Hepatitis E virus rapid detection method and system based on animal detection technology

Abstract

The invention relates to the technical field of molecular biology, and discloses a hepatitis E virus rapid detection method and system based on an animal detection technology, and the method comprises the following steps: carrying out nucleic acid extraction on a hepatitis E virus sample to obtain a nucleic acid extraction sample; establishing a PCR system of the nucleic acid extraction sample, and generating uniform microdroplets of the nucleic acid extraction sample and the PCR system; calculating a rupture coefficient of the uniform microdroplet, performing PCR amplification on the uniform microdroplet to obtain an amplified microdroplet, and performing signal detection on the amplified microdroplet by using a preset fluorescent dual-channel to obtain a fluorescent detection signal; generating a two-dimensional scatter diagram of the amplified droplets to determine FAM positive droplets in the amplified droplets; and analyzing the HEV genotype and variation site of the hepatitis E virus sample to establish an HEV strain evolutionary tree and risk-inducing factors of the hepatitis E virus sample, and generating an HEV geographical distribution heat map of the to-be-detected region by combining the HEV strain evolutionary tree and the risk-inducing factors. According to the invention, the accuracy of rapid detection of hepatitis E virus can be improved.

Description

TECHNICAL FIELD

[0001] The application relates to a rapid detection method and system for hepatitis E virus based on animal detection technology, and belongs to the technical field of molecular biology. BACKGROUND

[0002] The rapid detection of hepatitis E virus refers to the detection of samples (such as blood, urine or feces) in a short time (usually a few minutes to tens of minutes) using rapid detection reagents or equipment to determine whether there is a specific marker of hepatitis E virus (HEV). The rapid detection of hepatitis E virus is a convenient and rapid preliminary screening method, which helps to discover potential infections in time.

[0003] The traditional method of rapid detection of hepatitis E virus usually refers to a rapid detection kit (such as a test strip) based on immunochromatography technology, which can detect HEV antigens or antibodies in blood, urine or fecal samples, and can judge the results by observing the strip color development with the naked eye within a few minutes to tens of minutes. This method has relatively low sensitivity and specificity, may have false positives and false negatives, and can only provide qualitative (positive / negative) results, and cannot distinguish between acute infection and past infection, thereby resulting in poor accuracy of the rapid detection of hepatitis E virus. SUMMARY

[0004] The application provides a rapid detection method and system for hepatitis E virus based on animal detection technology, which aims to improve the accuracy of the rapid detection of hepatitis E virus.

[0005] To achieve the above-mentioned purpose, the application provides a rapid detection method for hepatitis E virus based on animal detection technology, which comprises the following steps:

[0006] Collecting a hepatitis E virus sample in a detection area, wherein the hepatitis E virus sample includes a pig-derived sample, a cow-derived sample, a sheep-derived sample and an environmental sample, extracting nucleic acid from the hepatitis E virus sample to obtain a nucleic acid extraction sample;

[0007] Establishing a PCR system for the nucleic acid extraction sample to generate uniform droplets of the nucleic acid extraction sample and the PCR system;

[0008] Calculating the breakage coefficient of the uniform droplets to perform PCR amplification on the uniform droplets to obtain amplified droplets, and using a preset fluorescent double channel to detect signals of the amplified droplets to obtain fluorescent detection signals;

[0009] Based on the fluorescent detection signals, a two-dimensional scatter plot of the amplified droplets is generated to determine FAM positive droplets in the amplified droplets;

[0010] Based on the FAM-positive droplets, the HEV genotype and mutation sites of the hepatitis E virus sample are analyzed to establish an HEV strain evolutionary tree and risk-inducing factors of the hepatitis E virus sample. Combined with the HEV strain evolutionary tree and risk-inducing factors, a HEV geographical distribution heat map of the test area is generated.

[0011] Optionally, extracting nucleic acid from the hepatitis E virus sample to obtain a nucleic acid extraction sample comprises:

[0012] determining a lysis buffer for the hepatitis E virus sample;

[0013] mixing the hepatitis E virus sample and the lysis buffer to obtain a mixed sample-lysis buffer;

[0014] Adding a magnetic bead suspension to the mixed sample-lysis buffer to obtain a magnetic bead mixture;

[0015] analyzing the magnetic bead uniformity coefficient of the magnetic bead mixture, and when the magnetic bead uniformity coefficient meets a preset magnetic bead uniformity threshold, washing the magnetic bead mixture to obtain a washed magnetic bead mixture;

[0016] The washed magnetic bead mixture is eluted to obtain a nucleic acid extraction sample of the hepatitis E virus sample.

[0017] Optionally, analyzing the magnetic bead uniformity coefficient of the magnetic bead mixture includes:

[0018] Acquiring a mixed solution image of the magnetic bead mixed solution;

[0019] Identify the number of magnetic beads in the mixed solution image;

[0020] Based on the number of magnetic beads, the magnetic bead uniformity coefficient of the magnetic bead mixture was calculated using the following formula:

[0021]

[0022] in, It represents the uniformity coefficient of magnetic beads in the magnetic bead mixture. Indicates the number of magnetic beads in the magnetic bead mixture. Indicates the first The Euclidean distance from a bead to its nearest neighbor bead, It represents the average Euclidean distance between the magnetic beads and the magnetic beads mixture. Indicates the effective radius of the magnetic bead.

[0023] Optionally, generating uniform droplets of the nucleic acid extraction sample and the PCR system comprises:

[0024] The cross structure of the nucleic acid extraction sample and the PCR system is designed;

[0025] According to the cross structure, a cross microfluidic chip of the nucleic acid extraction sample is configured;

[0026] A clogging state of the cross microfluidic chip is analyzed;

[0027] When the clogging state meets a preset clogging standard, a uniform microdroplet of the nucleic acid extraction sample and the PCR system is generated by using the cross microfluidic chip.

[0028] Optionally, the analysis of the clogging state of the cross microfluidic chip comprises:

[0029] Clogging test data of the cross microfluidic chip is obtained;

[0030] The clogging test data is used to determine a chip inlet pressure, a chip outlet pressure, and a fluid dynamic viscosity of the cross microfluidic chip;

[0031] Based on the chip inlet pressure, the chip outlet pressure, and the fluid dynamic viscosity, a clogging state of the cross microfluidic chip is calculated by using the following formula:

[0032]

[0033] wherein, represents the clogging state of the cross microfluidic chip, represents the chip inlet pressure of the cross microfluidic chip, represents the chip outlet pressure of the cross microfluidic chip, represents the maximum pressure difference of the cross microfluidic chip, represents the fluid dynamic viscosity of the cross microfluidic chip, represents the effective length of the microchannel of the cross microfluidic chip, represents the volume flow rate of the cross microfluidic chip, represents the constant pi, represents the hydraulic radius of the microchannel.

[0034] Optionally, the calculation of the breakage coefficient of the uniform microdroplet comprises:

[0035] A microscopic image of the uniform microdroplet is collected;

[0036] The microscopic image is binarized and segmented to obtain a segmented microscopic image;

[0037] The segmented microscopic image is identified to obtain complete microdroplets and broken microdroplets, so as to calculate a static breakage coefficient of the uniform microdroplet;

[0038] a time-stress coupling factor of the uniform microdroplet is defined;

[0039] a fracture coefficient of the uniform microdroplet is calculated in combination with the time-stress coupling factor and the static fracture coefficient.

[0040] Optionally, the calculating the fracture coefficient of the uniform microdroplet in combination with the time-stress coupling factor and the static fracture coefficient comprises:

[0041] a material stress coefficient and a fracture kinetics index of the uniform microdroplet are analyzed based on the time-stress coupling factor;

[0042] a fracture coefficient of the uniform microdroplet is calculated according to the static fracture coefficient, the material stress coefficient and the fracture kinetics index by using the following formula:

[0043]

[0044] wherein, represents the fracture coefficient of the uniform microdroplet, represents the static fracture coefficient of the uniform microdroplet, represents the material stress coefficient, represents the actual operation time, represents the reference time, represents the fracture kinetics index.

[0045] Optionally, the generating the two-dimensional scatter plot of the amplification microdroplet based on the fluorescent detection signal comprises:

[0046] background correction is performed on the fluorescent detection signal to obtain a corrected fluorescent detection signal;

[0047] standardization processing is performed on the corrected fluorescent detection signal to obtain a processed fluorescent detection signal;

[0048] a HEX fluorescent intensity and a FAM fluorescent intensity of the processed fluorescent detection signal are analyzed;

[0049] a two-dimensional data set of the processed fluorescent detection signal is constructed based on the HEX fluorescent intensity and the FAM fluorescent intensity;

[0050] a two-dimensional scatter plot of the amplification microdroplet is established according to the two-dimensional data set.

[0051] Optionally, the analyzing the HEV genotype and the mutation site of the hepatitis E virus sample based on the FAM positive microdroplet comprises:

[0052] the FAM positive microdroplets are merged to obtain merged FAM positive microdroplets;

[0053] extracting PCR amplification products of the combined FAM positive microdroplets to obtain HEV target gene fragments of the combined FAM positive microdroplets;

[0054] constructing a sequencing library of the HEV target gene fragments to obtain raw sequencing data of the HEV target gene fragments;

[0055] quality control of the raw sequencing data to obtain target sequencing data;

[0056] and identifying variant sites of the target sequencing data;

[0057] According to the variant sites, the HEV genotype of the hepatitis E virus sample is determined.

[0058] To solve the above problems, the application also provides a rapid detection system for hepatitis E virus based on animal detection technology, which comprises:

[0059] A sample nucleic acid extraction module is used to collect a hepatitis E virus sample in a to-be-tested area, wherein the hepatitis E virus sample comprises a pig-derived sample, a bovine-derived sample, a sheep-derived sample and an environmental sample, and nucleic acid extraction is performed on the hepatitis E virus sample to obtain a nucleic acid extraction sample;

[0060] A uniform microdroplet construction module is used to establish a PCR system of the nucleic acid extraction sample to generate uniform microdroplets of the nucleic acid extraction sample and the PCR system;

[0061] A fluorescence signal detection module is used to calculate a breakage coefficient of the uniform microdroplets, to perform PCR amplification on the uniform microdroplets to obtain amplified microdroplets, to perform signal detection on the amplified microdroplets by using a preset fluorescence double channel to obtain a fluorescence detection signal;

[0062] A positive microdroplet analysis module is used to generate a two-dimensional scatter plot of the amplified microdroplets based on the fluorescence detection signal to determine FAM positive microdroplets in the amplified microdroplets;

[0063] An HEV analysis module is used to analyze an HEV genotype and a variant site of the hepatitis E virus sample according to the FAM positive microdroplets to establish an HEV strain phylogenetic tree and a risk inducing factor of the hepatitis E virus sample, and to generate an HEV geographic distribution heat map of the to-be-tested area in combination with the HEV strain phylogenetic tree and the risk inducing factor.

[0064] Firstly, the method improves the detection efficiency and accuracy. The method realizes the rapid generation and PCR amplification of uniform microdroplets through microfluidic chip technology, greatly shortens the detection time, and the fluorescence double-channel signal detection technology can accurately detect the signal of the amplified microdroplets, thereby improving the detection accuracy. Secondly, the method can analyze the genotype and mutation site of the virus. Through the analysis of the FAM positive microdroplets, the genotype and mutation site of the hepatitis E virus can be determined, which provides an important basis for the tracing and transmission research of the virus. In addition, the method can establish the virus strain phylogenetic tree and risk inducing factor, which helps to deeply understand the transmission rule and risk factor of the virus. Finally, the method can generate a geographical distribution heat map of the virus, which provides an important reference for public health management. Therefore, the application can improve the accuracy of rapid detection of hepatitis E virus. BRIEF DESCRIPTION OF DRAWINGS

[0065] Figure 1 A flowchart of a rapid detection method of hepatitis E virus based on animal detection technology provided by an embodiment of the application is shown.

[0066] Figure 2 A schematic diagram of constructing an HEV strain phylogenetic tree for implementing the rapid detection method of hepatitis E virus based on animal detection technology provided by an embodiment of the application is shown.

[0067] Figure 3 A module schematic diagram for implementing the rapid detection method of hepatitis E virus based on animal detection technology provided by an embodiment of the application is shown.

[0068] The purposes, functional features and advantages of the application will be further described with reference to the embodiments and the accompanying drawings. DETAILED DESCRIPTION

[0069] It should be understood that the specific embodiments described herein are only used to explain the application and not to limit the application.

[0070] The embodiment of the application provides a rapid detection method of hepatitis E virus based on animal detection technology. The execution subject of the rapid detection method of hepatitis E virus based on animal detection technology includes but is not limited to at least one of electronic devices such as a server, a terminal and the like which can be configured to execute the method provided by the embodiment of the application. In other words, the rapid detection method of hepatitis E virus based on animal detection technology can be executed by software or hardware installed in a terminal device or a server device. The server includes but is not limited to a single server, a server cluster, a cloud server or a cloud server cluster and the like.

[0071] Embodiment 1

[0072] Reference Figure 1As shown, a flowchart of a rapid detection method for hepatitis E virus based on animal detection technology is provided in an embodiment of the present application. In this embodiment, the rapid detection method for hepatitis E virus based on animal detection technology comprises the following steps:

[0073] S1, collecting a hepatitis E virus sample in a to-be-detected area, wherein the hepatitis E virus sample comprises a pig-derived sample, a cow-derived sample, a sheep-derived sample, and an environmental sample, nucleic acid extraction is performed on the hepatitis E virus sample to obtain a nucleic acid extraction sample.

[0074] It should be explained that the to-be-detected area refers to a specific geographical range or place that needs to be monitored or investigated for hepatitis E virus (HEV), the pig-derived sample refers to a sample collected from a pig, the cow-derived sample refers to a sample collected from a cow, the sheep-derived sample refers to a sample collected from a sheep, and the environmental sample refers to a sample collected in the to-be-detected area and not directly derived from an animal individual, such as a slaughter platform, a knife, a device surface wiping sample, ground, and a drainage ditch sludge sample.

[0075] The present application performs nucleic acid extraction on the hepatitis E virus sample to obtain a nucleic acid extraction sample, which can provide a basis for subsequent hepatitis E virus analysis.

[0076] In detail, the nucleic acid extraction on the hepatitis E virus sample to obtain a nucleic acid extraction sample comprises the following steps:

[0077] Determining a lysis buffer for the hepatitis E virus sample;

[0078] Mixing the hepatitis E virus sample and the lysis buffer to obtain a mixed sample-lysis buffer;

[0079] Adding a magnetic bead suspension to the mixed sample-lysis buffer to obtain a magnetic bead mixture;

[0080] Analyzing a magnetic bead uniformity coefficient of the magnetic bead mixture, when the magnetic bead uniformity coefficient meets a preset magnetic bead uniformity threshold, washing the magnetic bead mixture to obtain a washed magnetic bead mixture;

[0081] Eluting the washed magnetic bead mixture to obtain a nucleic acid extraction sample of the hepatitis E virus sample.

[0082] The lysis buffer refers to a chemical solution used to lyse hepatitis E virus (HEV) sample cells or virus particles, comprising proteinase K, a detergent (such as SDS), and a chelating agent (such as EDTA). The mixed sample-lysis buffer refers to a preliminary reaction system formed by mixing the porcine, bovine, ovine, or environmental sample to be tested with the lysis buffer. The magnetic bead mixture refers to a suspension of magnetic beads (such as silica gel or silica magnetic beads) coated with magnetic beads that specifically bind to nucleic acids. The magnetic bead mixture refers to the suspension formed after adding the magnetic bead suspension to the mixed sample-lysis buffer. The magnetic bead uniformity coefficient refers to an index of the uniformity of the dispersion of magnetic beads in the mixture. The magnetic bead uniformity threshold refers to a preset standard value of the magnetic bead uniformity coefficient. The washing magnetic bead mixture refers to the step of washing the magnetic beads with a washing solution (such as a low-salt buffer) after removing unbound impurities by magnetic separation to purify the bound nucleic acids. The nucleic acid extraction sample refers to a pure viral nucleic acid solution released from the magnetic beads by heating or chemical elution after magnetic bead adsorption and washing.

[0083] Optionally, eluting the washed magnetic bead mixture to obtain the nucleic acid extraction sample of the hepatitis E virus sample refers to removing the centrifuge tube containing the washed magnetic beads from the magnetic stand and adding a specified volume of elution buffer (usually sterile deionized water or a low-salt buffer with an appropriate pH value that can promote the dissociation of nucleic acids from the magnetic beads) for elution.

[0084] Furthermore, the analyzing the magnetic bead uniformity coefficient of the magnetic bead mixture includes:

[0085] Acquiring a mixed solution image of the magnetic bead mixed solution;

[0086] Identify the number of magnetic beads in the mixed solution image;

[0087] Based on the number of magnetic beads, the magnetic bead uniformity coefficient of the magnetic bead mixture was calculated using the following formula:

[0088]

[0089] in, It represents the uniformity coefficient of magnetic beads in the magnetic bead mixture. Indicates the number of magnetic beads in the magnetic bead mixture. Indicates the first The Euclidean distance from a bead to its nearest neighbor bead, It represents the average Euclidean distance between the magnetic beads and the magnetic beads mixture. Indicates the effective radius of the magnetic bead.

[0090] The mixed liquid image refers to a picture of a magnetic bead mixed liquid (i.e., a state of magnetic beads suspended in a liquid) taken by an imaging device (such as a microscope or a camera), the magnetic bead quantity refers to the total number of magnetic beads identified and counted by image processing and analysis technology in the obtained mixed liquid image, the Euclidean distance refers to the straight-line distance between a particular magnetic bead (referred to as the cth magnetic bead) and another magnetic bead closest to it in the image, the Euclidean distance average value refers to the sum of the Euclidean distances from all magnetic beads to their respective nearest neighbors in the mixed liquid image divided by the total number of magnetic beads, and the effective action radius refers to the distance range at which significant interactions (such as magnetic attraction, electrostatic repulsion, steric hindrance, etc.) between magnetic beads begin to occur.

[0091] S2, a PCR system of the nucleic acid extraction sample is established to generate uniform droplets of the nucleic acid extraction sample and the PCR system.

[0092] The establishment of the PCR system of the nucleic acid extraction sample can ensure accurate detection of HEV in animal samples (such as pig liver and feces), and is suitable for subsequent droplet generation and absolute quantitative analysis.

[0093] The generation of uniform droplets of the nucleic acid extraction sample and the PCR system realizes rapid generation of uniform droplets, greatly shortening the detection time.

[0094] In detail, the generation of uniform droplets of the nucleic acid extraction sample and the PCR system includes:

[0095] The cross structure of the nucleic acid extraction sample and the PCR system is designed;

[0096] According to the cross structure, the cross microfluidic chip of the nucleic acid extraction sample is configured;

[0097] The clogging state of the cross microfluidic chip is analyzed;

[0098] When the clogging state meets the preset clogging standard, the uniform droplets of the nucleic acid extraction sample and the PCR system are generated using the cross microfluidic chip.

[0099] The cross structure refers to a structure formed by the vertical intersection of two fluid channels (such as a nucleic acid extraction sample channel and a PCR system channel) in a microfluidic chip. The cross microfluidic chip refers to a microfluidic chip designed based on the cross structure. The clogging state refers to the phenomenon of increased flow resistance caused by particulate matter (such as magnetic bead residues), bubbles, or abnormal fluid viscosity in the chip channel. The clogging standard refers to a judgment threshold for quantifying the degree of clogging. The uniform droplet refers to a droplet with stable volume and uniform size generated by optimizing the fluid dynamics (such as flow rate ratio, channel geometry) of the cross structure.

[0100] Optionally, the design of the cross structure of the nucleic acid extraction sample and the PCR system can be designed by using the multi-physical field simulation software COMSOL Multiphysics.

[0101] Further, the analysis of the clogging state of the cross microfluidic chip comprises:

[0102] Obtaining clogging test data of the cross microfluidic chip;

[0103] Determining the chip inlet pressure, chip outlet pressure, and fluid dynamic viscosity of the cross microfluidic chip through the clogging test data;

[0104] Based on the chip inlet pressure, chip outlet pressure, and fluid dynamic viscosity, the clogging state of the cross microfluidic chip is calculated using the following formula:

[0105]

[0106] Wherein, represents the clogging state of the cross microfluidic chip, represents the chip inlet pressure of the cross microfluidic chip, represents the chip outlet pressure of the cross microfluidic chip, represents the maximum pressure difference of the cross microfluidic chip, represents the fluid dynamic viscosity of the cross microfluidic chip, represents the effective length of the microchannel of the cross microfluidic chip, represents the volumetric flow rate of the cross microfluidic chip, represents the circumference ratio, represents the hydraulic radius of the microchannel.

[0107] The clogging test data refers to various parameter information collected when testing whether the cross microfluidic chip is clogged, which generally includes but is not limited to: the pressure at the chip inlet, the pressure at the chip outlet, the fluid flow through the chip, the viscosity of the fluid, etc., wherein the chip inlet pressure refers to the pressure applied at the inlet of the microfluidic chip fluid channel, the chip outlet pressure refers to the pressure at the outlet of the microfluidic chip fluid channel after the fluid flows through the microfluidic chip, the fluid dynamic viscosity refers to a physical quantity describing the internal friction or resistance to flow of the fluid, the maximum tolerable pressure difference of the chip refers to the maximum difference between the inlet pressure and the outlet pressure that the microfluidic chip can withstand in design, the effective length of the microchannel refers to the actual length of the channel through which the fluid flows in the microfluidic chip, the volumetric flow rate refers to the volume of fluid flowing through a certain cross section of the microfluidic chip channel per unit time, and the hydraulic radius of the microchannel refers to a parameter reflecting the influence of the geometric characteristics of the channel on the flow resistance.

[0108] S3, calculate the breakage coefficient of the uniform microdroplet to perform PCR amplification on the uniform microdroplet to obtain an amplified microdroplet, and use a preset fluorescent double-channel to detect a signal of the amplified microdroplet to obtain a fluorescent detection signal.

[0109] The calculation of the breakage coefficient of the uniform microdroplet can comprehensively evaluate the stability of the microdroplet and significantly improve the reliability of HEV detection.

[0110] In detail, the calculation of the breakage coefficient of the uniform microdroplet includes:

[0111] Collecting a microscopic image of the uniform microdroplet;

[0112] Performing binary segmentation on the microscopic image to obtain a segmented microscopic image;

[0113] Identifying the complete microdroplets and broken microdroplets in the segmented microscopic image to calculate the static breakage coefficient of the uniform microdroplet;

[0114] Defining a time-stress coupling factor of the uniform microdroplet;

[0115] And combining the time-stress coupling factor and the static breakage coefficient, calculating the breakage coefficient of the uniform microdroplet.

[0116] The micrograph refers to an image of a uniform microdroplet taken by a microscope (usually a high-resolution optical microscope or a fluorescence microscope), the segmented micrograph refers to a result image obtained after processing the original micrograph, the static breakage coefficient refers to an index for evaluating the breakage of the microdroplet by observing the integrity of the microdroplet in the segmented micrograph under relatively static or low flow conditions, the time-stress coupling factor refers to a parameter describing the mutual relationship between the stress received by the microdroplet in the dynamic flow process and the existence time of the microdroplet, and the breakage coefficient refers to an index for comprehensively evaluating the overall breakage tendency of the uniform microdroplet under specific working conditions (combining static and dynamic factors).

[0117] Optionally, the binarization segmentation of the micrograph to obtain the segmented micrograph can be implemented by binarization segmentation.

[0118] Further, the combination of the time-stress coupling factor and the static breakage coefficient to calculate the breakage coefficient of the uniform microdroplet comprises:

[0119] Based on the time-stress coupling factor, analyzing the material stress coefficient and the breakage kinetics index of the uniform microdroplet;

[0120] According to the static breakage coefficient, the material stress coefficient and the breakage kinetics index, the breakage coefficient of the uniform microdroplet is calculated by using the following formula:

[0121]

[0122] wherein, the breakage coefficient of the uniform microdroplet, the static breakage coefficient of the uniform microdroplet, the material stress coefficient, the actual operation time, the reference time, the breakage kinetics index.

[0123] The material stress coefficient refers to the ability of the liquid (for example, a solution containing a nucleic acid extraction sample or a PCR system) constituting the microdroplet to resist external stress (such as shear force, tensile force) to cause interface breakage or internal structure damage, the breakage kinetics index refers to the rate describing the microdroplet breakage process, the actual operation time refers to the duration of the microdroplet experiencing specific operation conditions (for example, flowing in a certain channel segment, staying in a certain reaction chamber) in the microfluidic chip, and the reference time refers to the time required for the microdroplet to complete one cycle in the chip.

[0124] It should be explained that the amplified microdroplets refer to microdroplets formed after PCR (polymerase chain reaction) amplification, the fluorescent double channel refers to two independent detection paths used in the fluorescent detection system, including FAM channel and HEX channel, the double channel allows simultaneous detection of two different signals, which helps to distinguish true positive signals from background noise, non-specific amplification or system errors. For example, one channel shows positive, while the other channel (negative control channel) has no signal, which can increase the reliability of the results, and the fluorescent detection signal refers to the quantified light intensity data detected from the amplified microdroplets by the fluorescent double channel system.

[0125] S4. Based on the fluorescent detection signal, a two-dimensional scatter plot of the amplified microdroplets is generated to determine the FAM positive microdroplets in the amplified microdroplets.

[0126] Based on the fluorescent detection signal, the two-dimensional scatter plot of the amplified microdroplets can generate a clear and intuitive two-dimensional scatter plot for displaying and analyzing the fluorescent detection signal of the amplified microdroplets, thereby assisting in judging the results of PCR amplification.

[0127] In detail, the two-dimensional scatter plot of the amplified microdroplets based on the fluorescent detection signal comprises:

[0128] The background correction is performed on the fluorescent detection signal to obtain a corrected fluorescent detection signal;

[0129] The corrected fluorescent detection signal is standardized to obtain a processed fluorescent detection signal;

[0130] The HEX fluorescence intensity and the FAM fluorescence intensity of the processed fluorescent detection signal are analyzed;

[0131] Based on the HEX fluorescence intensity and the FAM fluorescence intensity, a two-dimensional data set of the processed fluorescent detection signal is constructed;

[0132] According to the two-dimensional data set, a two-dimensional scatter plot of the amplified microdroplets is established.

[0133] Wherein, the corrected fluorescence detection signal refers to the data after removing the background noise from the original fluorescence signal. The background noise may come from non-specific signals such as instrument dark current, environmental light interference or reagent autofluorescence. The processed fluorescence detection signal refers to the data after further standardization of the corrected signal, eliminating systematic errors between different experimental batches or instruments. The HEX fluorescence intensity refers to the signal intensity of the HEX fluorescent dye (commonly used in the reference or control channel). The FAM fluorescence intensity refers to the signal intensity of the FAM fluorescent dye (commonly used in the target detection channel). The two-dimensional data set refers to a set of coordinate pairs composed of processed HEX and FAM fluorescence intensities. Each microdroplet corresponds to a data point (HEX intensity, FAM intensity). The two-dimensional scatter plot refers to plotting each microdroplet in the two-dimensional data set as a scatter point with HEX fluorescence intensity as the X-axis and FAM fluorescence intensity as the Y-axis.

[0134] Optionally, the two-dimensional scatter plot of the amplified microdroplets can be plotted by data analysis software (such as MATLAB, Python's matplotlib library, R language, etc.) or professional graphics software (such as GraphPad Prism, Excel, etc.) according to the two-dimensional data set.

[0135] The present application determines the FAM positive microdroplets in the amplified microdroplets, which can realize the quantitative analysis of FAM in the amplified microdroplets, and provide a basis for the later FAM expansion analysis. Wherein, the FAM positive microdroplets refer to microdroplets that produce fluorescence signal intensity exceeding the threshold after PCR amplification.

[0136] S5, according to the FAM positive microdroplets, analyzing the HEV genotype and variation site of the hepatitis E virus sample to establish the HEV strain phylogenetic tree and risk inducing factor of the hepatitis E virus sample, combining the HEV strain phylogenetic tree and risk inducing factor, generating the HEV geographic distribution heat map of the region to be tested.

[0137] The present application provides an important basis for virus tracing and transmission research according to the FAM positive microdroplets, analyzing the HEV genotype and variation site of the hepatitis E virus sample.

[0138] In detail, according to the FAM positive microdroplets, analyzing the HEV genotype and variation site of the hepatitis E virus sample includes:

[0139] The FAM positive microdroplets are combined to obtain combined FAM positive microdroplets;

[0140] The PCR amplification product of the combined FAM positive microdroplets is extracted to obtain the HEV target gene fragment of the combined FAM positive microdroplets;

[0141] constructing a sequencing library of the HEV target gene fragment to obtain raw sequencing data of the HEV target gene fragment;

[0142] quality control of the raw sequencing data to obtain target sequencing data;

[0143] and identifying variant sites of the target sequencing data;

[0144] determining the HEV genotype of the hepatitis E virus sample according to the variant sites.

[0145] wherein the merged FAM positive droplets refer to the product obtained by physically merging all previously identified FAM channel positive droplets, the PCR amplification product refers to the DNA fragment synthesized by PCR (polymerase chain reaction), the HEV target gene fragment refers to the specific part of the hepatitis E virus genome that the primers are designed to amplify in the PCR amplification step, the sequencing library refers to a collection of pre-processed products of target DNA (or cDNA after reverse transcription of RNA) fragments to meet the requirements of specific sequencing platforms (such as Illumina, PacBio, Oxford Nanopore, etc.), the raw sequencing data refers to the raw output data directly generated by the sequencer without any processing or correction, the target sequencing data refers to the sequence data with higher quality and can be used for subsequent analysis after a series of quality control (QC) and filtering steps, including removing adapter sequences, removing low-quality reads, removing short reads, etc., the polymorphic site refers to a specific genomic location where base changes occur in the sequencing data, the HEV genotype refers to the classification of hepatitis E virus according to the similarity of viral gene sequences (especially target gene fragment sequences), and the variant site refers to all sequence changes relative to the reference sequence or a certain benchmark (such as epidemic strain, vaccine strain).

[0146] Optionally, the construction of the sequencing library of the HEV target gene fragment can be performed according to the selected sequencing platform (such as Illumina, PacBio, Nanopore, etc.) to construct the library of the purified HEV gene fragment.

[0147] Optionally, the determination of the HEV genotype of the hepatitis E virus sample according to the variant sites can be performed by comparing these variant sites with the characteristic variant sites of known HEV genotypes (HEV 1-8) to determine the genotype of the sample.

[0148] The HEV strain phylogenetic tree of the hepatitis E virus sample and the risk inducing factor can understand the genetic background (phylogenetic tree) of a single HEV sample, and can also combine its molecular characteristics and epidemiological background to deeply explore the risk factors that may cause infection, transmission or disease severity. The HEV strain phylogenetic tree refers to a graphical representation method that displays the genetic relationship and evolutionary history between different hepatitis E virus (HEV) strains. In detail, the HEV strain phylogenetic tree can be referred to Figure 2 The HEV strain phylogenetic tree construction schematic diagram for implementing the rapid detection method of hepatitis E virus based on animal detection technology provided by an embodiment of the present application is as follows: first, starting from the starting point of "HEV genotype and variation site", enter the first key step "sequence alignment". In this stage, the target sequence needs to be compared with the known reference sequence to identify possible differences and similarities. After completing the sequence alignment, the reference sequence is selected according to the alignment result, which is crucial for subsequent multiple sequence alignment and determines the reference sequence to be used. Next is the "multiple sequence alignment" link, which aligns the selected reference sequence with other related sequences to better understand their genetic relationship. Through the results of multiple sequence alignment, the comparison result analysis can be carried out to find out which regions have variations or conservation. These information is very helpful for understanding the evolutionary history and transmission route of the virus. After analyzing the comparison results, the next important stage "phylogenetic tree construction" is entered. The evolutionary distance needs to be calculated, which is a way to measure the genetic difference between different sequences. The last step is "building tree structure", that is, converting the previously calculated evolutionary distance into a visual phylogenetic tree graph. This graph not only shows the relationship between the sequences, but also reveals their common ancestor and the direction and time scale of evolution; the risk inducing factor refers to a factor that increases the likelihood of an individual being infected with hepatitis E virus or affects the severity of the disease after infection. These factors can be related to the environment, behavior, host or virus itself. For example, through the phylogenetic tree, it can be identified which strains are genetically very similar (closely clustered). If these closely clustered strains come from patients in different regions or at different times, all pointing to the same source (for example, a certain specific farm, slaughterhouse, water source or imported food batch), then this source becomes a risk inducing factor.

[0149] Finally, the present application combines the HEV strain phylogenetic tree and risk inducing factors to generate the HEV geographic distribution heat map of the to-be-tested region, which converts complex virus genetics and epidemiology data into easily understood geographic spatial information. The HEV geographic distribution heat map refers to a visual tool that displays the distribution of hepatitis E virus (HEV) and the relative risk level in the to-be-tested region. In detail, the HEV geographic distribution heat map can match the genotype / variant site of each positive sample, the associated information of risk inducing factors, and the geographic location or internal region location of the corresponding to-be-tested region, and is established based on geographic information system (GIS) software (such as ArcGIS, QGIS) or data visualization tools.

[0150] Firstly, the method improves the detection efficiency and accuracy. The method realizes the rapid generation and PCR amplification of uniform droplets through microfluidic chip technology, greatly shortens the detection time, and the fluorescence double-channel signal detection technology can accurately detect the signal of the amplification droplet, thereby improving the detection accuracy. Secondly, the method can analyze the virus genotype and variant site. Through the analysis of FAM positive droplets, the genotype and variant site of hepatitis E virus can be determined, which provides an important basis for virus tracing and transmission research. In addition, the method can establish the virus strain phylogenetic tree and risk inducing factors, which helps to deeply understand the transmission rule and risk factors of the virus. Finally, the method can generate the geographic distribution heat map of the virus, which provides an important reference for public health management. Therefore, the present application can improve the accuracy of rapid detection of hepatitis E virus.

[0151] Embodiment 2:

[0152] As shown in Figure 3 FIG. 1 is a functional module diagram of a hepatitis E virus rapid detection system based on animal detection technology according to the present application.

[0153] The hepatitis E virus rapid detection system based on animal detection technology 300 according to the present application can be installed in an electronic device. According to the functions to be implemented, the hepatitis E virus rapid detection system based on animal detection technology can include a sample nucleic acid extraction module 301, a uniform droplet construction module 302, a fluorescence signal detection module 303, a positive droplet analysis module 304, and an HEV analysis module 305. The modules according to the present application can also be referred to as units, which refer to a series of computer program segments that can be executed by an electronic device processor and can complete a fixed function, and are stored in the memory of the electronic device.

[0154] In the embodiments of the present application, the functions of the modules / units are as follows:

[0155] The sample nucleic acid extraction module 301 is used to collect hepatitis E virus samples from the test area, wherein the hepatitis E virus samples include pig samples, cattle samples, sheep samples and environmental samples, and extract nucleic acid from the hepatitis E virus samples to obtain nucleic acid extraction samples;

[0156] The uniform droplet construction module 302 is used to establish the PCR system of the nucleic acid extraction sample and generate uniform droplets of the nucleic acid extraction sample and the PCR system;

[0157] The fluorescence signal detection module 303 is used to calculate the rupture coefficient of the uniform droplet, perform PCR amplification on the uniform droplet to obtain an amplified droplet, and perform signal detection on the amplified droplet using a preset fluorescence dual channel to obtain a fluorescence detection signal;

[0158] The positive droplet analysis module 304 is configured to generate a two-dimensional scatter plot of the amplified droplets based on the fluorescence detection signal to determine the FAM-positive droplets in the amplified droplets;

[0159] The HEV analysis module 305 is used to analyze the HEV genotype and mutation sites of the hepatitis E virus sample based on the FAM-positive droplets to establish the HEV strain evolution tree and risk-inducing factors of the hepatitis E virus sample, and combine the HEV strain evolution tree and risk-inducing factors to generate a HEV geographical distribution heat map of the test area.

[0160] In detail, the modules in the hepatitis E virus rapid detection system 300 based on animal detection technology in the embodiment of the present invention are used in the same manner as above. Figure 1 The same technical means are used as the rapid detection method for hepatitis E virus based on animal detection technology described in , and can produce the same technical effects, so they will not be repeated here.

[0161] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention.

[0162] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A rapid detection method for hepatitis E virus based on animal detection technology, characterized in that: The method comprises: Collecting hepatitis E virus samples from the area to be tested, wherein the hepatitis E virus samples include pig-derived samples, cattle-derived samples, sheep-derived samples, and environmental samples, and performing nucleic acid extraction on the hepatitis E virus samples to obtain nucleic acid extracted samples; establishing a PCR system for the nucleic acid extraction sample to generate uniform droplets of the nucleic acid extraction sample and the PCR system; Calculating the rupture coefficient of the uniform droplet, performing PCR amplification on the uniform droplet to obtain an amplified droplet, and performing signal detection on the amplified droplet using a preset fluorescent dual channel to obtain a fluorescent detection signal; generating a two-dimensional scatter plot of the amplified droplets based on the fluorescence detection signal to determine FAM-positive droplets in the amplified droplets; Based on the FAM-positive droplets, the HEV genotype and mutation sites of the hepatitis E virus sample are analyzed to establish an HEV strain evolutionary tree and risk-inducing factors of the hepatitis E virus sample. Combined with the HEV strain evolutionary tree and risk-inducing factors, a HEV geographical distribution heat map of the test area is generated.

2. The method for rapid detection of hepatitis E virus based on animal detection technology according to claim 1, characterized in that: The nucleic acid extraction of the hepatitis E virus sample to obtain a nucleic acid extraction sample comprises: determining a lysis buffer for the hepatitis E virus sample; mixing the hepatitis E virus sample and the lysis buffer to obtain a mixed sample-lysis buffer; Adding a magnetic bead suspension to the mixed sample-lysis buffer to obtain a magnetic bead mixture; analyzing the magnetic bead uniformity coefficient of the magnetic bead mixture, and when the magnetic bead uniformity coefficient meets a preset magnetic bead uniformity threshold, washing the magnetic bead mixture to obtain a washed magnetic bead mixture; The washed magnetic bead mixture is eluted to obtain a nucleic acid extraction sample of the hepatitis E virus sample.

3. The method for rapid detection of hepatitis E virus based on animal detection technology according to claim 2, characterized in that: The analyzing the magnetic bead uniformity coefficient of the magnetic bead mixture comprises: Acquiring a mixed solution image of the magnetic bead mixed solution; Identify the number of magnetic beads in the mixed solution image; Based on the number of magnetic beads, the magnetic bead uniformity coefficient of the magnetic bead mixture was calculated using the following formula: ； in, It represents the uniformity coefficient of magnetic beads in the magnetic bead mixture. Indicates the number of magnetic beads in the magnetic bead mixture. Indicates the first The Euclidean distance from a bead to its nearest neighbor bead, It represents the average Euclidean distance between the magnetic beads and the magnetic beads mixture. Indicates the effective radius of the magnetic bead.

4. The method for rapid detection of hepatitis E virus based on animal detection technology according to claim 3, characterized in that: The generating of uniform droplets of the nucleic acid extraction sample and the PCR system comprises: Designing a cross structure of the nucleic acid extraction sample and the PCR system; According to the cross structure, a cross microfluidic chip for extracting nucleic acid samples is configured; Analyzing the blockage state of the cross-microfluidic chip; When the blockage state meets a preset blockage standard, the cross microfluidic chip is used to generate uniform droplets of the nucleic acid extraction sample and the PCR system.

5. The method for rapid detection of hepatitis E virus based on animal detection technology according to claim 4, characterized in that: The analyzing the blockage state of the cross microfluidic chip includes: Obtaining blockage test data of the cross-microfluidic chip; Determining the chip inlet pressure, chip outlet pressure, and fluid dynamic viscosity of the cross-microfluidic chip based on the blockage test data; The blockage state of the cross-microfluidic chip is calculated based on the chip inlet pressure, the chip outlet pressure and the fluid dynamic viscosity.

6. The method for rapid detection of hepatitis E virus based on animal detection technology according to claim 5, characterized in that: The calculating the breakup coefficient of the uniform droplet comprises: collecting a microscopic image of the uniform droplet; performing binary segmentation on the microscopic image to obtain a segmented microscopic image; identifying intact droplets and broken droplets in the segmented microscopic image to calculate a static breakup coefficient of the uniform droplet; defining a time-stress coupling factor for the uniform droplet; The rupture coefficient of the uniform droplet is calculated by combining the time-stress coupling factor and the static rupture coefficient.

7. The method for rapid detection of hepatitis E virus based on animal detection technology according to claim 6, characterized in that: The calculating the rupture coefficient of the uniform droplet by combining the time-stress coupling factor and the static rupture coefficient comprises: analyzing the material stress coefficient and the fracture dynamics index of the uniform droplet based on the time-stress coupling factor; The rupture coefficient of the uniform droplet is calculated according to the static rupture coefficient, the material stress coefficient and the rupture dynamics index.

8. The method for rapid detection of hepatitis E virus based on animal detection technology according to claim 7, characterized in that: Generating a two-dimensional scatter plot of the amplified droplets based on the fluorescence detection signal comprises: performing background correction on the fluorescence detection signal to obtain a corrected fluorescence detection signal; performing standardization processing on the corrected fluorescence detection signal to obtain a processed fluorescence detection signal; Analyzing the HEX fluorescence intensity and FAM fluorescence intensity of the processed fluorescence detection signal; constructing a two-dimensional data set for processing the fluorescence detection signal based on the HEX fluorescence intensity and the FAM fluorescence intensity; A two-dimensional scatter plot of the amplified droplets is created based on the two-dimensional data set.

9. The method for rapid detection of hepatitis E virus based on animal detection technology according to claim 8, characterized in that: The method of analyzing the HEV genotype and mutation site of the hepatitis E virus sample based on the FAM-positive droplet comprises: Merging the FAM-positive droplets to obtain merged FAM-positive droplets; extracting the PCR amplification product of the merged FAM-positive droplets to obtain the HEV target gene fragment of the merged FAM-positive droplets; constructing a sequencing library of the HEV target gene fragment to obtain original sequencing data of the HEV target gene fragment; Performing quality control on the raw sequencing data to obtain target sequencing data; and identifying the variant sites of the target sequencing data; The HEV genotype of the hepatitis E virus sample is determined based on the mutation site.

10. A rapid detection system for hepatitis E virus based on animal detection technology, characterized in that: The system comprises: A sample nucleic acid extraction module is used to collect hepatitis E virus samples from the test area, wherein the hepatitis E virus samples include pig-derived samples, cattle-derived samples, sheep-derived samples, and environmental samples, and extract nucleic acid from the hepatitis E virus samples to obtain nucleic acid extracted samples; a uniform droplet construction module, used to establish a PCR system for the nucleic acid extraction sample and generate uniform droplets for the nucleic acid extraction sample and the PCR system; a fluorescence signal detection module, configured to calculate the rupture coefficient of the uniform droplet, perform PCR amplification on the uniform droplet to obtain an amplified droplet, and perform signal detection on the amplified droplet using a preset fluorescence dual channel to obtain a fluorescence detection signal; a positive droplet analysis module, configured to generate a two-dimensional scatter plot of the amplified droplets based on the fluorescence detection signal, so as to determine FAM-positive droplets in the amplified droplets; The HEV analysis module is used to analyze the HEV genotype and mutation sites of the hepatitis E virus sample based on the FAM-positive droplets to establish the HEV strain evolution tree and risk-inducing factors of the hepatitis E virus sample, and generate an HEV geographical distribution heat map of the test area in combination with the HEV strain evolution tree and risk-inducing factors.

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