CN113943781A - Rapid absolute quantification method for pathogenic microorganisms in large-volume liquid sample - Google Patents
Rapid absolute quantification method for pathogenic microorganisms in large-volume liquid sample Download PDFInfo
- Publication number
- CN113943781A CN113943781A CN202111208631.9A CN202111208631A CN113943781A CN 113943781 A CN113943781 A CN 113943781A CN 202111208631 A CN202111208631 A CN 202111208631A CN 113943781 A CN113943781 A CN 113943781A
- Authority
- CN
- China
- Prior art keywords
- reaction system
- liquid sample
- membrane
- microorganisms
- nucleic acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a rapid absolute quantification method of pathogenic microorganisms in a large-volume liquid sample, belonging to the technical field of microorganism detection. The method comprises the following steps: (1) filtering a liquid sample to be detected by using a nano-aperture membrane to enable target microorganisms to be intercepted on the surface of the filter membrane; (2) attaching a sealed small chamber to the surface of the filter membrane for intercepting the target microorganism, adding a loop-mediated isothermal amplification reaction system, and sealing; the reaction system contains hydrogel monomers, and the target microorganisms on the surface of the filter membrane are covered by the reaction system in a colloidal state through crosslinking gelling; (3) loop-mediated isothermal nucleic acid amplification reaction; (4) and analyzing and counting the fluorescence points by using a fluorescence imaging technology, and calculating the absolute concentration of the target microorganism. The invention utilizes a track etching membrane interception and hydrogel reaction system to carry out double nano-confinement on target microorganisms, so that bacteria and viruses are amplified at initial positions, amplification products cannot diffuse, amplification fluorescent spots with distinct shapes are formed, and the quantitative positioning detection of the microorganisms is realized.
Description
Technical Field
The invention relates to the technical field of microorganism detection, in particular to a method for rapidly detecting microorganisms in a large-volume liquid sample based on double nanometer confinement.
Background
The traditional pathogenic bacteria detection method mainly realizes quantitative detection by bacterial culture, and has long time consumption and low specificity. With the development of molecular biology analysis technology, the nucleic acid analysis technology based on genome represented by PCR technology and loop-mediated isothermal nucleic acid amplification technology is the most widely applied method for analyzing pathogenic microorganisms at present, and the biochemical information of pathogenic microorganisms is obtained by detecting and analyzing specific DNA or RNA fragments in the microorganisms.
In recent years, with the continuous development of molecular biology technology and microfluidic technology, digital PCR technology has come into play. The digital PCR technology is used as a breakthrough technology for quantitative analysis, and can absolutely quantify the number of nucleic acids in a sample. It promotes the PCR technology to a new height, and makes the quantitative analysis step enter a new stage.
In the existing digital PCR technology, a micro-droplet wrapping a target gene is usually generated by designing an instrument or a micro-chamber is formed by designing a micro-fluidic chip to separate the target gene, so that the target gene amplification detection is carried out, and the absolute quantitative detection is realized.
Similarly, the digital loop-mediated isothermal amplification technology is rapidly developed on the basis of the traditional loop-mediated isothermal amplification technology. In contrast, the method can be carried out under a constant temperature condition, has higher specificity and sensitivity, is expected to reduce the cost required by the detection technology, and greatly shortens the detection time.
Various digital nucleic acid amplification devices have been introduced on the market, such as Integrated Fluidic Circuit (IFC) chip-based devices, spin-centrifuge disk-based devices, slide-chip-based devices, and droplet-based devices. These digital nucleic acid amplification devices are expensive to design and often cannot be adapted and adapted to the field or substrate detection environment.
However, the detection limit of the digital nucleic acid amplification technology reported at present is high because it can only detect ultramicro samples (such as 2 microliters). In order to reduce the detection limit and avoid false positives, researchers usually need to perform a series of tedious pre-treatments such as centrifugation, filtration, impurity washing, bacterial elution, DNA extraction, purification, etc. on a large volume of samples, and finally perform digital amplification detection on the nucleic acid samples after purification and elution. This process is not only loaded down with trivial details, consuming time, needs the professional moreover in special laboratory, utilizes the maximization instrument to go on, can't be used for quick detection on the spot.
Therefore, how to realize rapid absolute quantification of target microorganisms in a large volume of liquid sample and simplify sample pretreatment is a problem to be solved by those skilled in the art.
Disclosure of Invention
The invention aims to provide a method for directly and rapidly detecting pathogenic microorganisms in a large-volume liquid sample, such as bacteria and virus microorganisms in the liquid sample of fruit juice, beer, milk tea, environmental water sources and the like, to overcome the defects in the prior art and realize absolute quantitative analysis of the microorganisms in the large-volume liquid sample.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a rapid absolute quantification method of pathogenic microorganisms in a large-volume liquid sample, which comprises the following steps:
(1) filtering a liquid sample to be detected by using a nano-aperture membrane to enable target microorganisms to be intercepted on the surface of the filter membrane, and then drying the filter membrane;
(2) attaching a sealed small chamber to the surface of the filter membrane for intercepting the target microorganism, adding a loop-mediated isothermal nucleic acid amplification reaction system containing a hydrogel monomer into the sealed small chamber, and sealing; crosslinking hydrogel monomers into gel to enable a reaction system to cover the target microorganisms on the surface of the filter membrane in a colloidal state, wherein the reaction system contains a primer designed and matched with the DNA or RNA of the target microorganisms;
(3) putting the whole amplification reaction system under a constant temperature condition to carry out nucleic acid isothermal amplification reaction;
(4) and after the reaction is finished, analyzing a fluorescence signal appearing in the amplification reaction system by utilizing a fluorescence imaging technology, counting fluorescence points, and calculating to obtain the absolute quantitative concentration of the target microorganism in the liquid sample to be detected.
The liquid sample to be detected can be a liquid real sample or a liquid sample after homogenizing and homogenizing.
The pathogenic microorganism can be bacteria or viruses, and specifically, the bacteria can be escherichia coli, salmonella or listeria, and can also be other strains. The virus can be MS2 virus, norovirus, African swine fever virus, new coronavirus, etc., and can also be other viruses.
When the nucleic acid substance of the target microorganism is RNA, reverse transcriptase is added to the reaction system to reverse-transcribe the RNA into cDNA, and LAMP amplification is performed.
The invention integrates a filtering membrane with a nano-aperture with a hydrogel reaction system to realize the double nano-confinement of LAMP amplification of target microorganisms, firstly, the initial position of the microorganisms is limited by nano-apertures on the filtering membrane by utilizing filtering of the filtering membrane, then the hydrogel reaction system is formed on the surface of the filtering membrane which intercepts bacteria or viruses, the microorganisms are subjected to in-situ separation by utilizing the three-dimensional network structure of the hydrogel as a natural physically separated micro-chamber, so that the microorganisms start to be amplified in situ at the initial position, amplified products of the microorganisms cannot be diffused out, and the final result is that a single microorganism forms an amplification fluorescent point with a distinct shape, thereby realizing absolute quantitative detection.
In the step (1), the target microorganisms in the liquid sample are intercepted by the nano-aperture membrane, so that on one hand, the dispersed microorganisms in the large-volume liquid sample are enriched, and on the other hand, pathogenic bacteria are positioned on the surface of the nano-aperture membrane for the next in-situ nucleic acid amplification reaction.
The nano-aperture of the nano-aperture membrane should be very uniform and smaller than the size of bacteria and viruses. Preferably, the nano-aperture membrane is a track etching membrane, and the nano-aperture of the nano-aperture membrane is 50-500 nm. The track etching membrane is a microporous filter membrane prepared by a track etching method, the pore diameter distribution is uniform and the quantity is tight, in the filtering process, the solution uniformly passes through the filter membrane at a constant speed, and the proper pore diameter ensures that only one pathogenic microorganism is intercepted in one filter pore without overlapping.
More preferably, when the target microorganism is a bacterium, the nano-pore size of the track-etched membrane is 400 nm; when the target microorganism is a virus, the nanopore size of the track-etched membrane is 100 nm.
The thickness of the nano-aperture membrane is 1-50 μm.
Further, the liquid sample is filtered using a filter. Specifically, the method comprises the following steps: firstly, the nano-aperture membrane is arranged on a filter head of a filter, a sample is extracted by a filter injector, and the sample is injected into the filter at a constant speed so as to be uniformly distributed on nano-apertures of the filter membrane. And then taking down the filtering injector, sucking and blowing the filter membrane until blow-drying, taking out the filter membrane, attaching the filter membrane to a glass slide, and attaching the filter membrane to the glass slide by using the small sealing chamber.
Furthermore, the invention compares the influence of filtering membranes made of different materials on the LAMP amplification result, and the target microorganism is positioned on the filtering membrane to directly perform LAMP amplification detection in the method, so that the material of the membrane can influence the amplification result. For example, the strong fluorescence of the film itself can affect the brightness and dark ratio of the display result; for example, poor membrane biocompatibility can lead to its inability to serve as a substrate for solid phase LAMP amplification.
Preferably, the material of the nano-pore membrane is polycarbonate or polyethylene terephthalate. Research shows that when LAMP amplification of target microorganisms is performed on a polycarbonate film or polyethylene terephthalate, amplification products show bright and concentrated spots under irradiation of exciting light, and each amplification spot is clearly visible.
In the step (2), a hydrogel system containing lysozyme or reverse transcriptase is directly added into the sealed small chamber, so that the whole amplification system covers the target microorganisms on the surface of the filter membrane, hydrogel monomers in the reaction system are crosslinked into gel at room temperature, and the three-dimensional network structure of the hydrogel plays a role in limiting the microorganisms. The microorganisms are lysed at the initial site and a nucleic acid in situ amplification reaction occurs.
In the invention, the loop-mediated isothermal nucleic acid amplification reaction system containing lysozyme or reverse transcriptase is prepared and then covered on the target microorganism of the filter membrane, so that the target microorganism is prevented from contacting with a cracking reagent and an amplification reaction reagent in advance to start reaction in the preparation process of the reaction system.
Further, when the pathogenic microorganism is a bacterium, the loop-mediated isothermal nucleic acid amplification reaction system contains lysozyme.
Preferably, the loop-mediated isothermal nucleic acid amplification reaction system comprises: 1 × Isothermal Amplification Buffer, 6mM total MgSO41.4mM dNTPs, 640U/mL Bst 2.0WarmStart polymerase, a primer mixture, a fluorescent dye and a hydrogel monomer; also comprises 0.1mg/mL lysozyme and 1.0mg/mL BSA;
1.0mg/mLBSA and 0.1mg/mL lysozyme are added into the system as cell lysate of the whole amplification system. The lysozyme in the system can crack the target bacteria trapped on the nano-membrane pores in situ without DNA extraction.
When the pathogenic microorganism is a virus, the loop-mediated isothermal nucleic acid amplification reaction system contains reverse transcriptase.
Preferably, the loop-mediated isothermal nucleic acid amplification reaction system comprises: 1 × Isothermal Amplification Buffer, 6mM total MgSO41.4mM dNTPs, 640U/mL Bst 2.0WarmStart polymerase, a primer mixture, a fluorescent dye and a hydrogel monomer; also included is 300U/mL WarmStart reverse transcriptase.
The primer mixture is an LAMP primer designed and matched with a target bacterial DNA or virus reverse transcription cDNA, and preferably, the primer mixture in a reaction system comprises: 1.6 μ M FIB and BIP, 0.2 μ M F3 and B3, 0.8 μ M LF and LB.
Preferably, the hydrogel monomer comprises four-arm-polyethylene glycol-acrylate and sulfydryl-polyethylene glycol-sulfydryl, the two monomers are dissolved in water at room temperature to form gel through crosslinking, and the gel forming time is only 2-4 min. Therefore, when the loop-mediated isothermal nucleic acid amplification reaction system is prepared, the hydrogel monomer is added last. The molar ratio of the two is 1: 1-4.
More preferably, the four-arm-polyethylene glycol-acrylate of molecular weight 10000 and the mercapto-polyethylene glycol-mercapto of molecular weight 3400 are mixed in a molar ratio of 1: 2.
Preferably, the fluorescent Dye is any one of SYBR Green Dye, Eva Green Dye and Lamp Dye.
Preferably, the sealed cells are sealed with a waterproof membrane. The whole system is arranged in a sealed chamber, so that the water in the hydrogel can be prevented from evaporating due to the temperature rise, and the accuracy of the detection result is improved.
In the step (3), the whole nano-confinement loop-mediated isothermal nucleic acid amplification system is placed in a sealed chamber for isothermal amplification.
Preferably, the conditions of the loop-mediated isothermal nucleic acid amplification reaction include: firstly, at 65-70 ℃, for 15-30 min; and secondly, heating at 75-80 ℃ for 3-7 min.
Researches show that after the loop-mediated isothermal amplification of the first step is carried out, the heating step of the second step can enhance the brightness ratio of fluorescence imaging after amplification, so that the visual effect of the detection result is more vivid.
More preferably, the conditions of the loop-mediated isothermal nucleic acid amplification reaction include: step one, at 65 ℃, 20 min; second, heat at 80 ℃ for 5 min.
In step (4), after amplification is completed, a laser emitter (such as a laser of a fluorescence microscope or an LED irradiation lamp) is used for irradiating the reaction system, so that the amplified clusters after reaction burst fluorescence, and a photo is taken and fluorescence points are counted by using counting software.
The amplification product will emit fluorescence under the irradiation of the exciting light, and each amplification point will emit a corresponding fluorescence light group, which can be clearly displayed under the fluorescence imaging technology. Experimental results show that the microorganisms filtered on the membrane can be subjected to in-situ cracking or reverse transcription to further perform amplification, the amplification product is still at the initial position, one microorganism only corresponds to one fluorescence point, the fluorescence points can be counted through counting software, and finally the concentration of the target pathogenic microorganism in the sample to be detected is obtained through conversion.
In the invention, the target microorganism is intercepted and paved on the surface of the filter membrane, namely the target microorganism is positioned in a two-dimensional plane of a hydrogel system to carry out LAMP amplification reaction, thereby facilitating the subsequent counting analysis of fluorescent points and improving the counting accuracy. Since a two-dimensional plane image is usually observed under a fluorescence microscope, if fluorescent dots are dispersed in a three-dimensional space of a reaction system, the upper and lower fluorescent dots are easily overlapped, and the focusing is not clear.
The final result of the method is judged as visual fluorescent spot analysis, the experimental result can be observed only by providing an excitation light source to excite fluorescence theoretically, and the method is expected to realize the rapid detection of the microorganisms in the areas with deficient resources and crude conditions. Preferably, the laser emitter is an LED flashlight.
Preferably, the counting software is Image J software.
The invention has the following beneficial effects:
(1) the invention provides a technical method for double-nano-confinement loop-mediated isothermal nucleic acid amplification, wherein a first heavy nano-confinement is to use a filter membrane etched with nano-pores to retain target microorganisms on the membrane, so that the initial positions of the microorganisms are limited by the nano-pores on the filter membrane; the second nanometer confinement is that the whole amplification system utilizes the three-dimensional network structure of the hydrogel as a natural physically separated micro-chamber, and the combination of the two steps of methods ensures that the microorganisms begin to amplify at the initial position and the amplified products cannot diffuse out, and the final result is the formation of amplification fluorescent spots with clear shapes, thereby realizing the separation of single microorganism and carrying out quantitative and localized detection on pathogenic microorganisms.
(2) The amplification method can complete the nano-confinement amplification analysis of the microorganism to be detected without a complex and expensive micro-current control equipment device, has extremely quick and simple operation steps, is simple and easy to operate, can detect a large-volume sample at one time, does not need to carry out DNA extraction or reverse transcription on target bacteria or viruses in advance, and greatly shortens the detection time.
(3) The method can be applied to the detection of various pathogenic bacteria and viruses, has no requirement on target microorganisms, can carry out quantitative and positioning detection on the target microorganisms only by designing proper pore size and matched primers, and provides an economic and efficient rapid detection means for the detection of the microorganisms in a large-volume liquid real sample or a pretreated liquid sample.
(4) The detection method provided by the invention can be applied to direct detection of liquid samples, can also be applied to rapid detection of liquid treatment liquid after pretreatment, has the advantages of high reaction speed and low detection cost, and can be well applied to rapid detection on site or in basic level.
Drawings
FIG. 1 is an electron microscope image of a polycarbonate nanoporous film.
FIG. 2 is a graph showing the results of experiments on films of different materials integrated with an amplification system, wherein (a) is a graph showing the results of experiments on a Polycarbonate (PC) film, (b) is a graph showing the results of experiments on a polyethylene terephthalate (PET) film, and (c) is a graph showing the results of experiments on a conventional commercial film.
FIG. 3 is a graph showing the results of the optimization comparison of whether or not 80 ℃ heating was performed, wherein (a) heating was performed at 80 ℃ and (b) heating was not performed at 80 ℃.
FIG. 4 is a graph comparing the results of experiments with different coatings on Polycarbonate (PC) films, wherein a, b, c are coated with black, gold, and gray coatings, respectively, on the PC film, and d is the PC film without coating.
FIG. 5 is a comparison graph of fluorescence imaging for detecting general Escherichia coli (a) in a sample by a loop-mediated isothermal nucleic acid amplification method in a solution and for rapidly detecting general Escherichia coli (b) in a sample by a double-nanotopography loop-mediated isothermal nucleic acid amplification method based on an etched filter membrane.
FIG. 6 is a comparison of the detection limits of the loop-mediated isothermal nucleic acid amplification system in hydrogel (a) and the double nanotopography-based loop-mediated isothermal nucleic acid amplification system based on etched filter membranes (b).
FIG. 7 is a fluorescence imaging contrast diagram of a double-nanotopography loop-mediated isothermal nucleic acid amplification method based on an etched filter membrane for rapidly detecting Listeria monocytogenes in a sample, wherein (a) is a detection result of the sample containing Listeria monocytogenes, and (b) is a negative control.
FIG. 8 is a comparison graph of fluorescence imaging for rapid detection of new coronavirus RNA in a sample by a dual-nano-confinement loop-mediated isothermal nucleic acid amplification method based on an etched filter membrane, wherein (a) is a detection result of new coronavirus RNA in the sample, and (b) is a negative control.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Those skilled in the art should understand that they can make modifications and equivalents without departing from the spirit and scope of the present invention, and all such modifications and equivalents are intended to be included within the scope of the present invention.
The following embodiment performs measurement by preparing a standard sample, and judges the accuracy of the detection method of the present invention based on the concentration of DNA obtained by the measurement and the actual concentration of DNA in the standard sample.
The primers used in the examples were all from Biotechnology engineering (Shanghai) Inc.; 10 × Isothermal Amplification Buffer, Bst 2.0WarmStart in Amplification SystemTMDNA polymerase, MgSO4dNTPs were purchased from New England Biolab, USA; 4-arm-polyethylene glycol-acrylate (molecular weight 10000), mercapto-polyethylene glycol-mercapto (molecular weight 3400) were purchased from Laysan Bio corporation, usa; lysozyme, SYBR Green, was purchased from ThermoFisher Scientific, USA; sealed chambers (frame-seal) were purchased from Bio-rad, USA; polyethylene terephthalate (PET) membranes, Polycarbonate (PC) membranes and aqueous PES microfiltration membranes were purchased from whatman, Inc. and 13mm in diameter.
Example 1 Rapid detection of E.coli in samples by NanoLimited Domain Loop-mediated isothermal nucleic acid amplification method based on etched filtration Membrane
1. The corresponding primer sequences of E.coli are as follows:
F3:5'-GCCATCTCCTGATGACGC-3'；
B3:5'-ATTTACCGCAGCCAGACG-3'；
LF:5'-CTTTGTAACAACCTGTCATCGACA-3'；
LB:5'-ATCAATCTCGATATCCATGAAGGTG-3'；'
FIP:5'-CATTTTGCAGCTGTACGCTCGCAGCCCATCATGAATGTTGCT-3'；
BIP:5'-CTGGGGCGAGGTCGTGGTATTCCGACAAACACCACGAATT-3'；
preparing a standard sample: the original concentration of the Escherichia coli DNA is 100 CFU/mL;
2. the gasket is sequentially loaded in the filter, the filtering membrane of the nano hole is etched, the sacrificial membrane for preventing the filtering membrane from deforming is used, then the filtering injector extracts 1mL of standard sample, the sample is injected into the filter at a constant speed, bacteria liquid is uniformly distributed on the nano hole of the filtering membrane, target bacteria are intercepted in the nano hole, then the filtering injector is taken down and the filtering membrane is blown by suction until the drying is carried out, the pointed tweezers are used for taking out the filtering membrane to be attached to a glass slide, and a small sealing chamber is attached.
In the embodiment, membranes made of different materials are selected to intercept target bacteria and used for subsequent LAMP amplification, and the influence of factors such as fluorescence and biocompatibility of the membranes on amplification results is researched. Specifically, a polyethylene terephthalate (PET) film (with a nanopore diameter of 400nm), a Polycarbonate (PC) film (with a nanopore diameter of 400nm) and a traditional commercial water-based PES (polyether sulfone) microporous filter membrane (with a nanopore diameter of 220nm) are adopted, wherein an electron microscope image of the polycarbonate nanopore membrane (PCTE membrane) is shown in FIG. 1, and the nanopore diameter is 400 nm.
3. An amplification system (25. mu.L) containing hydrogel was prepared: 1.6mg 4-arm-polyethylene glycol-acrylate, 1.1mg mercapto-polyethylene glycol-mercapto, 2.5. mu.L 10 × Isotermal Amplification Buffer, 6mM total MgSO41.4mM dNTPs, 640U/mL Bst 2.0WarmStart polymerase, 1.6. mu.M FIB and BIP, 0.2. mu. M F3 and B3, 0.8. mu.M LF and LB, 1 XSSYBR Green, 1.0mg/mL BSA, 0.1mg/mL lysozyme.
4. The amplification system prepared in 3 above was dropped into a sealed chamber, and a waterproof film was attached to form a completely closed space. The whole amplification system is amplified for 20min under the constant temperature condition of 65 ℃, and then heated for 5min at 80 ℃.
Meanwhile, the amplification reaction condition is set as a control group which is amplified for 20min under the constant temperature condition of 65 ℃.
5. And observing the amplified reaction system by using a fluorescence microscope, taking a picture of a fluorescence point of the amplified system, counting the fluorescence point by using Image J software, and quantitatively calculating the original concentration of the object to be detected in the hydrogel amplified system.
The experimental results for the different membrane selections are shown in fig. 2, the amplification system shows bright and concentrated spots on the Polycarbonate (PC) membrane and the polyethylene terephthalate (PET) membrane (fig. 2a, b), and each amplified spot is clearly visible, whereas in the conventional commercial membrane (fig. 2c), the amplification of the amplification system is affected and the spots are few and dark. Therefore, Polycarbonate (PC) films are preferred for the present invention.
The experimental result for optimizing the amplification condition is shown in fig. 3, and after constant-temperature amplification, a heating optimization process at 80 ℃ for 5min is performed, so that the brightness-dark ratio of fluorescence imaging is obviously enhanced, and the final detection result is more vivid in visualization effect.
Comparative example 1 Effect of modifying coating on Polycarbonate (PC) film on amplification results
It is known that different filter materials can affect the amplification result of the experiment. Furthermore, in order to investigate the influence of modifying different coatings on the same material of the filter membrane on the whole amplification process and the amplification result, the method of the present invention investigated the influence of modifying a coating on a Polycarbonate (PC) membrane on the amplification result.
Specifically, black dye, gold dye and gray dye were applied to Polycarbonate (PC) films to form black, gold and gray coatings, respectively, as in example 1.
As shown in FIG. 4, no amplification spots were detected on the black coating (FIG. 4a), less amplification spots were observed on the gold coating (FIG. 4b), the gray coating (FIG. 4c) and the amplification spots were scattered. On the unmodified coated PC membrane (fig. 4d), bright and concentrated amplification spots were present, and the amplification amount was the largest, so we finally selected the unmodified coated PC membrane as the filtration membrane in the present invention.
Comparative example 2 Loop-mediated isothermal nucleic acid amplification method in solution for detecting common Escherichia coli in sample
1. The primer sequences for E.coli were the same as in example 1.
Preparing a standard sample: coli DNA was originally at a concentration of 50 CFU/. mu.L.
2. Preparing a loop-mediated isothermal nucleic acid amplification reaction system: 2.5 μ L10 × Isothermal Amplification Buffer, 6mM total MgSO41.4mM dNTPs, 640U/mL Bst 2.0WarmStart polymerase, 1.6. mu.M FIB and BIP, 0.2. mu. M F3 and B3, 0.8. mu.M LF and LB, 1X SYBR Green, 1.0 mg/mLBA, 0.1mg/mL lysozyme. That is, the two hydrogel monomers of the amplification reaction system in example 1 were removed, and the rest remained the same.
3. mu.L of the standard sample was added to the reaction system. The whole amplification system was amplified for 20min at a constant temperature of 65 ℃ and then heated for 5min at 80 ℃ under the same conditions as in example 1.
4. Observing the reaction system after amplification by a fluorescence microscope and taking a fluorescent dot picture of the amplification system.
As shown in FIG. 5(a), the amplification results of the reaction in the solution were only qualitative and not quantitative in the reaction system of 25. mu.L.
After the double nano-confinement treatment using the nanopore-etched filter and the hydrogel under the conditions of example 1, the amplified products of the bacteria on the membrane were fixed at the initial positions to form bright and focused spots, as shown in fig. 5 (b).
Therefore, compared with the common loop-mediated isothermal nucleic acid amplification method, the detection result of the method has higher visualization degree, low experiment cost and simple operation.
Comparative example 3 Loop-mediated isothermal nucleic acid amplification method in hydrogel for detecting common Escherichia coli in sample
1. The primer sequences for E.coli were the same as in example 1.
Preparing a series of standard samples:
the concentration of the escherichia coli standard sample a is 1 CFU/. mu.L;
the concentration of the escherichia coli standard sample b is 10 CFU/. mu.L
The concentration of the escherichia coli standard sample c is 20 CFU/. mu.L;
the d concentration of the escherichia coli standard sample is 40 CFU/mu L;
e, the concentration of the escherichia coli standard sample e is 80 CFU/mu L;
2. a loop-mediated isothermal nucleic acid amplification reaction system containing a hydrogel was prepared as in example 1.
3. mu.L of the standard sample was added to the reaction system. The whole amplification system was amplified for 20min at a constant temperature of 65 ℃ and then heated for 5min at 80 ℃ under the same conditions as in example 1.
4. Observing the amplified reaction system by a fluorescence microscope, shooting a fluorescent dot picture of the amplified system, counting the fluorescent dots, and further calculating the detection limit.
The result is shown in FIG. 6, the detection limit of the loop-mediated isothermal nucleic acid amplification in the hydrogel is 1000CFU/mL, while the detection limit of the etching filtration membrane based nano-confinement loop-mediated isothermal nucleic acid amplification method of the invention can be as low as 0.2CFU/mL, which greatly improves the detection limit.
Example 2a nano-confinement loop-mediated isothermal nucleic acid amplification method based on an etched filter membrane rapidly detects listeria monocytogenes in a sample.
1. The corresponding primer sequences of the listeria monocytogenes are as follows:
F3:5'-TTGCGCAACAAACTGAAGC-3'；
B3:5'-GCTTTTACGAGAGCACCTGG-3'；
LF:5'-TAGGACTTGCAGGCGGAGATG-3'；
LB:5'-GCCAAGAAAAGGTTACAAAGATGG-3'；
FIP:5'-CGTGTTTCTTTTCGATTGGCGTCTTTTTTTCATCCATGGCACCACC-3'；
BIP:5'-CCACGGAGATGCAGTGACAAATGTTTTGGATTTCTTCTTTTTCTCCACAAC-3'；
preparing a standard sample: the original concentration of the listeria monocytogenes DNA is 100 Cell/mL;
2. the method comprises the following steps of sequentially loading a gasket, a filter membrane with etched nano holes and a sacrificial membrane for preventing the filter membrane from deforming in a filter, then filtering 500 mu L of a standard sample to the filter membrane with the etched nano holes, intercepting target bacteria in the nano holes, repeatedly pumping and blowing the filter, drying the filter membrane, placing the filter membrane on a glass slide, and attaching a small sealing chamber. A negative control group was also prepared.
3. An amplification system (25. mu.L) containing hydrogel was prepared: 1.6mg 4-arm-polyethylene glycol-acrylate, 1.1mg mercapto-polyethylene glycol-mercapto, 2.5. mu.L 10 × Isotermal Amplification Buffer, 6mM total MgSO41.4mM dNTPs, 640U/mL Bst 2.0WarmStart polymerase, 1.6. mu.M FIB and BIP, 0.2. mu. M F3 and B3, 0.8. mu.M LF and LB, 1X SYBR Green, 1.0 mg/mLBA, 0.1mg/mL lysozyme.
4. The amplification system prepared in 3 above was dropped into a sealed chamber, and a waterproof film was attached to form a completely closed space. The whole amplification system is amplified for 20min under the constant temperature condition of 65 ℃, and then heated for 5min at 80 ℃.
5. And observing the amplified reaction system by using a fluorescence microscope, taking a picture of a fluorescence point of the amplified system, counting the fluorescence point by using Image J software, and quantitatively calculating the original concentration of the object to be detected in the hydrogel amplified system.
The results of the experiment are shown in FIG. 7(a), and absolute qualitative and quantitative detection can be performed, and bright and focused fluorophores can be formed, and direct counting can be performed by counting software. As shown in FIG. 7(b), no false positive occurred, and the method can avoid the interference of false positive well.
Example 3 a nanotrenological loop-mediated isothermal nucleic acid amplification method based on etched filtration membranes rapidly detects new coronavirus in a sample.
1. The corresponding primer sequences of the new coronavirus are as follows:
F3:5'-AACACAAGCTTTCGGCAG-3'；
B3:5'-GAAATTTGGATCTTTGTCATCC-3'；
LF:5'-TTCCTTGTCTGATTAGTTC-3'；
LB:5'-ACCTTCGGGAACGTGGTT-3'；
FIP:5'-CGCATTGGCATGGAAGTCACTTTGATGGCACCTGTGTAG-3'；
BIP:5'-TGCGGCCAATGTTTGTAATCAGCCAAGGAAATTTTGGGGAC-3'；
preparing a standard sample: the concentration of the new coronavirus is 100 copy/mL;
2. the method comprises the following steps of sequentially loading a gasket, a filter membrane (with the aperture of 100nm) with nano holes etched and a sacrificial membrane for preventing the filter membrane from deforming in a filter, then filtering 1mL of standard sample to the filter membrane with the nano holes etched in the track, intercepting target viruses in the nano holes, repeatedly pumping and blowing the filter, drying the filter membrane, placing the filter membrane on a glass slide, and attaching a small sealing chamber. A negative control group was also prepared.
3. An amplification system (25. mu.L) containing hydrogel was prepared: 1.6mg 4-arm-polyethylene glycol-acrylate, 1.1mg mercapto-polyethylene glycol-mercapto, 2.5. mu.L 10 × Isotermal Amplification Buffer, 6mM total MgSO41.4mM dNTPs, 640U/mL Bst 2.0WarmStart polymerase, 300U/mL WarmStart reverse transcriptase, 1.6. mu.M FIB and BIP, 0.2. mu. M F3 and B3, 0.4. mu.M LF and LB, 1X SYBR Green.
4. The amplification system prepared in 3 above was dropped into a sealed chamber, and a waterproof film was attached to form a completely closed space. The whole amplification system is amplified for 15min under the constant temperature condition of 65 ℃, and then heated for 5min at 80 ℃.
5. And observing the amplified reaction system by using a fluorescence microscope, taking a picture of a fluorescence point of the amplified system, counting the fluorescence point by using Image J software, and quantitatively calculating the original concentration of the object to be detected in the hydrogel amplified system.
The results of the experiment are shown in FIG. 8(a), and absolute qualitative and quantitative detection can be performed, and bright and aggregated fluorophores can be formed, and direct counting can be performed by counting software. As shown in FIG. 8(b), no false positive occurred, and the method can avoid the interference of false positive well.
Claims (10)
1. A method for rapid absolute quantification of pathogenic microorganisms in a bulk liquid sample, comprising the steps of:
(1) filtering a liquid sample to be detected by using a nano-aperture membrane to enable target microorganisms to be intercepted on the surface of the filter membrane, and then drying the filter membrane;
(2) attaching a sealed small chamber to the surface of the filter membrane for intercepting the target microorganism, adding a loop-mediated isothermal nucleic acid amplification reaction system containing a hydrogel monomer into the sealed small chamber, and sealing; crosslinking hydrogel monomers into gel to enable a reaction system to cover the target microorganisms on the surface of the filter membrane in a colloidal state, wherein the reaction system contains a primer designed and matched with the DNA or RNA of the target microorganisms;
(3) putting the whole amplification reaction system under a constant temperature condition to carry out nucleic acid isothermal amplification reaction;
(4) and after the reaction is finished, analyzing a fluorescence signal appearing in the amplification reaction system by utilizing a fluorescence imaging technology, counting fluorescence points, and calculating to obtain the absolute quantitative concentration of the target microorganism in the liquid sample to be detected.
2. The method according to claim 1, wherein the nanopore membrane is made of polycarbonate or polyethylene terephthalate.
3. The method for rapid absolute quantification of a pathogenic microorganism in a bulk liquid sample according to claim 1, wherein the nanoporous membrane is a track-etched membrane having a nanopore size of 50 to 500 nm.
4. The method for rapid absolute quantification of a pathogenic microorganism in a bulk liquid sample according to claim 1, wherein the pathogenic microorganism is a bacterium or a virus, and when the pathogenic microorganism is a bacterium, lysozyme is contained in the loop-mediated isothermal nucleic acid amplification reaction system; when the pathogenic microorganism is a virus, the loop-mediated isothermal nucleic acid amplification reaction system contains reverse transcriptase.
5. The method for rapid absolute quantification of a pathogenic microorganism in a bulk liquid sample according to claim 1, wherein the loop-mediated isothermal nucleic acid amplification reaction system comprises: 1 × Isothermal Amplification Buffer, 6mM total MgSO41.4mM dNTPs, 640U/mL Bst 2.0WarmStart polymerase, a primer mixture, a fluorescent dye and a hydrogel monomer;
when the target microorganism is bacteria, the reaction system also comprises 1.0mg/mL BSA and 0.1mg/mL lysozyme;
when the target microorganism is a virus, the reaction system further comprises 300U/mL of WarmStart reverse transcriptase.
6. The method for rapid absolute quantification of pathogenic microorganisms in a bulk liquid sample according to claim 1, wherein in the step (2), the hydrogel monomer comprises four-arm polyethylene glycol acrylate and sulfhydryl-polyethylene glycol-sulfhydryl, and the molar ratio of the two is 1: 1-4.
7. The method for rapid absolute quantification of a pathogenic microorganism in a bulk liquid sample according to claim 1, wherein in step (2), the sealed chamber is sealed with a waterproof membrane.
8. The method for rapid absolute quantification of a pathogenic microorganism in a bulk liquid sample according to claim 1, wherein in step (3), the conditions of the loop-mediated isothermal nucleic acid amplification reaction comprise: firstly, at 65-70 ℃, for 15-30 min; and secondly, heating at 75-80 ℃ for 3-7 min.
9. The method for rapid absolute quantification of a pathogenic microorganism in a bulk liquid sample according to claim 8, wherein the conditions of the loop-mediated isothermal nucleic acid amplification reaction comprise: step one, at 65 ℃, 20 min; second, heat at 80 ℃ for 5 min.
10. The method for rapid absolute quantification of pathogenic microorganisms in a bulk liquid sample according to claim 1, wherein in step (4), the reaction system is irradiated with a flashlight, a photograph is taken and fluorescence spot counting is performed with counting software.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111208631.9A CN113943781B (en) | 2021-10-18 | 2021-10-18 | Rapid absolute quantification method for pathogenic microorganisms in large-volume liquid sample |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111208631.9A CN113943781B (en) | 2021-10-18 | 2021-10-18 | Rapid absolute quantification method for pathogenic microorganisms in large-volume liquid sample |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113943781A true CN113943781A (en) | 2022-01-18 |
| CN113943781B CN113943781B (en) | 2023-06-30 |
Family
ID=79330897
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111208631.9A Active CN113943781B (en) | 2021-10-18 | 2021-10-18 | Rapid absolute quantification method for pathogenic microorganisms in large-volume liquid sample |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113943781B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101824493A (en) * | 2010-05-18 | 2010-09-08 | 天津朝海科技有限公司 | Kit for detecting hepatitis B virus |
| CN106771217A (en) * | 2016-12-05 | 2017-05-31 | 百奥森（江苏）食品安全科技有限公司 | A kind of single method for quick for increasing listeria spp |
| CN110846384A (en) * | 2019-11-01 | 2020-02-28 | 浙江大学 | Digital loop-mediated isothermal amplification detection method based on track etching membrane and application |
| CN112266986A (en) * | 2020-12-22 | 2021-01-26 | 博奥生物集团有限公司 | Virus nucleic acid extraction or preservation reagent, primer probe combination, virus amplification reagent, kit and application thereof |
| CN112410405A (en) * | 2020-11-24 | 2021-02-26 | 浙江大学 | Rapid detection method for bacteria in complex sample |
-
2021
- 2021-10-18 CN CN202111208631.9A patent/CN113943781B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101824493A (en) * | 2010-05-18 | 2010-09-08 | 天津朝海科技有限公司 | Kit for detecting hepatitis B virus |
| CN106771217A (en) * | 2016-12-05 | 2017-05-31 | 百奥森（江苏）食品安全科技有限公司 | A kind of single method for quick for increasing listeria spp |
| CN110846384A (en) * | 2019-11-01 | 2020-02-28 | 浙江大学 | Digital loop-mediated isothermal amplification detection method based on track etching membrane and application |
| CN112410405A (en) * | 2020-11-24 | 2021-02-26 | 浙江大学 | Rapid detection method for bacteria in complex sample |
| CN112266986A (en) * | 2020-12-22 | 2021-01-26 | 博奥生物集团有限公司 | Virus nucleic acid extraction or preservation reagent, primer probe combination, virus amplification reagent, kit and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| CHANGYU YI 等: "Nanoporous hydrogel for direct digital nucleic acid amplification in untreated complex matrices for single bacteria counting", 《BIOSENSORS AND BIOELECTRONICS》 * |
| DE-GUO WANG 等: "Two Methods for Increased Specificity and Sensitivity in Loop-Mediated Isothermal Amplification", 《MOLECULES》 * |
| XINGYU LIN 等: "Asymmetric Membrane for Digital Detection of Single Bacteria in Milliliters of Complex Water Samples", 《ACS NANO》 * |
| YAJUAN DONG 等: "Comparative evaluation of 19 reverse transcription loop‑mediated isothermal amplification assays for detection of SARS‑CoV‑2", 《SCIENTIFIC REPORTS》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113943781B (en) | 2023-06-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20200277653A1 (en) | Analyte Enrichment Methods and Compositions | |
| JP7125418B2 (en) | Target molecule density determination in fluorescence images | |
| US20050202433A1 (en) | Novel high density arrays and methods for analyte analysis | |
| Li et al. | Recent advancements in nucleic acid detection with microfluidic chip for molecular diagnostics | |
| CN112410405B (en) | Rapid detection method for bacteria in complex sample | |
| CN105301237A (en) | Method for detecting nucleic acid by colloidal gold chromatography technology and reagent kit | |
| US20210180116A1 (en) | Assay using multi-layer membrane to detect microbiological target and method of manufacturing multi-layer membrane | |
| JP2009000070A (en) | Method for large-scale parallel analysis of nucleic acid | |
| CN112126587B (en) | Nucleic acid detection chip device, nucleic acid detection chip and preparation method thereof | |
| CN113825842A (en) | Method for detecting target molecule | |
| WO2013104864A1 (en) | Method for directly detecting and identifying a microorganism in a biological sample via optical means | |
| US20210349080A1 (en) | Multi-faceted method for detecting and analyzing target molecule by molecular aptamer beacon (mab) | |
| JP2020108358A (en) | Method for detecting target object with high sensitivity | |
| CN113943781B (en) | Rapid absolute quantification method for pathogenic microorganisms in large-volume liquid sample | |
| JP2017528147A (en) | Partial lysis and assay methods | |
| CN101824468A (en) | Primer group for detecting Yersinia pestis, rapid diagnosis kit and detection method | |
| CN102286636B (en) | Dengue virus detection and parting method, special chip and kit | |
| CN116814742A (en) | Construction method of single cell space transcriptome sequencing library | |
| Cao et al. | Establishment of scalable nanoliter digital LAMP technology for the quantitative detection of multiple myeloproliferative neoplasm molecular markers | |
| CN117327841B (en) | Method for jointly detecting monkey pox virus and chikungunya virus by capillary modified LAMP method | |
| WO2024098470A1 (en) | Nucleic acid absolute quantification detection system and nucleic acid absolute quantification method | |
| CN115725787A (en) | HPV detection method based on hydrogel loop-mediated isothermal amplification | |
| US9638612B2 (en) | Selective release of sub-group of biological units | |
| CN115572758A (en) | Microorganism detection device and detection method | |
| CN117867179A (en) | LAMP primer group for detecting dengue virus, microfluidic chip and detection method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |