuccinimide (NHS) 10-30 uL are added; even more preferably 5 mg/mL EDC 30 uL,5 mg/mL NHS 15 uL.
In the step S2, the concentration of the phage solution to be added is preferably 10 6-109 PFU/mL, and more preferably 10 9 PFU/mL.
The concentration of the magnetic nanoparticle-Phage complex MNP-Phage obtained in the step S2 is preferably 0.5-2 mg/mL, and more preferably 1mg/mL.
Preferably, the step S3 specifically includes:
step S3.1: washing the carboxyl-modified 150 nm-sized magnetic nanoparticles with 500-1000 uL MEST buffer (10 mM,0.05%Tween 20,pH 6.0); after magnetic separation, re-suspending with 500-1000 uL MES buffer (10 mM, pH 6.0), adding 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), placing on a rotary mixer, rotating and mixing at 300-500 rpm/min, and activating at 25-37deg.C for 15-30 min; washing with 500-1000 uL PBST buffer solution after activation, removing supernatant by magnetic separation, adding streptavidin, supplementing with PBS buffer solution to 500-1000 uL, and incubating; after the incubation is finished, removing the supernatant after magnetic separation, adding PBS buffer (10 mM,1%BSA,pH 7.4) containing 500-1000 uL of 1% bovine serum albumin, and incubating at 37 ℃ for 30-60 min; removing the supernatant after magnetic separation, washing with 500-1000 uL PBST buffer solution (10 mM,0.05%Tween 20,pH 7.4), and re-suspending with 500-1000 uL ultrapure water to obtain streptavidin-modified magnetic nanoparticle (MNP-SA), and preserving at 4deg.C for use;
Step S3.2: adding biotinylated ATP aptamer, gDNA-5, binding buffer and ultrapure water into a centrifuge tube, heating at 95-98deg.C for 5-8 min, immediately ice-bathing for 5-8 min, placing on a rotary mixer, mixing at 300-500 rpm/min, and hybridizing at 37deg.C for 1-1.5 h; adding 500-1000 uL of MNP-SA solution obtained in the step S3.1, and incubating at 37 ℃ for 0.5-1 h; removing the supernatant after magnetic separation, washing with 500-1000 uL PBST buffer solution, adding 500-1000 uL ultrapure water for resuspension, and obtaining the magnetic nanoparticle-bivalent aptamer complex MNP-BAPT.
Further preferably, in the step S3.1, 5 mg/mL of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride 10-30 uL and 5 mg/mL of N-hydroxysuccinimide 10-30 uL are added; even more preferably 5 mg/mL EDC 10 uL,5 mg/mL NHS 10 uL.
In the step S3, the dosage of adding streptavidin is preferably 10 mg/mL 5-10 uL; further preferably 10 mg/mL 5 uL.
In the step S3, the concentration of the obtained streptavidin-modified magnetic nano particles, namely MNP-SA, is preferably 0.5-2 mg/mL; still more preferably 1mg/mL.
In the step S3, the biotinylated ATP aptamer, gDNA-5, 10×binding buffer and ultrapure water are added into a centrifuge tube, and the amount of the biotinylated ATP aptamer, 100uM gDNA-5, 10×binding buffer and ultrapure water is preferably 100uM, in a volume ratio of 5:5:3:17, mixing evenly.
In the step S3, the concentration of the magnetic nanoparticle-divalent aptamer complex, namely MNP-BAPT, is preferably 0.5-2 mg/mL, and more preferably 1mg/mL.
Preferably, in the step S4, the MNP-phase solution is incubated with the bacterial liquid; the MNP-phase concentration is preferably 200 ug/mL, and the dosage is preferably 100uL; the target strain concentration is preferably 10 1-108 CFU/mL, and the dosage is preferably 100 uL. Uniformly mixing MNP-phase solution and bacterial liquid according to the volume ratio of 1:1 for incubation;
In the step S4, the incubation is finished, the incubation is washed by PBST buffer solution, the supernatant is removed by magnetic separation, and the volume ratio of the ultrapure water to the MNP-BAPT solution is 19:1, adding and incubating;
In the step S4, the concentration of the MNP-BAPT solution is preferably 200-800 ug/mL; still more preferably 500 ug/mL.
In the step S4, the concentration of PfAgo enzyme in the reaction system is preferably 200L-800ug/mL; still more preferably 500 ug/mL.
In the step S4, the concentrations of gDNA-1, gDNA-2 and gDNA-3 in the reaction system are preferably 2 uM-10 uM; still more preferably 5 uM.
In the step S4, the shearing reaction time is preferably 10-60 min; still more preferably 30min.
Preferably, in the step S4, the incubation is finished, the supernatant is collected by magnetic separation, and the volume ratio of the supernatant to the supernatant is 35:5:1:1:1:5:1:1 adding supernatant, pfAgo enzyme, gDNA-1, gDNA-2, gDNA-3, reaction buffer, fluorescent probe 1 and fluorescent probe 2; still more preferably, the total system is 100 uL, which is 70 uL supernatant, 10 uL 500 ug/mL PfAgo,5 uM gDNA-1, gDNA-2, gDNA-3 each 2 uL,10 uL 10 Xreaction buffer, and 2 uL 5 uM fluorescent probe 1 and fluorescent probe 2.
Preferably, in the step S5, the reaction solution in the step S4 of 20-100 uL is added to a 384-well plate, and a multifunctional enzyme-labeled instrument is used for reading fluorescent signals. Each sample was measured 3 times independently and the average of 3 fluorescence intensities was taken.
The living bacteria detection kit or the living bacteria quantitative detection method is used for quantitatively detecting food-borne pathogenic bacteria.
According to the invention, the bacteriophage specificity is utilized to identify and lyse the pathogenic bacteria living bacteria, and DNA and ATP released by the lysis are used as detection markers, so that the nucleic acid extraction-free is realized, the operation steps are reduced, the specificity and accuracy of the living bacteria identification are improved, and the detection cost is reduced; according to the invention, pfAgo is used as nucleic acid guiding enzyme, so that the accurate identification of the target strain nucleic acid sequence is realized, no PAM region is required to be designed, no RNA is required to participate, the primer design step is simplified, and the RNA degradation risk is avoided; the invention utilizes the ability of PfAgo protein secondary cutting, realizes accurate identification of target strain nucleic acid sequence and realizes signal reading by cutting fluorescent probe; the more the target strain is, the more DNA and ATP are released, and the released DNA can guide the subsequent PfAgo enzyme to cut the fluorogenic substrate, so that signal reading is realized. More importantly, the ATP bivalent aptamer constructed by the invention can release guide DNA (deoxyribonucleic acid) while capturing ATP molecules, and the DNA can also guide PfAgo enzyme to cut a fluorescent probe so as to realize secondary signal reading; therefore, DNA and ATP released from the target strain living bacteria cooperatively trigger PfAgo enzyme in a single tube to cut the fluorescent probe, and double fluorescent signals generate a synergistic enhancement effect, so that the detection sensitivity can be greatly improved without nucleic acid amplification. The invention comprehensively utilizes the technologies, and finally establishes the method for quantitatively detecting the living bacteria with no extraction, no amplification and high sensitivity. Compared with the PCR, RT-PCR and a series of isothermal amplification technologies or detection methods combined with CRISPR/Cas which are widely used at present, the invention has the advantages of high sensitivity, accurate detection result, no need of nucleic acid extraction and amplification steps, no RNA participation, simple primer design and the like.
The beneficial effects of the invention are as follows:
(1) The invention provides a PfAgo enzyme-mediated biosensing system and an enhancement system based on DNA and ATP collaborative triggering, which are used for high-sensitivity quantitative detection of living bacteria without DNA extraction and amplification, and the system comprises the following steps: 1) Designing gDNA; 2) Preparing MNP-phase complex; 3) Preparing MNP-BAPT complex; 4) PfAgo enzyme cutting; 5) Fluorescent reading.
(2) The method can accurately detect living bacteria, DNA and ATP cooperatively trigger fluorescent signals in a single tube, and the double fluorescent signals produce a synergistic enhancement effect, and has the characteristics of high sensitivity, accurate detection result, no need of nucleic acid extraction and amplification, no need of RNA participation, no need of PAM restriction on primer design and the like.
(3) The phage in the invention can specifically capture and proliferate by utilizing the living target strain, and finally, the target strain is cracked to release DNA and ATP, and the detection method is designed by utilizing the characteristics to realize the nucleic acid extraction-free of the living bacteria, so that the operation steps are simplified, and the accuracy of the detection result is improved.
(4) According to the invention, the PfAgo enzyme is utilized to specifically cut the target strain nucleic acid sequence, a PAM region is not required to be designed in the cutting process, RNA participation is not required, the primer design step is simplified, the high cutting activity of the target strain nucleic acid sequence is maintained, and meanwhile, the problem of inaccurate detection caused by easy degradation of RNA is avoided. By utilizing the secondary cutting capability of PfAgo enzyme, the fluorescent probe is cut under the guidance of the generated secondary gDNA, and fluorescent signal reading is realized while the accurate identification of the target strain nucleic acid sequence is realized.
(5) The bivalent aptamer constructed in the invention can specifically and rapidly recognize ATP molecules, release guide DNA while recognizing ATP molecules, and continuously cut fluorescent probes under PfAgo enzyme mediation to realize secondary signal reading.
(6) DNA and ATP cooperatively trigger PfAgo enzyme in a single tube to cut a fluorescent probe, and double fluorescent signals generate a synergistic enhancement effect, so that the detection sensitivity is greatly improved.
Drawings
FIG. 1 is a schematic diagram of the principle of quantitative detection of living bacteria by the method of the present invention.
FIG. 2 is a schematic diagram of the results of the quantitative detection of viable bacteria using the method of the present invention in example 1; wherein A is the linear range of the biosensor for independently detecting salmonella DNA, independently detecting salmonella ATP and simultaneously detecting salmonella DNA and ATP; b is a detection standard curve.
FIG. 3 is a schematic diagram showing the results of the verification of the lytic ability of the phage of example 2 against live bacteria (A) and dead bacteria (B) of Salmonella.
FIG. 4 is a schematic diagram showing the results of the enzyme cleavage activity assay of PfAgo in example 3; wherein A is the result of PfAgo electrophoresis of cleaved fluorescent probe 3 (lane 1: containing gDNA-4; lane 2: containing fluorescent probe 3; lane 3: containing gDNA-4 and fluorescent probe 3; lane 4: containing PfAgo enzyme and fluorescent probe 3; lane 5: containing PfAgo enzyme, gDNA-4 and fluorescent probe 3); b is a graph of the quantitative relationship between the DNA concentration and the fluorescence intensity of Salmonella.
FIG. 5 is a schematic diagram showing the results of the verification of ATP-triggering PfAgo enzyme cleavage activity in example 4; wherein A is the result of hybridization electrophoresis of bivalent aptamers (lane 1: containing one ATP aptamer; lane 2: containing gDNA-5; lane 3: containing two hybridization complexes of ATP aptamers and gDNA-5); b is a graph of the quantitative relationship between the concentration of ATP standard and the fluorescence intensity.
FIG. 6 is a graphical representation of the results of concentration optimization of MNP-BAPT used in the PfAgo enzyme cleavage reaction system of example 5.
FIG. 7 is a schematic diagram showing the results of optimizing the concentration of PfAgo enzyme used in the PfAgo enzyme cleavage reaction system in example 6.
FIG. 8 is a graph showing the results of optimizing the concentration of gDNA used in the PfAgo enzyme cleavage reaction system in example 7.
FIG. 9 is a schematic diagram of the results of the time optimization of the PfAgo enzyme cleavage reaction in example 8.
FIG. 10 is a schematic diagram showing the quantitative detection results of Salmonella using real-time fluorescence PCR in example 9.
FIG. 11 is a schematic diagram showing the results of a specific assay for quantitatively detecting live Salmonella bacteria using the method of the present invention in example 10; wherein A is the fluorescence intensity value of 5 food-borne pathogenic bacteria of salmonella, listeria monocytogenes, staphylococcus aureus, escherichia coli and vibrio parahaemolyticus respectively detected by adopting the method; b is the fluorescence intensity value of the mixture of salmonella and other 4 food-borne pathogenic bacteria respectively detected by adopting the method.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples, but embodiments of the present invention are not limited thereto. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. The test methods for specific experimental conditions are not noted in the examples below, and are generally performed under conventional experimental conditions or under experimental conditions recommended by the manufacturer. The reagents and starting materials used in the invention may be prepared by commercial or conventional methods unless otherwise specified.
According to the invention, phage is utilized to carry out nucleic acid extraction, pfAgo enzyme is utilized as nucleic acid guiding enzyme, target strain nucleic acid sequence is accurately identified, and bivalent aptamer is utilized to capture ATP molecules, so that a PfAgo-mediated biosensing system and an enhancement system based on DNA and ATP synergistic triggering are constructed, and the extraction-free amplification-free high-sensitivity living bacteria quantitative detection method is obtained. FIG. 1 is a schematic diagram of the principle of quantitative detection of living bacteria by the method of the present invention. The specific principle is as follows: MNP-phase specifically captures and lyses target strains, releasing DNA and ATP. PfAgo enzyme can be guided by gDNA-1, gDNA-2 and gDNA-3 to target the target strain nucleic acid sequence for cleavage, so that a new small DNA molecular fragment is generated, which is marked as gDNA-4, gDNA-4 can be used as secondary gDNA to secondarily activate PfAgo enzyme, and fluorescent probe 1 complementarily paired with the secondary gDNA is precisely cleaved and fluorescent signals are generated. Meanwhile, the bivalent aptamer is constructed by designing the hybridization of gDNA-5 and the ATP aptamer, and then the bivalent aptamer is coupled on the surface of the magnetic nanoparticle to obtain MNP-BAPT. When the target strain is specifically lysed by phage, ATP is released, at which point MNP-BAPT can capture ATP and release gDNA-5, the gDNA-5 is assembled with the PfAgo enzyme, and the PfAgo enzyme is activated to cleave the complementarily paired fluorescent probe 2, producing a fluorescent signal. In a single tube, DNA and ATP react simultaneously to cooperatively trigger the cleavage activity of PfAgo enzyme, producing a double fluorescent signal. The concentration of the target strain in the sample matrix is transduced into DNA concentration and ATP concentration by the sensing system, and finally, the concentration of the target strain is read out by fluorescence signals under PfAgo enzyme mediation through the synergistic effect of the DNA and the ATP. The content of the living bacteria of the target strain in the sample matrix is in direct proportion to the fluorescence signal intensity, so that the quantitative detection of the living bacteria is realized.
Specifically, the invention relates to an extraction-free, amplification-free and high-sensitivity quantitative detection method for living bacteria, which comprises the following steps of:
S1: design gDNA: the gDNA is a single-stranded oligonucleotide sequence with the length of 15-22 nt; gDNA is designed according to the target strain nucleic acid sequence, and the designed gDNA-1, gDNA-2 and gDNA-3 are respectively complementary with different positions of the target strain DNA sequence, so that PfAgo enzyme is guided to cut the target strain DNA sequence; gDNA-1, gDNA-2 and gDNA-3 guide PfAgo enzyme to target the nucleic acid sequence of the target strain for shearing, and a single-stranded oligonucleotide sequence of 16 nt is cut at the moment to be gDNA-4; designing a single-stranded oligonucleotide sequence complementary to the biotinylated ATP aptamer according to the biotinylated ATP aptamer, wherein the single-stranded oligonucleotide sequence is gDNA-5; gDNA-4 and gDNA-5 are respectively complementary to two designed fluorescent probes, gDNA-4 is complementary to the designed fluorescent probe 1 (FAM-ssDNA 1 -BHQ 1), gDNA-5 is complementary to the designed fluorescent probe 2 (FAM-ssDNA 2 -BHQ 1), gDNA-4 and gDNA-5 cooperatively guide PfAgo enzyme to cleave the fluorescent probe, so that fluorescence is observed under excitation light irradiation; the 5 gDNA sequences are different;
s2: preparation of magnetic nanoparticle-phage complexes: the carboxyl-modified magnetic nanoparticles were washed with MEST buffer. After magnetic separation, resuspended in MES buffer, freshly prepared 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were added and placed on a rotary mixer for a certain period of time, at which time the carboxyl groups on the magnetic nanoparticles had been activated. Washing with MEST buffer solution after activation, removing the supernatant after magnetic separation, adding phage solution, and incubating, wherein the phage is coupled to the surface of the magnetic nanoparticles by carbodiimide reaction between self amine groups and carboxyl groups on the magnetic nanoparticles under the action of EDC and NHS crosslinking agents. At the end of incubation, the supernatant was removed after magnetic separation, and incubation was continued with the addition of PBS buffer containing bovine serum albumin to block unbound sites on the magnetic nanoparticles. After magnetic separation, the supernatant was removed, washed with PBST buffer, and resuspended with PBS buffer to obtain the magnetic nanoparticle-Phage complex, MNP-Phage.
S3: preparation of magnetic nanoparticle-divalent aptamer complexes: the carboxyl-modified magnetic nanoparticles were washed with MEST buffer. After magnetic separation, resuspended in MES buffer, and freshly prepared 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were added, and reacted on a rotary mixer, the reaction was completed with PBST buffer, at which time the carboxyl groups on the magnetic nanoparticles had been activated. After magnetic separation, the magnetic nanoparticles were resuspended with PBS buffer and incubated with streptavidin, at which time streptavidin was coupled to the magnetic nanoparticle surface by covalent coupling. At the end of incubation, the supernatant was removed after magnetic separation, and incubation was continued with the addition of PBS buffer containing bovine serum albumin to block unbound sites on the magnetic nanoparticles. After magnetic separation, the supernatant was removed, washed with PBST buffer and resuspended in ultrapure water to obtain streptavidin-modified magnetic nanoparticles, MNP-SA.
Biotinylated ATP aptamer, gDNA-5, binding buffer and ultrapure water were added to the centrifuge tube. Heating at 95 ℃ for 5 min ℃, immediately carrying out ice bath, placing on a rotary mixer, and carrying out hybridization at 37 ℃ to obtain the biotinylation bivalent aptamer. The MNP-SA solution was added to the centrifuge tube and incubated at 37 ℃. The supernatant is removed by magnetic separation and washing, and ultra-pure water is added for resuspension, so that the magnetic nanoparticle-bivalent aptamer complex, namely MNP-BAPT, is obtained.
S4: pfAgo enzyme cleavage: adding MNP-phase solution into a centrifuge tube containing target strains with different concentrations, incubating at 37 ℃, capturing and lysing live bacteria by Phage, and releasing target strain DNA and ATP into supernatant. Collecting supernatant containing target strain DNA and ATP after magnetic separation; adding MNP-BAPT, pfago enzyme, gDNA-1, gDNA-2, gDNA-3, reaction buffer solution, fluorescent probe 1 and fluorescent probe 2 into the supernatant to form a reaction system, and shearing by PfAgo enzyme; wherein gDNA-1, gDNA-2 and gDNA-3 guide PfAgo enzyme to cut on the complementary target DNA corresponding to the 10 th and 11 th positions of gDNA, and a new DNA fragment of 16nt is recorded as gDNA-4 after complete cutting; gDNA-4 is used as a secondary gDNA to excite PfAgo enzyme to carry out secondary cutting, and fluorescent probe 1 complementary to gDNA-4 is cut to generate a fluorescent signal; wherein MNP-BAPT can capture ATP in the supernatant and release gDNA-5 in the bivalent aptamer hybridization chain, gDNA-5 excites PfAgo enzyme to cut, and fluorescent probe 2 complementary to gDNA-5 is cut to generate a new fluorescent signal; ATP and DNA in the supernatant cooperatively trigger PfAgo to cut the fluorescent probes 1 and 2, and the double fluorescent signals generate a synergistic effect;
S5: reading signals: and (3) adding the reaction solution in 20 uL S4 to a 384-well plate, and reading fluorescent signals by using a multifunctional enzyme-labeled instrument, wherein the content of the living bacteria of the target strain in the sample matrix is in direct proportion to the intensity of the fluorescent signals, so that the quantitative detection of the living bacteria is realized.
Example 1: validity verification for quantitatively detecting living bacteria by adopting the method
S1: design gDNA: the target strain used in this example was a laboratory preservation strain, salmonella typhimurium (Salmonella TyphimuriumATCC, 14028), and gDNA-1, gDNA-2, and gDNA-3, each having a length of 16 nt, were complementarily paired with the Salmonella according to the conserved sequence of the Salmonella. gDNA-1 and gDNA-2 are designed to be complementary to one strand of the target strain duplex and gDNA-3 is designed to be complementary to the other strand of the target strain duplex. The guided PfAgo enzymes of gDNA-1, gDNA-2 and gDNA-3 exert a cleavage function, and a single-stranded oligonucleotide sequence of 16 nt, named gDNA-4, can be completely sheared on the complementary target DNA corresponding to the 10 th and 11 th positions of gDNA, and gDNA-4 can guide PfAgo to carry out secondary cleavage on a fluorescent probe 1 (FAM-ssDNA 1 -BHQ 1) complementary to gDNA-4, thereby generating a fluorescent signal. A single-stranded oligonucleotide sequence complementary to the biotinylated ATP aptamer is designed according to the biotinylated ATP aptamer, gDNA-5 is hybridized with two ATP aptamer chains to form a bivalent aptamer, when ATP exists, the bivalent aptamer captures ATP to release gDNA-5, and gDNA-5 can guide PfAgo to cleave a complementary strand fluorescent probe 2 (FAM-ssDNA 2 -BHQ 1) so as to generate a fluorescent signal.
The sequence of the sheared gDNA-4 is:
as shown in the sequence SEQ ID NO.4, 5'-p-GCACCGTCAAAGGAAC-3';
The sequences of gDNA-1, gDNA-2, gDNA-3 and gDNA-5 used are as follows:
gDNA-1: as shown in the sequence SEQ ID NO.1, 5'-p-TTGACGGTGCGATGAA-3';
gDNA-2: as shown in the sequence SEQ ID NO.2, 5'-p-CAGCTTTACGGTTCCT-3';
gDNA-3: as shown in the sequence SEQ ID NO.3, 5'-p-TCAAAGGAACCGTAAA-3';
gDNA-5: as shown in the sequence SEQ ID NO.5, 5'-p-TCCGCAATTTTTTTCCGCAAT-3';
the salmonella conserved sequences used are as follows:
5’-ATAAACTTCATCGCACCGTCAAAGGAACCGTAAAGCTGGC-3’；
3’-TATTTGAAGTAGCGTGGCAGTTTCCTTGGCATTTCGACCG-5’；
the sequence of the fluorescent probe used is as follows:
Fluorescent probe 1: FAM-TTACGGTTCCTTTGACGGTGCGATG-BHQ1 as shown in SEQ ID NO. 7;
Fluorescent probe 2: FAM-ATTGCGGAAAAAAATTGCGGA-BHQ1 as shown in SEQ ID NO. 8;
The ATP aptamer sequences used were:
5'-ACCTGGGGGAGTATTGCGGAGGAAGGT-3' as shown in SEQ ID NO. 6;
S2: preparation of magnetic nanoparticle-phage complexes: carboxyl-modified magnetic nanoparticles (COOH-MNP, diameter 1000 nm) were purchased from Ocean NanoTech company in this example. Phage LPST10 used was phage stored for the field isolation laboratory. 1mg of carboxyl-modified magnetic nanoparticle (COOH-MNP 1000) was taken, washed 3 times in a centrifuge tube with 1 mL MEST (10 mM,0.05%Tween 20,pH 6.0) buffer, and the supernatant was removed by magnetic separation. 30uL of freshly prepared 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 5 mg/mL) and 15uL of N-hydroxysuccinimide (NHS, 5 mg/mL) were added and adjusted to 1 mL with 4-morpholinoethanesulfonic acid (MES, pH 6.0) buffer. The centrifuge tube was placed on a rotary mixer and activated 15min at 25 ℃. The end of activation was washed 3 times with 500 uL MEST buffer and the supernatant was removed after magnetic separation. 100 uL phage solution (10 9 PFU/mL) was added to the centrifuge tube and adjusted to 1 mL with phosphate (PBS, 10mM,pH 7.4) buffer. Incubation at 37℃with 2h allowed the phage to bind well to the COOH-MNP 1000 surface sites. At the end of incubation, the supernatant was removed after magnetic separation, 1 mL PBS (10 mM,1%BSA,pH 7.4) buffer containing 1% bovine serum albumin (BSA, m/v) was added and incubated at 37 ℃ for 30min to block remaining unbound sites on the COOH-MNP 1000 surface. After magnetic separation, the supernatant was removed, washed 3 times with 1 mL PBST (10 mM,0.05%Tween 20,pH 7.4) buffer and unbound phage and excess BSA were washed away. Adding 1 mL PBS buffer solution to resuspend to obtain the magnetic nanoparticle-Phage complex with the final concentration of 1 mg/mL, namely MNP-Phage, and storing at 4 ℃ for standby.
S3: preparation of magnetic nanoparticle-divalent aptamer complexes: carboxyl-modified magnetic nanoparticles (COOH-MNP, diameter 150 nm) were purchased from Ocean NanoTech company in this example. The 10 x binding buffer used was prepared by: contains 500 mM Tris-HCl, 50 mM KCl, 1M NaCl and 10 mM MgCl2 and adjusts the pH of the binding buffer to 7.4. 500 ug carboxyl-modified magnetic nanoparticles (COOH-MNP 150) were taken, added with 1 mL MEST buffer and washed 3 times in a centrifuge tube. After magnetic separation, COOH-MNP 150 was resuspended in 480 uL MES buffer and 10 uL of the newly configured 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 5 mg/mL) and 10 uL N-hydroxysuccinimide (NHS, 5 mg/mL) were added in a total volume of 500 uL, placed on a rotary mixer and activated at 25℃for 15 min. At the end of activation, the cells were washed 3 times with 1 mL PBST buffer. After magnetic separation, the supernatant was removed, COOH-MNP 150 was resuspended in 495 uL PBS buffer, 5 uL streptavidin (SA, 10 mg/mL) was added and incubated at 37℃for 2.5 h. At the end of incubation, the supernatant was removed after magnetic separation, 1 mL PBS buffer containing 1% bovine serum albumin (BSA, m/v) was added and incubated at 37 ℃ for 30 min to block remaining unbound sites on the COOH-MNP 150 surface. After magnetic separation the supernatant was removed, the PBST buffer was washed 3 times and unbound SA and excess BSA were washed away. Adding 500 uL ultrapure water for resuspension to obtain the streptavidin-modified magnetic nanoparticle with the final concentration of 1 mg/mL, namely MNP-SA, and preserving at 4 ℃ for later use.
5 UL of 100 uM biotinylated ATP aptamer, 5 uL of 100 uM gDNA-5,3 uL of 10 Xbinding buffer and 17uL of ultrapure water were added to the centrifuge tube. Immediately after heating at 95℃to 5 min, ice bath 5 min was followed by hybridization at 37℃to 1h on a rotary mixer to give a bivalent aptamer. 500 uL MNP-SA solution was added to the centrifuge tube and incubated at 37℃for 30 min. After magnetic separation the supernatant was removed, and the unbound bivalent aptamer was washed 3 times with PBST buffer. Adding 500 uL ultrapure water for resuspension to obtain a magnetic nanoparticle-bivalent aptamer compound with the final concentration of 1 mg/mL, namely MNP-BAPT, and storing at 4 ℃ for later use.
S4: pfAgo enzyme cleavage: in this example PfAgo enzyme was purchased from Hylegen company and the 10 Xreaction buffer used was prepared by: contains 15mM Tris-HCl (pH 8.0), 250 mM NaCl and 0.5. 0.5 mM MnCl 2, and the pH of the reaction buffer is adjusted to 7.4. 100 uL 200 ug/mL MNP-phase was added to a centrifuge tube containing 100 uL different concentrations of Salmonella culture, and incubated 15 min at 37 ℃. The end of incubation was washed 3 times with 200 uL PBST buffer. After magnetic separation, the supernatant was removed, 95 uL ultrapure water and 5 uL of 500ug/mL MNP-BAPT solution were added, incubated at 37℃for 40min, and after the incubation, the supernatant was collected by magnetic separation. 70 uL supernatant, 10 uL of PfAgo enzyme (500 ug/mL), 2 uL of 5 uM gDNA-1, gDNA-2, gDNA-3, 10 uL of 10 Xreaction buffer, 2 uL of 5 uM fluorescent probe 1 and 2 were added into the centrifuge tube in sequence, the total volume of the reaction solution was 100 uL, and the reaction was carried out at 95℃for 30 min.
S5: fluorescence reading: the reaction solution obtained in step S4 of 20 uL was applied to 384-well plates, and fluorescent signals were read at 492: 492 nm with a multifunctional microplate reader. Each sample was measured 3 times independently and the average of 3 fluorescence intensities was taken. The quantitative relationship between the two is obtained by taking the salmonella concentration as an abscissa and taking the fluorescence intensity value as an ordinate. As shown in fig. 2A and 2B, the linear range of the single detection of salmonella DNA by the biosensor is 500-10 7 CFU/mL, the linear equation is y= 696.40x-1266.76 (x is the salmonella concentration logarithmic value), and R 2 =0.99. The biosensor separately detects ATP generated by salmonella lysis, the linear range is 10 3-107 CFU/mL, the linear equation is y= 1174.46x-3410.67 (x is salmonella concentration logarithmic value), and R 2 =0.98. When salmonella DNA and ATP are detected simultaneously, the linear range of detection is 10 2-107 CFU/mL, the linear equation is y= 1274.24x-2453.31 (x is salmonella concentration logarithmic value), R 2 =0.98, and the detection limit is 20 CFU/mL. Compared with the method for separately detecting DNA and ATP, the method has higher detection sensitivity for simultaneously detecting DNA and ATP, which indicates that DNA and ATP can cooperatively trigger PfAgo enzyme to cut and realize fluorescence signal enhancement.
Example 2: verification of the ability of phage to lyse live and dead salmonella
The salmonella bacteria liquid cultured to the logarithmic phase (10 8 CFU/mL) is inactivated by an autoclave, 20 uL of 10 8 CFU/mL phage solution is added, inoculated in a 5mL TSB culture medium, cultured for 12 hours at 37 ℃, centrifuged for 15min at 8000 g at 4 ℃, and the supernatant is filtered by a 0.22 um filter membrane to obtain the bacteria liquid. Pouring a small amount of TSA culture medium into the lower layer of the plate, mixing the filtered bacterial liquid 200uL with 3.8 mL semi-solid culture medium, pouring the mixture into the plate, adding 10 uL phage after solidification, culturing by a double-layer plate method, and observing the growth condition of plaques. The salmonella live bacteria liquid with the same concentration is cultured by the double-layer flat plate method. As shown in FIG. 3, phage can only produce plaques on solid media containing live Salmonella bacteria, indicating that phage can specifically lyse live Salmonella bacteria, but not dead Salmonella bacteria.
Example 3: pfAgo enzyme cleavage Activity verification in the method of the present invention
We have first devised a fluorescent probe 3 comprising 59 bases, the fluorescent probe 3 covering the base sequence of the fluorescent probe 1 described in the present invention. The sequence of fluorescent probe 3 is: as shown in SEQ ID NO.9, FAM-TACGTTACGTTACGTTACGTTACGTTACGGTTCCTTTGACGGTGCGATGTTACGTTACG-BHQ1, the underlined part was identical to the single-stranded oligonucleotide sequence in fluorescent probe 1. As shown in FIG. 4A, the cleavage on the complementary fluorescent probe 3 corresponding to positions 10 and 11 of gDNA-4, the fifth lane produced two bands containing 35 and 24 bases, respectively, indicating that PfAgo enzyme can cleave at specific positions. As shown in FIG. 4A, fluorescent probe 3 can be cleaved only if PfAgo and gDNA-4 are simultaneously present and involved in the reaction. Furthermore, we used the method of the invention to detect fluorescence changes in the presence of different concentrations of Salmonella DNA alone. As shown in FIG. 4B, as the DNA concentration increases, the fluorescence intensity gradually increases, indicating that PfAgo enzyme is capable of cleaving Salmonella DNA.
Example 4: ATP triggering PfAgo enzyme cleavage Activity verification in the method of the present invention
We first constructed a bivalent aptamer as shown in FIG. 5A, the first lane being gDNA-5 containing 21 bases, the sequence of gDNA-5 being: 5'-TCCGCAATTTTTTTCCGCAAT-3' as shown in SEQ ID NO. 5. The second lane is an ATP aptamer comprising 27 bases, the sequence of the ATP aptamer being: 5'-ACCTGGGGGAGTATTGCGGAGGAAGGT-3' as shown in SEQ ID NO. 6. The bivalent aptamer is formed by hybridization of two ATP aptamer chains with a gDNA-5 single strand. The third lane contains 75 bases, indicating that the bivalent aptamer construction was successful. To verify that the presence of ATP, the bivalent aptamer was triggered to release gDNA-5 and activate PfAgo enzyme to cleave fluorescent probe 2, we incubated 95 uL ATP solutions at different concentrations (10 1-106 nM) with 5 uL 500 ug/mL MNP-BAPT for 40 min at 37℃and the blank with ultrapure water. After magnetic separation, 88 uL supernatant was taken, 10 uL of PfAgo enzyme (500 ug/mL), 2 uL of 5 uM fluorescent probe 2 was added, the total volume of the reaction solution was 100 uL, and the reaction was carried out at 95℃for 30 min. 20 uL reaction solution was applied to 384 well plates and fluorescent signals were read at 492 nm using a multifunctional microplate reader. Each sample was measured 3 times independently and the average of 3 fluorescence intensities was taken. The linear relationship between the ATP concentration and the fluorescence intensity value is obtained by taking the ATP concentration as an abscissa and taking the fluorescence intensity value as an ordinate. As shown in FIG. 5B, as the ATP concentration increases, the fluorescence intensity value increases. It was shown that the constructed bivalent aptamer released gDNA-5 and stimulated PfAgo enzyme cleavage activity in the presence of ATP.
Example 5: the influence of MNP-BAPT concentration on the detection performance of quantitative living bacteria by the method of the invention is explored
S1: design gDNA: in this example, the gDNA design and the fluorescent probe design were the same as those in example 1.
S2: preparation of magnetic nanoparticle-phage complexes: in this example MNP-phase was prepared as in example 1.
S3: preparation of magnetic nanoparticle-divalent aptamer complexes: MNP-BAPT in this example was prepared as in example 1.
S4: pfAgo enzyme cleavage: in this example PfAgo enzyme was purchased from Hylegen company and the 10 Xreaction buffer used was prepared by: contains 15 mM Tris-HCl (pH 8.0), 250 mM NaCl and 0.5. 0.5 mM MnCl 2, and the pH of the reaction buffer is adjusted to 7.4. 100 uL 200 ug/mL MNP-phase was added to a centrifuge tube containing 100 uL different concentrations of Salmonella culture, and incubated 15min at 37 ℃. The end of incubation was washed 3 times with 200 uL PBST buffer. After magnetic separation, the supernatant was removed, 95 uL ultrapure water and 5 uL MNP-BAPT solutions of different concentrations (200 ug/mL, 500ug/mL, 800 ug/mL) were added, 40: 40 min was incubated at 37 ℃, incubation was completed, and the supernatant was collected by magnetic separation. 70 uL supernatant, 10uL of PfAgo enzyme (500 ug/mL) and 2 uL of 5 uM gDNA-1, gDNA-2, gDNA-3, 10uL of 10 Xreaction buffer solution, 2 uL of 5 uM fluorescent probe 1 and 2 were added into the centrifuge tube in sequence, the total volume of the reaction solution was 100 uL, and the reaction was carried out at 95℃for 30 min.
S5: fluorescence reading: the reaction solution obtained in step S4 of 20 uL was applied to 384-well plates, and fluorescent signals were read at 492: 492 nm with a multifunctional microplate reader. Each sample was measured 3 times independently and the average of 3 fluorescence intensities was taken. The quantitative relationship between the two is obtained by taking the salmonella concentration as an abscissa and taking the fluorescence intensity value as an ordinate. As shown in FIG. 6, when the concentration of MNP-BAPT was 500 ug/mL, the fluorescence intensity value was always higher in the range of salmonella concentration of 10 2-107 CFU/mL than that of MNP-BAPT at 200 ug/mL. At a concentration of 800 ug/mL for MNP-BAPT, the fluorescence intensity value was always lower in the range of 10 4-107 CFU/mL for Salmonella than for 500 ug/mL for MNP-BAPT, which may be an aggregation of MNP at high concentrations, affecting the PfAgo enzyme shearing process, resulting in a decrease in fluorescence intensity. Therefore, the detection performance of the detection method is optimal when the concentration of MNP-BAPT is 500 ug/mL.
Example 6: the influence of PfAgo enzyme concentration on the detection performance of the quantitative living bacteria by the method is explored
S1: design gDNA: in this example, the gDNA design and the fluorescent probe design were the same as those in example 1.
S2: preparation of magnetic nanoparticle-phage complexes: in this example MNP-phase was prepared as in example 1.
S3: preparation of magnetic nanoparticle-divalent aptamer complexes: MNP-BAPT in this example was prepared as in example 1.
S4: pfAgo enzyme cleavage: in this example PfAgo enzyme was purchased from Hylegen company and the 10 Xreaction buffer used was prepared by: contains 15 mM Tris-HCl (pH 8.0), 250 mM NaCl and 0.5. 0.5mM MnCl 2, and the pH of the reaction buffer is adjusted to 7.4. 100 uL of 500 ug/mL MNP-phase was added to a centrifuge tube containing 100 uL different concentrations of Salmonella culture, and incubated at 37℃for 15 min. The end of incubation was washed 3 times with 200 uL PBST buffer. After magnetic separation, the supernatant was removed, 95 uL ultrapure water and 5 uL of 500 ug/mL MNP-BAPT solution were added, incubated at 37℃for 40min, and after the incubation, the supernatant was collected by magnetic separation. 70 uL supernatant, 10 uL PfAgo enzymes (200 ug/mL, 500 ug/mL, 800 ug/mL), 2 uL 5 uM gDNA-1, gDNA-2, gDNA-3, 10 uL 10 Xreaction buffer, 2 uL 5 uM fluorescent probe 1 and fluorescent probe 2 were added sequentially into the centrifuge tube, the total volume of the reaction solution was 100 uL, and 30min was reacted at 95 ℃.
S5: fluorescence reading: the reaction solution obtained in step S4 of 20 uL was applied to 384-well plates, and fluorescent signals were read at 492: 492 nm with a multifunctional microplate reader. Each sample was measured 3 times independently and the average of 3 fluorescence intensities was taken. The quantitative relationship between the two is obtained by taking the salmonella concentration as an abscissa and taking the fluorescence intensity value as an ordinate. As shown in FIG. 7, fluorescence intensity was low at a concentration of Salmonella in the range of 10 2-107 CFU/mL and at a concentration of PfAgo enzyme of 200 ug/mL. When PfAgo enzyme concentration is 800 ug/mL, the fluorescence intensity value and PfAgo enzyme concentration is 500 ug/mL, and it is estimated that gDNA assembly PfAgo enzyme in the reaction system is saturated at this time, so that the detection performance of the detection method is optimal when PfAgo enzyme concentration is 500 ug/mL.
Example 7: the influence of gDNA concentration on the detection performance of the quantitative living bacteria by the method of the invention is explored
S1: design gDNA: in this example, the gDNA design and the fluorescent probe design were the same as those in example 1.
S2: preparation of magnetic nanoparticle-phage complexes: in this example MNP-phase was prepared as in example 1.
S3: preparation of magnetic nanoparticle-divalent aptamer complexes: MNP-BAPT in this example was prepared as in example 1.
S4: pfAgo enzyme cleavage: in this example PfAgo enzyme was purchased from Hylegen company and the 10 Xreaction buffer used was prepared by: contains 15 mM Tris-HCl (pH 8.0), 250 mM NaCl and 0.5. 0.5 mM MnCl 2, and the pH of the reaction buffer is adjusted to 7.4. 100 uL of 500ug/mL MNP-phase was added to a centrifuge tube containing 100 uL different concentrations of Salmonella culture, and incubated at 37℃for 15 min. The end of incubation was washed 3 times with 200 uL PBST buffer. After magnetic separation, the supernatant was removed, 95 uL ultrapure water and 5 uL of 500ug/mL MNP-BAPT solution were added, incubated at 37℃for 40min, and after the incubation, the supernatant was collected by magnetic separation. 70 uL supernatant, 10 uL of PfAgo enzyme (500 ug/mL) and 2 uL g DNA-1, 2g DNA-2, 3g DNA-10 uL 10 Xreaction buffer (2 uM, 5 uM, 10 uM) were added into the centrifuge tube in sequence, the total volume of the reaction solution was 100 uL, and the reaction was carried out at 95℃for 30 min.
S5: fluorescence reading: the reaction solution obtained in step S4 of 20 uL was applied to 384-well plates, and fluorescent signals were read at 492: 492 nm with a multifunctional microplate reader. Each sample was measured 3 times independently and the average of 3 fluorescence intensities was taken. The quantitative relationship between the two is obtained by taking the salmonella concentration as an abscissa and taking the fluorescence intensity value as an ordinate. As shown in FIG. 8, the fluorescence intensity was lower at a salmonella concentration in the range of 10 2-107 CFU/mL and a gDNA concentration of 2 uM. When the gDNA concentration was 10 uM, the fluorescence intensity value was not significantly changed from that when the gDNA concentration was 5 uM, and it was presumed that the gDNA and PfAgo assembly in the reaction system was saturated at this time, so that the gDNA concentration was 5 uM, and the detection performance of the detection method was optimal.
Example 8: the influence of PfAgo enzyme shearing reaction time on the detection performance of the quantitative living bacteria by the method is explored
S1: design gDNA: in this example, the gDNA design and the fluorescent probe design were the same as those in example 1.
S2: preparation of magnetic nanoparticle-phage complexes: in this example MNP-phase was prepared as in example 1.
S3: preparation of magnetic nanoparticle-divalent aptamer complexes: MNP-BAPT in this example was prepared as in example 1.
S4: pfAgo enzyme cleavage: in this example PfAgo enzyme was purchased from Hylegen company and the 10 Xreaction buffer used was prepared by: contains 15 mM Tris-HCl (pH 8.0), 250 mM NaCl and 0.5. 0.5 mM MnCl 2, and the pH of the reaction buffer is adjusted to 7.4. 100 uL of 500ug/mL MNP-phase was added to a centrifuge tube containing 100 uL different concentrations of Salmonella culture, and incubated at 37℃for 15 min. The end of incubation was washed 3 times with 200 uL PBST buffer. After magnetic separation, the supernatant was removed, 95 uL ultrapure water and 5 uL of 500ug/mL MNP-BAPT solution were added, incubated at 37℃for 40min, and after the incubation, the supernatant was collected by magnetic separation. 70 uL supernatant, 10 uL of PfAgo enzyme 500ug/mL, 2 uL of 5 uM gDNA-1, gDNA-2, gDNA-3, 10 uL of 10 Xreaction buffer, 2 uL of 5 uM fluorescent probe 1 and 2 fluorescent probe 2 were added sequentially into the centrifuge tube, the total volume of the reaction solution was 100 uL, and the reaction times were different at 95 ℃ (10 min/30 min/60 min).
S5: fluorescence reading: the reaction solution obtained in step S4 of 20 uL was applied to 384-well plates, and fluorescent signals were read at 492: 492 nm with a multifunctional microplate reader. Each sample was measured 3 times independently and the average of 3 fluorescence intensities was taken. The quantitative relationship between the two is obtained by taking the salmonella concentration as an abscissa and taking the fluorescence intensity value as an ordinate. As shown in FIG. 9, fluorescence intensity was high at a salmonella concentration in the range of 10 2-104 CFU/mL and a shear reaction time of 30 min. When the salmonella concentration is in the range of 10 4-107 CFU/mL and the shearing reaction time is 10min, the fluorescence intensity is lower, and meanwhile, compared with shearing for 60min, the fluorescence intensity is not changed remarkably after shearing for 30 min. Therefore, when the PfAgo shear reaction time is determined to be 30min, the detection performance of the detection method is optimal.
Example 9: sensitivity verification for quantitatively detecting salmonella by using the method of the invention
In the embodiment, the real-time fluorescence PCR primer and the probe are synthesized by referring to the 'national institute of Electrical and quarantine industry Standard-SN/T1870-2016'. The forward primer sequence is: 5'-GCGGCGTTGGAGAGTGATA-3', the reverse primer sequence is: 5'-AGCAATGGAAAAAGCAGGATG-3', the probe is: FAM-CATTTCTTAAACGGCGGTGTCTTTCCCT-TAMRA. The real-time fluorescence PCR reaction system is as follows: 2.5uL 10 XPCR buffer, 1uL forward primer (10 uM), 1uL reverse primer (10 uM), 1uL probe (10 uM), 1uL dNTP,0.5uLTaqDNA polymerase, 2uL template DNA, sterile ultra pure water make up to 25uL. The reaction system is evenly mixed and then transferred to a qPCR instrument for amplification. The amplification procedure was: pre-denaturation at 95℃for 3 min, denaturation at 94℃for 5s, annealing at 60℃for 40 s, FAM fluorescence was collected, 40 cycles were performed, and the reaction product was stored at 4 ℃. The linear range of salmonella detected by using the real-time fluorescence PCR is 10 2-107 CFU/mL, the linear equation is y= -3.45x+42.95 (x is the salmonella concentration logarithmic value), as shown in figure 10, the result of the real-time fluorescence PCR is similar to the method of the invention, and the linear range is consistent with the method of the invention, which shows that the method of the invention has good sensitivity.
Example 10: the method of the invention quantifies the specificity test of salmonella live bacteria
S1: design gDNA: in this example, the gDNA design and the fluorescent probe design were the same as those in example 1.
S2: preparation of magnetic nanoparticle-phage complexes: in this example MNP-phase was prepared as in example 1.
S3: preparation of magnetic nanoparticle-divalent aptamer complexes: MNP-BAPT in this example was prepared as in example 1.
S4: pfAgo enzyme cleavage: in this example PfAgo enzyme was purchased from Hylegen company and the 10 Xreaction buffer used was prepared by: the reaction buffer was adjusted to pH 7.4 containing 15mM Tris-HCl (pH 8.0), 250 mM NaCl, and 0.5 mM MnCl2. 100uL 500ug/mL MNP-phase was added to a centrifuge tube containing 100uL 10 6 CFU/mL of culture medium of different strains (Salmonella, E.coli, staphylococcus aureus, listeria monocytogenes, vibrio parahaemolyticus) in each tube, and incubated 15 min at 37 ℃. The end of incubation was washed 3 times with 200 uL PBST buffer. After magnetic separation, the supernatant was removed, 95 uL ultrapure water and 5 uL of 500ug/mL MNP-BAPT solution were added, incubated at 37℃for 40min, and after the incubation, the supernatant was collected by magnetic separation. 70 uL supernatant, 10 uL of PfAgo enzyme (500 ug/mL), 2 uL of 5 uM gDNA-1, gDNA-2, gDNA-3, 10 uL of 10 Xreaction buffer, 2 uL of 5 uM fluorescent probe 1 and 2 were added into the centrifuge tube in sequence, the total volume of the reaction solution was 100uL, and the reaction was carried out at 95℃for 30 min.
S5: fluorescence reading: the reaction solution obtained in step S4 of 20 uL was applied to 384-well plates, and fluorescent signals were read at 492: 492 nm with a multifunctional microplate reader. Each sample was measured 3 times independently and the average of 3 fluorescence intensities was taken. The quantitative relationship between the two is obtained by taking the concentration of each test strain as an abscissa and the fluorescence intensity value as an ordinate. As shown in FIG. 11A, the change in fluorescence intensity generated when the test strain is Salmonella is significantly greater than that of other strains, indicating that the method has good specificity for quantitatively detecting viable bacteria in the sample matrix. As shown in FIG. 11B, after different test strains are mixed with salmonella, the fluorescence intensity changes are not significantly different, and the good specificity and the good applicability of the method for salmonella detection are further shown.
The technical solution of the present invention is explained by the above embodiments, but the present invention is not limited to the above embodiments, i.e. it does not mean that the present invention must be implemented depending on the above specific embodiments. Any modifications, or equivalent substitutions of materials for the invention, which are made by those skilled in the art based on the present invention, fall within the scope of protection of the patent.
Claims (10)
1. A live bacteria detection kit, comprising:
The PfAgo enzyme is guided to target the gDNA of bacteria, specifically gDNA-1 shown as a sequence SEQ ID NO.1, gDNA-2 shown as a sequence SEQ ID NO.2 and gDNA-3 shown as a sequence SEQ ID NO. 3;
Guiding PfAgo enzyme to cut gDNA of the fluorescent probe 1, wherein the gDNA is partial DNA fragments cut by bacteria, specifically gDNA-4 shown as sequence SEQ ID NO.4, of gDNA-1, gDNA-2 and gDNA-3 guiding PfAgo enzyme;
Guiding PfAgo enzyme to cut gDNA of the fluorescent probe 2, wherein the gDNA is an oligonucleotide sequence hybridized with an ATP aptamer to form a bivalent aptamer, and specifically gDNA-5 shown as a sequence SEQ ID NO. 5;
the method comprises the steps of constructing a bivalent aptamer for ATP, wherein the bivalent aptamer is obtained by hybridization of two ATP aptamer sequences and one oligonucleotide sequence, specifically an ATP aptamer shown as a sequence SEQ ID NO.6 and specifically an oligonucleotide sequence shown as a sequence SEQ ID NO. 5;
The fluorescent probe with the fluorescent group and the quenching group is specifically a fluorescent probe 1 shown as SEQ ID NO.7 and a fluorescent probe 2 shown as SEQ ID NO. 8.
2. The live bacteria detection kit of claim 1, wherein the bacteria is salmonella typhimurium ATCC14028.
3. The quantitative detection method of the viable bacteria is characterized by comprising the following steps of:
s1: design gDNA:
s2: preparation of magnetic nanoparticle-phage complexes: washing the carboxyl magnetic nano particles with the size of 1000nm by using MEST buffer solution; removing the supernatant by magnetic separation, re-suspending by using MES buffer solution, adding 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, and placing on a rotary mixer for activation; washing the activated cells with MEST buffer solution, removing the supernatant by magnetic separation, and adding phage solution for incubation; at the end of incubation, the supernatant was removed by magnetic separation and blocked by addition of PBS (1% BSA) buffer; washing the closed end with PBST buffer solution, removing the supernatant by magnetic separation, and adding PBS buffer solution for resuspension to obtain a magnetic nanoparticle-Phage complex, namely MNP-Phage;
S3: preparation of magnetic nanoparticle-divalent aptamer complexes:
S3.1: washing the 150 nm-sized carboxyl magnetic nanoparticles with MEST buffer; removing the supernatant by magnetic separation, re-suspending by using MES buffer solution, adding 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, and placing on a rotary mixer for activation; washing the activated cells with PBST buffer, removing the supernatant by magnetic separation, re-suspending the cells with PBS buffer, and adding streptavidin for incubation; after the incubation is finished, removing the supernatant by magnetic separation, and adding PBS buffer solution for sealing; washing the end of the sealing by using PBST buffer solution, removing the supernatant by magnetic separation, and adding ultrapure water for resuspension to obtain streptavidin-modified magnetic nano particles, namely MNP-SA;
S3.2: adding the biotinylated ATP aptamer, gDNA-5, a binding buffer solution and ultrapure water into a centrifuge tube, heating, immediately performing ice bath, and placing on a rotary mixer for hybridization; adding MNP-SA solution for incubation after hybridization; washing with PBST buffer solution after incubation, removing supernatant by magnetic separation, and adding ultrapure water for resuspension to obtain a magnetic nanoparticle-bivalent aptamer complex, namely MNP-BAPT;
S4: pfAgo enzyme cleavage: incubating the MNP-phase solution with the bacterial liquid; washing with PBST buffer solution after incubation, removing supernatant by magnetic separation, and adding ultrapure water and MNP-BAPT solution for incubation; after the incubation is finished, collecting supernatant by magnetic separation, sequentially adding PfAgo enzymes, gDNA-1, gDNA-2, gDNA-3, a reaction buffer, a fluorescent probe 1 and a fluorescent probe 2, and reacting;
S5: fluorescence reading;
and (5) finishing quantitative detection of viable bacteria.
4. The quantitative detection method of viable bacteria according to claim 3, wherein in the step S1, three 15-22 nt gDNA complementary thereto are respectively designed according to the bacterial nucleic acid sequence to be detected, and are respectively named gDNA-1, gDNA-2 and gDNA-3; under the guidance of gDNA-1, gDNA-2 and gDNA-3, activating PfAgo enzyme to cut, and then releasing a 15-22 nt new DNA fragment serving as secondary gDNA, which is named gDNA-4; an oligonucleotide sequence 15-22 nt which hybridizes to the ATP aptamer to form a bivalent aptamer designated gDNA-5; wherein, the base at the 5' end of the gDNA is modified by a phosphate group; the gDNA-4 is complementary to the sequence of the 16-25 nt fluorescent probe 1; the gDNA-5 is complementary to the sequence of the 16-25 nt fluorescent probe 2.
5. The method according to claim 3, wherein in the step S2, the carboxyl-modified 1000 nm-sized magnetic nanoparticles are washed with MEST buffer; after magnetic separation, re-suspending with 500-1000 uL MES buffer, adding 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, placing on a rotary mixer, rotating and mixing at 300-500 rpm/min, and activating at 25-37 ℃ for 15-30 min; washing with MEST buffer solution after activation, removing supernatant after magnetic separation, adding 50-200 uL phage solution, and incubating at 37deg.C for 1-3 h; after the incubation is finished, removing the supernatant after magnetic separation, adding 500-1000 uL of PBS buffer solution containing 1% bovine serum albumin, and incubating at 37 ℃ for 30-60 min; after magnetic separation, the supernatant is removed, and the magnetic nanoparticle-Phage complex, namely MNP-Phage, is obtained by washing with PBST buffer solution and re-suspending with 500-1000 uL PBS buffer solution.
6. The quantitative detection method of viable bacteria according to claim 3 or 5, wherein in the step S2, 5mg/mL of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride 10-30 uL and 5mg/mL of N-hydroxysuccinimide 10-30 uL are added;
In the step S2, the concentration of the phage solution is 10 6-109 PFU/mL;
In the step S2, the concentration of the magnetic nanoparticle-Phage complex, namely MNP-Phage, is 0.5-2 mg/mL.
7. The method for quantitative detection of viable bacteria according to claim 3, wherein the step S3 specifically comprises:
Step S3.1: washing the carboxyl-modified 150 nm-sized magnetic nanoparticles with MEST buffer; after magnetic separation, re-suspending by using MES buffer solution, adding 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, placing on a rotary mixer, rotating and uniformly mixing at 300-500 rpm/min, and activating at 25-37 ℃ for 15-30 min; washing with PBST buffer solution after activation, removing supernatant by magnetic separation, adding streptavidin, supplementing to 500-1000 uL with PBS buffer solution, and incubating; after the incubation is finished, removing the supernatant after magnetic separation, adding 500-1000 uL of PBS buffer solution containing 1% bovine serum albumin, and incubating at 37 ℃ for 30-60 min; removing the supernatant after magnetic separation, washing with PBST buffer solution, and re-suspending with 500-1000 uL ultrapure water to obtain streptavidin-modified magnetic nanoparticles (MNP-SA), and preserving at 4 ℃ for later use;
Step S3.2: adding biotinylated ATP aptamer, gDNA-5, binding buffer and ultrapure water into a centrifuge tube, heating at 95-98deg.C for 5-8 min, immediately ice-bathing for 5-8 min, placing on a rotary mixer, mixing at 300-500 rpm/min, and hybridizing at 37deg.C for 1-1.5 h; adding 500-1000 uL of MNP-SA solution obtained in the step S3.1, and incubating at 37 ℃ for 0.5-1 h; after magnetic separation, the supernatant is removed, PBST buffer solution is used for washing, 500-1000 uL ultrapure water is added for resuspension, and the magnetic nanoparticle-bivalent aptamer complex, namely MNP-BAPT, is obtained.
8. The quantitative detection method of viable bacteria according to claim 3 or 7, wherein 5 mg/mL of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride 10-30 uL and 5 mg/mL of N-hydroxysuccinimide 10-30 uL are added to the step S3.1;
in the step S3.1, 10 mg/mL of streptavidin 5-10 uL is added for incubation;
In the step S3.1, the concentration of the streptavidin modified magnetic nano particles, namely MNP-SA, is 0.5-2 mg/mL;
in the step S3.2, the volume ratio of the biotinylated ATP aptamer, gDNA-5, the binding buffer and the ultrapure water is 5:5:3:17, uniformly mixing, adding into a centrifuge tube, and immediately carrying out ice bath after heating;
in the step S3, the concentration of the magnetic nanoparticle-bivalent aptamer complex, namely MNP-BAPT, is 0.5-2 mg/mL.
9. The quantitative detection method of viable bacteria according to claim 3, wherein in the step S4, the MNP-phase solution and the bacterial liquid are mixed uniformly in a volume ratio of 1:1 for incubation;
In the step S4, the incubation is finished, the incubation is washed by PBST buffer solution, the supernatant is removed by magnetic separation, and the volume ratio of the ultrapure water to the MNP-BAPT solution is 19:1, adding and incubating;
in the step S4, the concentration of the MNP-BAPT solution is 200-800ug/mL;
in the step S4, the concentration of PfAgo enzyme in the reaction system is 200-800ug/mL;
In the step S4, the concentrations of gDNA-1, gDNA-2 and gDNA-3 in the reaction system are 2 uM-10 uM;
In the step S4, the shearing reaction time is 10-60 min;
In the step S4, the incubation is finished, the supernatant is collected by magnetic separation, and the volume ratio of the supernatant to the supernatant is 35:5:1:1:1:5:1:1 adding supernatant, pfAgo enzyme, gDNA-1, gDNA-2, gDNA-3, reaction buffer, fluorescent probe 1 and fluorescent probe 2;
And step S5, adding the reaction solution obtained in the step S4 of 20-100 uL to a 384-well plate, reading fluorescent signals by using a multifunctional enzyme-labeled instrument, independently measuring 3 times for each sample, and taking the average value of the 3 times of fluorescent intensity.
10. The kit for detecting live bacteria according to any one of claims 1 to 2 or the method for quantitatively detecting live bacteria according to any one of claims 3 to 9 for quantitatively detecting food-borne pathogenic bacteria.
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