CN117778546A - Agricultural product microorganism rapid detection system and method thereof - Google Patents
Agricultural product microorganism rapid detection system and method thereof Download PDFInfo
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Abstract
The invention relates to the technical field of microorganism detection, in particular to a rapid detection system and a rapid detection method for agricultural product microorganisms, which aim to solve the problem that the detection time is long because a plate colony counting method is adopted for microorganism detection.
Description
Technical Field
The invention relates to the technical field of microorganism detection, in particular to a rapid microorganism detection system and a rapid microorganism detection method for agricultural products.
Background
The agricultural product surface is colonized with a plurality of microorganisms and keeps dynamic balance, and the microorganisms have very important ecological functions in the aspects of substance synthesis, degradation, carbon nitrogen element circulation and the like. In the agricultural product field, detection of surface microorganisms can analyze various index conditions of agricultural products.
The plate colony counting method is to disperse the microbe in the sample to be measured into single cell, to take some diluted sample liquid to spread on the plate, to culture, to form macroscopic colony, one single colony should represent one single cell in the original sample; counting the colony number, and calculating the bacteria content in the sample according to the dilution times and the sampling inoculation amount. Detection of microorganisms by plate colony counting requires a long time, which makes it difficult to rapidly detect microorganisms.
Disclosure of Invention
Therefore, the invention provides a rapid detection system and a rapid detection method for agricultural product microorganisms, which are used for solving the problems mentioned in the background.
The invention provides a rapid detection system for agricultural product microorganisms, which comprises;
a microorganism collection module; obtaining a microbial sample;
a sample pretreatment module; for removing interfering substances and sterilizing;
nucleic acid DNA extraction module: for extracting nucleic acids from a microbial sample;
a sequencing library construction module; the method is used for constructing a sequencing library by adopting a pathogenic microorganism detection kit;
a sequencing module; the method comprises the steps of sequencing a constructed library to obtain nucleic acid DNA sequence information;
the detection result acquisition module; and the detection result of the microorganism sample is obtained according to the nucleic acid DNA sequence information.
Further, the detection method includes:
s1, acquiring a microorganism sample;
s2, sterilizing the agricultural product microorganism sample;
s3, extracting nucleic acid in the microbial sample; introducing an amine group into a target object to modify the target object; injecting dimethyl dithiodipropimido and the nucleic acid obtained by the lysis treatment of the microbial sample onto the modified target object to generate a complex of the dimethyl dithiodipropimido and the nucleic acid; treating the complex of dimethyl dithiodipropionate and the nucleic acid with an elution buffer to extract the nucleic acid in the microbial sample; subjecting the microbial sample to lysis treatment to release nucleic acid in the microbial sample, subjecting the microbial sample to lysis treatment, subjecting the microbial sample subjected to the lysis treatment to purification treatment to remove impurities in the microbial sample, and subjecting the microbial sample subjected to the lysis treatment to purification treatment;
s4, constructing a sequencing library by adopting a pathogenic microorganism detection kit; the method comprises the steps of obtaining a pathogenic microorganism detection kit, wherein the pathogenic microorganism detection kit comprises a restriction enzyme mixed solution for identifying four bases, taq DNA polymerase, T4 DNA ligase, a connection promoter, dNTP, ATP, a connector, high-fidelity DNA polymerase and dithiothreitol; constructing a genome DNA sequencing library by adopting a pathogenic microorganism detection kit, and carrying out enzyme digestion on sample DNA to obtain fragmented DNA; directly carrying out terminal repair and dA tail addition on the fragmented DNA to obtain DNA with terminal repair and dA tail addition; connecting the DNA with the end repaired and dA tail with a joint to obtain a joint connection product with barcode; carrying out PCR amplification on the joint connection product with the barcode to obtain the genome DNA sequencing library;
s5, sequencing the constructed library by using a second generation sequencing platform to obtain nucleic acid sequence information; constructing different libraries according to the attribute of the microbial sample to be detected, wherein the libraries comprise whole genome library establishment, target segment capturing library establishment or transcriptome library establishment; sequencing the extracted nucleic acid in the microbial sample by adopting a second generation sequencer, and recording a base signal, wherein the sequencing comprises sequencing while synthesizing or sequencing while connecting; performing data processing on the base signals, wherein the data processing comprises sequence alignment, local alignment or base quality correction;
s6, constructing a microbial genome database, and comparing and analyzing the nucleic acid sequence information with the microbial genome database to determine the types and the contents of the microorganisms in the microbial samples.
Further, in the sterilization in S1, a solution containing 75% alcohol is used for the sterilization treatment.
Further, the target object in S3 includes a thin film device, a magnetic bead, a ring resonator, and a nanoparticle.
Further, the method for performing lysis treatment on the microorganism sample in the step S3 comprises the following steps of; mechanical method, repeated freeze thawing method, high temperature boiling method, enzymatic degradation method, guanidine salt degradation method, alkaline degradation method, CTAB degradation method, and phenol degradation method.
Further, the method for purifying the microorganism sample in the step S3 comprises the following steps: organic solvent-ethanol precipitation, silicon membrane adsorption, magnetic bead, anion exchange, and nucleic acid automatic extraction.
Compared with the prior art, the method has the advantages that nucleic acid in a microorganism sample is extracted through a nucleic acid extraction module, a sequencing library is constructed through a sequencing library construction module by adopting a pathogenic microorganism detection kit, and the constructed library is sequenced through the sequencing module, so that nucleic acid sequence information is obtained; the detection result acquisition module acquires a detection result of the microbial sample according to the nucleic acid sequence information; compared with the prior art, the method does not need to culture the microbial sample, only needs to extract the microbial sample, then sequences the nucleic acid in the extracted microbial sample to obtain the nucleic acid sequence information, and can obtain the detection result of the microbial sample according to the nucleic acid sequence information, thereby greatly shortening the time consumed by detection and realizing the rapid detection of the microbial sample.
Detailed Description
In order that the objects and advantages of the invention will become more apparent, the invention will be further described with reference to the following examples; it should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Preferred embodiments of the present invention are described below. It should be understood by those skilled in the art that these embodiments are merely for explaining the technical principles of the present invention, and are not intended to limit the scope of the present invention.
It should be noted that, in the description of the present invention, terms such as "upper," "lower," "left," "right," "inner," "outer," and the like indicate directional or positional relationships, and are merely used for convenience of description, but do not indicate or imply that the devices or elements must have a particular orientation, be constructed and operated in a particular orientation, and thus should not be construed as limiting the invention.
Furthermore, it should be noted that, in the description of the present invention, unless explicitly specified and limited otherwise, the terms "mounted," "connected," and "connected" are to be construed broadly, and may be either fixedly connected, detachably connected, or integrally connected, for example; can be mechanically or electrically connected; can be directly connected or indirectly connected through an intermediate medium, and can be communication between two elements. The specific meaning of the above terms in the present invention can be understood by those skilled in the art according to the specific circumstances.
To further illustrate the invention in detail.
The invention relates to a rapid detection system for microorganisms of agricultural products, which comprises;
a microorganism collection module; obtaining a microbial sample;
a sample pretreatment module; for removing interfering substances and sterilizing;
nucleic acid DNA extraction module: for extracting nucleic acids from a microbial sample;
a sequencing library construction module; the method is used for constructing a sequencing library by adopting a pathogenic microorganism detection kit;
a sequencing module; the method comprises the steps of sequencing a constructed library to obtain nucleic acid DNA sequence information;
the detection result acquisition module; and the detection result of the microorganism sample is obtained according to the nucleic acid DNA sequence information.
The detection method comprises the following steps:
s1, acquiring a microorganism sample;
s2, sterilizing the agricultural product microorganism sample;
s3, extracting nucleic acid in the microbial sample; introducing an amine group into a target object to modify the target object; injecting dimethyl dithiodipropimido and the nucleic acid obtained by the lysis treatment of the microbial sample onto the modified target object to generate a complex of the dimethyl dithiodipropimido and the nucleic acid; treating the complex of dimethyl dithiodipropionate and the nucleic acid with an elution buffer to extract the nucleic acid in the microbial sample; subjecting the microbial sample to lysis treatment to release nucleic acid in the microbial sample, subjecting the microbial sample to lysis treatment, subjecting the microbial sample subjected to the lysis treatment to purification treatment to remove impurities in the microbial sample, and subjecting the microbial sample subjected to the lysis treatment to purification treatment; dimethyl dithiodipropimid is used as an amino reactive cross-linking agent for cells, proteins and nucleic acids because it can form a reversible cross-linked structure. Nucleic acids can be rapidly extracted from microbial samples by rapid and strong intercoupling between dimethyl dithiodipropionate and nucleic acids, unlike the interaction between dimethyl dithiodipropionate and proteins. Specific steps for extracting nucleic acid in a microbial sample are provided, and nucleic acid can be extracted from the microbial sample rapidly. The lysis treatment is mainly to treat the microorganism sample in a physical, chemical or biological way so as to achieve the purpose of releasing nucleic acid, wherein the mechanical method destroys the structure of the microorganism through mechanical force; repeatedly freezing and thawing the microorganism for more than three times by a repeated freezing and thawing method, so that the microorganism structure swells and finally breaks to release nucleic acid; directly adding a microorganism sample into pure water for boiling by a high-temperature boiling method, centrifuging at a high speed to precipitate impurities, and positioning released nucleic acid in supernatant; the enzymatic degradation method is characterized in that lysozyme or protease is added into a microbial sample to destroy the structure of the microbial sample, degrade proteins in chromosomes and promote the separation of nucleic acids; the guanidine salt splitting method adopts guanidine salts such as guanidine isothiocyanate, creatine guanidine, guanidine hydrochloride and the like to split a microorganism sample, and inactivates nuclease to prevent nucleic acid from being degraded by the nuclease; alkaline lysis method employs strong base such as Sodium Dodecyl Sulfate (SDS) to precipitate acidic polysaccharide and nucleic acid in low ionic strength solution; the phenol extraction method is to extract nucleic acid by using protein strong denaturant such as phenol. The purification treatment is to eliminate the impurities after the microbial sample is cracked by using the biological characteristics of nucleic acid molecules and a chemical or physical method so as to achieve the process of purifying the nucleic acid, and the organic solvent-ethanol precipitation method is to separate out the nucleic acid from an ethanol solution for precipitation, remove the impurities such as protein and the like by using the organic solvent, and precipitate the nucleic acid by using precooled ethanol or isopropanol; the silicon film adsorption method is to utilize the high affinity of a nucleic acid skeleton with negative charges and silicon dioxide with positive charges, make the nucleic acid closely combine with the silicon film through the action of sodium ions in a high hydrochloric acid environment, and elute the nucleic acid in a low-salt neutral or weak alkaline environment after the impurities are removed by washing; the magnetic bead method is to adopt biological magnetic beads with active groups with the surface coated with the characteristic of reversibly adsorbing nucleic acid, adsorb the nucleic acid under certain ionic strength and pH, achieve the combination and separation of solid and liquid phases under the action of an external magnetic field, and then obtain the nucleic acid with high purity through elution; the anion exchange method utilizes positive charges carried on the surface of anion exchange resin and negative charges carried on a nucleic acid skeleton, combines nucleic acid under low alkalinity, and elutes nucleic acid under high hydrochloric acid; the automatic extraction method of nucleic acid adopts the principle of magnetic bead method or silicon film method to realize high flux, thus saving time cost and labor cost. Specific methods of nucleic acid in a microbial sample are provided.
S4, constructing a sequencing library by adopting a pathogenic microorganism detection kit; the method comprises the steps of obtaining a pathogenic microorganism detection kit, wherein the pathogenic microorganism detection kit comprises a restriction enzyme mixed solution for identifying four bases, taq DNA polymerase, T4 DNA ligase, a connection promoter, dNTP, ATP, a connector, high-fidelity DNA polymerase and dithiothreitol; constructing a genome DNA sequencing library by adopting a pathogenic microorganism detection kit, and carrying out enzyme digestion on sample DNA to obtain fragmented DNA; directly carrying out terminal repair and dA tail addition on the fragmented DNA to obtain DNA with terminal repair and dA tail addition; connecting the DNA with the end repaired and dA tail with a joint to obtain a joint connection product with barcode; carrying out PCR amplification on the joint connection product with the barcode to obtain the genome DNA sequencing library; the mixed solution of the restriction enzymes is a combination of a plurality of enzymes in MspI, aluI, cviQI, mseI, mlucI, haeIII, and the connection promoter consists of polyethylene glycol 6000, bovine serum albumin and triton X-100; the genome DNA sequencing library is constructed by adopting the pathogenic microorganism detection kit, so that the construction speed of the sequencing library can be increased, and the time consumed for detecting the microorganism sample is further shortened.
S5, sequencing the constructed library by using a second generation sequencing platform to obtain nucleic acid sequence information; constructing different libraries according to the attribute of the microbial sample to be detected, wherein the libraries comprise whole genome library establishment, target segment capturing library establishment or transcriptome library establishment; sequencing the extracted nucleic acid in the microbial sample by adopting a second generation sequencer, and recording a base signal, wherein the sequencing comprises sequencing while synthesizing or sequencing while connecting; performing data processing on the base signals, wherein the data processing comprises sequence alignment, local alignment or base quality correction; sequencing the nucleic acid in the extracted microbial sample by extracting the nucleic acid in the microbial sample, and constructing a sequencing library by adopting a pathogenic microorganism detection kit; sequencing the constructed library by utilizing second generation sequencing to obtain nucleic acid sequence information; obtaining a detection result of the microbial sample according to the nucleic acid sequence information; compared with the prior art, the method does not need to culture a microbial sample, only needs to extract the microbial sample, then sequences the nucleic acid in the extracted microbial sample to obtain nucleic acid sequence information, and can obtain the detection result of the microbial sample according to the nucleic acid sequence information, so that the time consumed by detection is greatly shortened, the rapid detection of the microbial sample is realized, and the Illumina platform is provided with a plurality of series from Mini-Seq with low flux to HiSeq X Ten with ultrahigh flux, and the sequencing technology of the Illumina platform is based on the sequencing-by-synthesis technology of a reversible terminator, and the sequencing process of the Illumina platform is mainly divided into three parts: library establishment, bridge amplification clustering reaction and SBS process. The library building comprises cutting a template to the fragment length required by a sequencing platform, and adding a sequencing primer, a tag sequence and a cluster generating primer specific to the sequencing platform at two ends of the fragmented template; and (5) enriching the library by using the cluster generating primer to complete library establishment. Different test samples, such as genomic DNA, transcriptome mRNA and small RNA, or different sequencing purposes, such as whole genome sequencing, whole exon sequencing, single cell genome sequencing and chromatin co-precipitation sequencing, all construct different libraries. The clustering reaction is carried out in a special micro-fluid cluster station or cBet instrument, the constructed library is subjected to alkaline denaturation to form a single chain, then the single chain library is introduced into a glass flow cell, the surface of the flow cell is provided with a sequence which is completely complementary with a bridge amplification primer on a connector, and the single chain library is randomly hybridized with an anchoring sequence on the surface of the glass flow cell through the heating and cooling processes. The SBS process is similar to sanger sequencing, dNTPs are added, subsequent base extension can be prevented, during the sequencing reaction, a sequencing primer hybridizes with a single-stranded template on a glass flow cell, dNTPs carrying specific fluorescence are added in 4, each cycle is single-base extension under the action of DNA polymerase and a reversible terminator, one of the dNTPs carrying specific fluorescence in 4 is added in a product, unbound dNTPs can be eluted, the added base type is determined by collecting signals generated by fluorescent groups, then the fluorescent groups and the termination groups are removed through chemical action, the bases at the tail ends of the chains are changed into an extensible state, the base extension process and the signal collection process are repeated, the sequencing flux is improved, and the sequencing cost is reduced by adopting a second generation sequencing technology to sequence nucleic acids in the extracted microbial samples.
S6, constructing a microbial genome database, and comparing and analyzing the nucleic acid sequence information with the microbial genome database to determine the types and the contents of microorganisms in the microbial samples, wherein the genome database is a database which integrates the nucleotide sequences, the single nucleotide polymorphisms, the structures, the properties and the related descriptions of all known nucleic acids and comprises information such as scientific names, source species classification names, references and the like of the nucleic acids; the type and content of the microorganism in the microorganism sample can be determined by comparing the nucleic acid sequence information with a microorganism genome database.
In the step S1, a solution containing 75% alcohol is used for sterilization treatment, so as to effectively kill microorganisms including bacteria, viruses, etc., and ensure that the collected agricultural product microorganism sample is not affected by external pollution in the subsequent steps. The concentration of 75% alcohol is widely recognized as a relatively effective disinfectant that is capable of destroying cell membranes, proteins, and nucleic acids of microorganisms, thereby completely inactivating the microorganisms. This helps ensure that contaminants from the original sample are obtained in the subsequent extraction of nucleic acids rather than from the external environment.
The target object in S3 includes a thin film device, magnetic beads, a ring resonator, and nanoparticles, wherein the thin film device is used as a carrier or support structure for nucleic acid extraction, the nucleic acid can interact with a compound or group on the thin film, so that the subsequent processing steps are more efficient, the magnetic beads are generally used for separating, enriching or purifying target substances (nucleic acids), the magnetic beads are introduced into a sample, the magnetic beads can be used for separating the particles from other components by using a magnetic field, thereby realizing selective extraction of the nucleic acids, the ring resonator can be used for specific detection or sensing applications, the ring resonator can generate specific signal changes by interacting with the target substances, the ring resonator can be used for detecting the existence of microorganisms or other molecules, the nanoparticles can be used for labeling or enhancing detection signals, the nano particles can be combined with the target substances, the detection sensitivity is improved, and the target substances are easier to detect.
The method for performing lysis treatment on the microorganism sample in the step S3 comprises the following steps of; mechanical, repeated freeze thawing, high temperature boiling, enzymatic degradation, guanidine salt cleavage, alkali cleavage, CTAB cleavage, phenol cleavage, mechanical, physical disruption of microbial cell walls by mechanical forces (e.g., stirring, grinding, etc.), releasing the nucleic acids inside; repeated freezing and thawing to destroy cell wall and release nucleic acid; a high-temperature boiling method in which a microbial sample is boiled at a high temperature to destroy cell walls and release nucleic acids; enzymatic degradation methods, in which a microbial cell wall is degraded with a specific enzyme (e.g., protease, nuclease, etc.), thereby releasing nucleic acids; guanidine salt lysis methods, using guanidine salts (e.g., guanidine chloride) to disrupt microbial cell walls, facilitating release of nucleic acids; alkaline lysis, which uses alkaline conditions to destroy the microbial cell wall, resulting in release of nucleic acids; CTAB cleavage method, in which a surfactant such as CTAB (cetyltrimethylammonium bromide) is used to destroy cell membranes and release nucleic acids; phenol cleavage, in which the cell wall of the microorganism is cleaved with a solvent such as phenol and chloroform to isolate nucleic acids.
The method for purifying the microorganism sample in the step S3 comprises the following steps: organic solvent-ethanol precipitation, silicon membrane adsorption, magnetic bead, anion exchange, and nucleic acid automatic extraction; an organic solvent-ethanol precipitation method in which nucleic acid is precipitated by adding an organic solvent (usually, an alcohol such as ethanol) to thereby isolate nucleic acid; a silicon membrane adsorption method, wherein nucleic acid is combined with a silicon membrane or a silica gel column by utilizing the affinity of the material, and impurities are removed through an elution step, so that pure nucleic acid is finally obtained; the magnetic bead method uses magnetic particles with surface modification, the particles can specifically bind nucleic acid, and the nucleic acid and the magnetic beads are separated together under the action of a magnetic field to remove impurities; anion exchange methods utilize anion exchange resins or membranes to separate nucleic acids by the principles of adsorption and desorption. The nucleic acid is adsorbed on the anion exchange material, while impurities are removed; the automatic extraction method of nucleic acid utilizes automatic equipment to extract and purify nucleic acid, and improves the operation efficiency and consistency.
When the kit is used, nucleic acid in a microbial sample is extracted through a nucleic acid extraction module, a sequencing library is constructed through a sequencing library construction module by adopting a pathogenic microorganism detection kit, and the constructed library is sequenced through the sequencing module, so that nucleic acid sequence information is obtained; the detection result acquisition module acquires a detection result of the microbial sample according to the nucleic acid sequence information; compared with the prior art, the method does not need to culture the microbial sample, only needs to extract the microbial sample, then sequences the nucleic acid in the extracted microbial sample to obtain the nucleic acid sequence information, and can obtain the detection result of the microbial sample according to the nucleic acid sequence information, thereby greatly shortening the time consumed by detection and realizing the rapid detection of the microbial sample.
The technical solution of the present invention has been described in connection with the preferred embodiments, but it is easily understood by those skilled in the art that the scope of the present invention is not limited to these specific embodiments. Equivalent modifications and substitutions for related technical features may be made by those skilled in the art without departing from the principles of the present invention, and such modifications and substitutions will be within the scope of the present invention.
The foregoing description is only of the preferred embodiments of the invention and is not intended to limit the invention; various modifications and variations of the present invention will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (6)
1. A rapid microbial detection system for agricultural products, said system comprising;
a microorganism collection module; obtaining a microbial sample;
a sample pretreatment module; for removing interfering substances and sterilizing;
nucleic acid DNA extraction module: for extracting nucleic acids from a microbial sample;
a sequencing library construction module; the method is used for constructing a sequencing library by adopting a pathogenic microorganism detection kit;
a sequencing module; the method comprises the steps of sequencing a constructed library to obtain nucleic acid DNA sequence information;
the detection result acquisition module; and the detection result of the microorganism sample is obtained according to the nucleic acid DNA sequence information.
2. The method for detecting a rapid microbial detection system for agricultural products according to claim 1, wherein the method for detecting comprises:
s1, acquiring a microorganism sample;
s2, sterilizing the agricultural product microorganism sample;
s3, extracting nucleic acid in the microbial sample; introducing an amine group into a target object to modify the target object; injecting dimethyl dithiodipropimido and the nucleic acid obtained by the lysis treatment of the microbial sample onto the modified target object to generate a complex of the dimethyl dithiodipropimido and the nucleic acid; treating the complex of dimethyl dithiodipropionate and the nucleic acid with an elution buffer to extract the nucleic acid in the microbial sample; subjecting the microbial sample to lysis treatment to release nucleic acid in the microbial sample, subjecting the microbial sample to lysis treatment, subjecting the microbial sample subjected to the lysis treatment to purification treatment to remove impurities in the microbial sample, and subjecting the microbial sample subjected to the lysis treatment to purification treatment;
s4, constructing a sequencing library by adopting a pathogenic microorganism detection kit; the method comprises the steps of obtaining a pathogenic microorganism detection kit, wherein the pathogenic microorganism detection kit comprises a restriction enzyme mixed solution for identifying four bases, taq DNA polymerase, T4 DNA ligase, a connection promoter, dNTP, ATP, a connector, high-fidelity DNA polymerase and dithiothreitol; constructing a genome DNA sequencing library by adopting a pathogenic microorganism detection kit, and carrying out enzyme digestion on sample DNA to obtain fragmented DNA; directly carrying out terminal repair and dA tail addition on the fragmented DNA to obtain DNA with terminal repair and dA tail addition; connecting the DNA with the end repaired and dA tail with a joint to obtain a joint connection product with barcode; carrying out PCR amplification on the joint connection product with the barcode to obtain the genome DNA sequencing library;
s5, sequencing the constructed library by using a second generation sequencing platform to obtain nucleic acid sequence information; constructing different libraries according to the attribute of the microbial sample to be detected, wherein the libraries comprise whole genome library establishment, target segment capturing library establishment or transcriptome library establishment; sequencing the extracted nucleic acid in the microbial sample by adopting a second generation sequencer, and recording a base signal, wherein the sequencing comprises sequencing while synthesizing or sequencing while connecting; performing data processing on the base signals, wherein the data processing comprises sequence alignment, local alignment or base quality correction;
s6, constructing a microbial genome database, and comparing and analyzing the nucleic acid sequence information with the microbial genome database to determine the types and the contents of the microorganisms in the microbial samples.
3. The method for rapid detection of microorganisms in agricultural products according to claim 2, wherein the sterilization process is performed using a solution containing 75% alcohol at the time of the sterilization in S1.
4. The method for rapid detection of microorganisms in agricultural products according to claim 2, wherein the target object in S3 comprises a thin film device, magnetic beads, ring resonators, nanoparticles.
5. The method for detecting the rapid detection system for microorganisms of agricultural products according to claim 2, wherein the method for performing the lysis treatment on the microorganism sample in S3 comprises the steps of; mechanical method, repeated freeze thawing method, high temperature boiling method, enzymatic degradation method, guanidine salt degradation method, alkaline degradation method, CTAB degradation method, and phenol degradation method.
6. The method for rapid detection of microorganisms in agricultural products according to claim 2, wherein the method for purifying the microorganism sample in S3 comprises: organic solvent-ethanol precipitation, silicon membrane adsorption, magnetic bead, anion exchange, and nucleic acid automatic extraction.
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