Typing detection system for sewage diarrhea virus

Abstract

The present invention relates to the technical field of virus detection, and discloses a sewage diarrhea virus typing detection system, which comprises a pathogen enrichment unit, an improved polyethylene glycol precipitation method is adopted to carry out pathogen enrichment on a sewage sample; and the nucleic acid extraction unit is used for extracting nucleic acid in the enriched sample by using a magnetic bead extraction method. According to the typing detection system for the diarrhea viruses in the sewage, simultaneous detection and typing of various diarrhea viruses such as norovirus, rotavirus, adenovirus, astrovirus, parareovirus and fiveleaf virus in the sewage can be realized, the detection range is widened, powerful support is provided for comprehensive monitoring of virus pollution conditions in the sewage, and the system is suitable for popularization and application. According to the present invention, the multiple fluorescent RT-PCR technology is adopted, and the efficient sample enrichment and nucleic acid extraction method is combined, such that the detection sensitivity is improved, the low-concentration viral nucleic acid can be detected, and the potential viral threat is not missed;

Description

Technical Field

[0001] This invention relates to the field of virus detection technology, and in particular to a typing detection system for sewage diarrhea viruses. Background Technology

[0002] In today's society, diarrhea viruses have become important pathogens causing diarrheal diseases in humans worldwide, seriously threatening human health. These viruses are widely present in sewage. Therefore, monitoring and early warning of diarrhea viruses in sewage is particularly important, as it can help us to detect potential epidemic risks in a timely manner and take effective prevention and control measures.

[0003] However, current traditional methods for detecting diarrhea viruses in wastewater have many shortcomings:

[0004] 1) The viral content in sewage is complex, with a wide variety of viruses and large differences in concentration. A single detection method is difficult to detect all diarrhea viruses comprehensively and accurately.

[0005] 2) Traditional detection methods are typically cumbersome and time-consuming, making it difficult to meet the demand for rapid and efficient detection. During virus detection, the complex sample processing can easily lead to the loss or degradation of viral nucleic acids, affecting the accuracy and reliability of the results. Furthermore, it makes it difficult to classify viral subtypes and detect virulence genes, thus failing to provide sufficient information for virus tracing and epidemiological research.

[0006] 3) Traditional detection methods also have limitations in terms of sensitivity and specificity, and are prone to false negative or false positive results, which affect the accurate assessment of the degree of viral contamination in sewage. Summary of the Invention

[0007] Given the complexity of viral content in existing wastewater, the variety of viral types, and the significant differences in concentration, single detection methods are insufficient to comprehensively and accurately detect all diarrhea viruses. Furthermore, detection methods are typically cumbersome and time-consuming, failing to meet the demand for rapid and efficient detection. Moreover, they are prone to false negative or false positive results, affecting the accurate assessment of the degree of viral contamination in wastewater. Therefore, this invention is proposed.

[0008] Therefore, the purpose of this invention is to provide a typing and detection system for diarrhea viruses in sewage. Its objective is to simultaneously detect and type multiple diarrhea viruses in sewage, including norovirus, rotavirus, adenovirus, astrovirus, paraenterovirus, and zaruzin virus, thus broadening the detection range and providing strong support for comprehensive monitoring of viral contamination in sewage. Through multiplex fluorescent RT-PCR technology, combined with efficient sample enrichment and nucleic acid extraction methods, the system improves detection sensitivity, enabling the detection of low concentrations of viral nucleic acid and ensuring that no potential viral threats are missed.

[0009] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a typing and detection system for sewage diarrhea virus, comprising a pathogen enrichment unit: using a modified polyethylene glycol precipitation method to enrich pathogens in sewage samples;

[0010] Nucleic acid extraction unit: Extracts nucleic acids from enriched samples using magnetic bead extraction method;

[0011] Primer and probe design unit: Primers and probes were designed for the conserved sequences of nucleic acids of seven viruses: norovirus G1, norovirus G2, rotavirus, adenovirus, astrovirus, human paraenteritis virus and zarovirus.

[0012] Internal control virus addition unit: Adds chili pepper mottle virus as an ideal internal control virus for wastewater pathogen detection;

[0013] Multiplex Detection System Establishment Unit: Based on the designed primers and probes, establish a multiplex detection system;

[0014] Fluorescent PCR cycling condition setting unit;

[0015] Threshold setting and quality control standard unit: Set the threshold to be just above the highest point of fluorescence signal of the normal negative control, or adjust it according to the instrument noise level;

[0016] Primer pool design unit: Based on the gene characteristics of seven pathogens, gene-specific comparison analysis, gene variation region analysis, and pathogen subtype characteristic analysis were performed.

[0017] Targeted sequencing cDNA synthesis unit: Using wastewater samples that are positive for multiplex fluorescent RT-PCR as templates, single strands of cDNA are synthesized using reverse transcriptase and random primers;

[0018] Targeted sequencing multiplex PCR amplification unit: using synthesized cDNA single strands as templates, amplification is performed using the "diarrhea pathogen primer pool";

[0019] Product purification unit: After the PCR reaction, magnetic beads are used to purify the product;

[0020] Sequencing library construction and sequencing unit: The NGS library was prepared and sequenced using BGI single-end sequencing (S100).

[0021] As a preferred embodiment of the typing and detection system for sewage diarrhea viruses described in this invention, the modified polyethylene glycol precipitation method includes the following steps:

[0022] Adjust the pH of the wastewater sample to neutral to ensure the stability of the pathogens during the subsequent enrichment process;

[0023] A certain concentration of polyethylene glycol solution with a molecular weight of 6,000-20,000 was added to the sewage sample, and the amount added was 5%-15% of the sewage sample volume, in order to promote the aggregation of pathogens.

[0024] At a specific temperature, such as 4℃-25℃, slowly stir for a certain period of time, such as 1-4 hours, to allow the pathogens to fully combine with polyethylene glycol to form a precipitate;

[0025] After centrifugation, the precipitate is collected to remove most of the impurities from the wastewater and improve the efficiency of subsequent nucleic acid extraction.

[0026] As a preferred embodiment of the typing and detection system for sewage diarrhea viruses described in this invention, the magnetic bead extraction method includes the following steps:

[0027] The enriched sample is mixed with magnetic beads containing specific binding groups. The magnetic beads can specifically bind to viral nucleic acids. Under the action of an external magnetic field, the magnetic beads are separated from the liquid, thereby separating the nucleic acid from the sample.

[0028] The magnetic beads bound to nucleic acids were washed multiple times with a washing solution to remove residual impurities and further improve the purity of the nucleic acids.

[0029] Finally, the nucleic acid was eluted from the magnetic beads using elution buffer to obtain high-purity viral nucleic acid, providing a high-quality template for subsequent multiplex fluorescent RT-PCR detection.

[0030] As a preferred embodiment of the typing and detection system for sewage diarrhea virus described in this invention, the primers and probes are designed with rigorous gene-specific alignment analysis and gene variation region analysis to ensure that they can specifically identify the nucleic acid sequence of the target virus and have no cross-reaction with non-target sequences.

[0031] As a preferred embodiment of the typing and detection system for sewage diarrhea viruses described in this invention, the multiplex detection system includes three PCR amplification reaction systems, A, B, and C, each system targeting a specific virus type for detection.

[0032] Tube A detects the following virus types: Norovirus G1, Norovirus G2, and Rotavirus.

[0033] B tube detects virus types: human paraenterovirus, adenovirus, astrovirus;

[0034] C-tube detection of virus types: Zaruzin virus, chili pepper mottled virus.

[0035] In a preferred embodiment of the typing and detection system for sewage diarrhea viruses described in this invention, the fluorescent PCR cycling condition setting unit sets the cycling conditions of the fluorescent PCR instrument, specifically as follows:

[0036] One cycle: 50℃, 10min;

[0037] One cycle: 97℃, 1 min;

[0038] 45 cycles: 97℃, 5 sec; 58℃, 30 sec, with fluorescence collected at 58℃.

[0039] As a preferred embodiment of the typing and detection system for sewage diarrhea virus described in this invention, the threshold setting and quality control standard are established in the quality control standard unit, that is, the blank control and negative control have no amplification curve, and the positive control has an S-shaped amplification curve in the detection channel, the experiment is valid, otherwise the experimental results are invalid.

[0040] As a preferred embodiment of the typing and detection system for sewage diarrhea viruses described in this invention, the primer pool design unit selects highly conserved regions of each pathogen as target genes, designs a pair of primers for each target gene, and the amplicon length ranges from 1000 to 500 bp, ultimately forming a primer set, namely the "diarrhea pathogen primer pool", consisting of 14 pairs of primers. The primers in the primer pool are optimized to ensure that no cross-reaction or primer dimer occurs between the primers during multiplex PCR amplification, thereby improving amplification efficiency and specificity.

[0041] As a preferred embodiment of the genotyping and detection system for sewage diarrhea viruses described in this invention, the PCR reaction system includes specific concentrations of dNTPs, Mg2+, Taq DNA polymerase, and other components to ensure the smooth progress of the amplification reaction. The amplification program is optimized and includes steps such as pre-denaturation, denaturation, annealing, and extension to ensure that each target gene is sufficiently amplified.

[0042] As a preferred embodiment of the genotyping and detection system for sewage diarrhea viruses described in this invention, the sequencing library construction and sequencing unit further includes an analysis process for the sequencing data, as detailed below:

[0043] Data preprocessing: The raw sequencing data after sequencing is subjected to quality assessment, and impurities such as low-quality sequences and adapter sequences are removed to obtain high-quality clean data;

[0044] Sequence alignment analysis: The clean data is compared with known diarrhea virus gene databases. Bioinformatics tools such as BWA and Bowtie are used to perform sequence alignment to determine the types of diarrhea viruses contained in the sewage samples.

[0045] Virus typing: Based on the comparison results and combined with the genetic characteristics of different viruses, the detected viruses are accurately typed to determine the specific type of the virus;

[0046] Virulence gene detection: Further analysis of virulence-related gene fragments in the viral gene sequence to assess the potential harm of the virus.

[0047] In a preferred embodiment of the genotyping and detection system for sewage diarrhea viruses described in this invention, the targeted sequencing process is as follows:

[0048] Using sewage samples that tested positive for diarrhea virus nucleic acid by multiplex fluorescent RT-PCR as templates, cDNA single strands were synthesized using reverse transcriptase and random primers;

[0049] Using cDNA single strands as templates, the target region in the sample was amplified using the constructed "diarrhea pathogen primer pool". The kit used was the ATOPlex DNA multiplex PCR universal library preparation module.

[0050] After the PCR reaction, magnetic beads were used to purify the product. The kit used was the MGIEAsyDNA Purification Magnetic Bead Kit. The magnetic beads specifically bind to the product, and impurities are separated under the action of an external magnetic field. After washing and elution steps, the purified amplification product is obtained, which improves the accuracy and reliability of sequencing results.

[0051] As a preferred embodiment of the genotyping and detection system for sewage diarrhea viruses described in this invention, the sequencing library construction and sequencing steps are as follows:

[0052] Sequencing library construction: The purified amplification products are processed by end repair and barcode adapter addition to construct a library suitable for high-throughput sequencing platforms. End repair makes the DNA fragment ends flush, which facilitates subsequent adapter ligation. Adding barcode adapters gives each sample a unique identifier, which makes it easier to distinguish data from different samples after sequencing.

[0053] Sequencing: The NGS library was prepared and sequenced using BGI Genomics' single-end sequencing (S100). During the sequencing process, sequencing depth and data quality were controlled to ensure that sufficient effective data could be obtained for accurate detection and typing of the virus.

[0054] After sequencing is completed, the sequencing data is monitored and preliminarily analyzed in real time to promptly identify and address any potential data quality issues, ensuring the accuracy and reliability of the sequencing results and providing strong data support for the monitoring and early warning of diarrhea viruses in sewage.

[0055] Post-sequencing result verification involves using commercially available reagents to retest positive samples detected by sequencing, ensuring the accuracy of the results. By comparing the test results of the self-developed kit with those of commercially available reagents, the targeted sequencing results are verified and calibrated, improving the reliability of the detection system. Simultaneously, multiple comparisons and analyses of the sequencing results are performed, combined with clinical sample information and results from other testing methods, to further confirm the infection status and epidemic trend of the virus, providing a scientific basis for public health decision-making.

[0056] Compared with the prior art, the present invention has at least the following beneficial effects:

[0057] This invention enables the simultaneous detection and typing of multiple diarrhea viruses in wastewater, including norovirus, rotavirus, adenovirus, astrovirus, paraenterovirus, and zarozygovirus, broadening the detection range and providing strong support for comprehensive monitoring of viral contamination in wastewater. Through multiplex fluorescent RT-PCR technology, combined with efficient sample enrichment and nucleic acid extraction methods, the detection sensitivity is improved, enabling the detection of low concentrations of viral nucleic acid and ensuring no potential viral threats are missed. Furthermore, the use of specific primers and probes ensures precise pairing with the target viral nucleic acid sequence, effectively avoiding cross-reactions with other pathogens and guaranteeing the accuracy of the detection results.

[0058] In terms of the detection process, this method organically combines pathogen enrichment of wastewater samples, nucleic acid extraction, multiplex fluorescent RT-PCR detection, and targeted sequencing. This simplifies the operation, reduces the professional skills required of operators, minimizes human error, and improves the repeatability and stability of the detection. The results can not only qualitatively determine the presence of specific diarrheal viruses in wastewater, but also achieve viral subtype classification and virulence gene detection through targeted sequencing, providing richer information for virus tracing, epidemiological research, and the formulation of prevention and control strategies.

[0059] Meanwhile, the application of this system in detecting diarrhea viruses in sewage can monitor the viral contamination status of sewage in a timely and accurate manner, providing scientific evidence for public health departments so that they can quickly take corresponding prevention and control measures, thereby effectively preventing and controlling the outbreak and spread of viral diarrhea, and ensuring public health and social stability. Attached Figure Description

[0060] Figure 1 This is a sensitivity experiment diagram of rotavirus, norovirus GI type, and GII type nucleic acid fluorescent RT-PCR for the genotyping detection system for sewage diarrhea viruses of the present invention;

[0061] Figure 2 The above diagram shows the sensitivity of adenovirus, human paraenteritis virus and astrovirus fluorescent RT-PCR experiments for the typing and detection system of sewage diarrhea viruses of this invention.

[0062] Figure 3 This is a fluorescence RT-PCR sensitivity experiment diagram of the chili pepper mottle virus and zaruzin virus in the genotyping detection system for sewage diarrhea viruses of the present invention;

[0063] Figure 4 This invention provides a specificity test for the sewage diarrhea virus typing and detection system. Figure 1 ;

[0064] Figure 5 This invention provides a specificity test for the sewage diarrhea virus typing and detection system. Figure 2 ;

[0065] Figure 6 This invention provides a specificity test for the sewage diarrhea virus typing and detection system. Figure 3 ;

[0066] Figure 7 This is a graph showing the repeatability detection results of rotavirus fluorescent RT-PCR using the genotyping detection system for sewage diarrhea viruses of the present invention.

[0067] Figure 8 This is a graph showing the repeatability detection results of norovirus GI fluorescent RT-PCR using the sewage diarrhea virus typing detection system of the present invention;

[0068] Figure 9 This is a graph showing the repeatability detection results of norovirus GII fluorescent RT-PCR using the genotyping detection system for sewage diarrhea viruses of the present invention.

[0069] Figure 10 This is a graph showing the repeatability detection results of astrovirus fluorescent RT-PCR for the genotyping detection system for sewage diarrhea viruses of the present invention;

[0070] Figure 11 This is a graph showing the repeatability detection results of human paraenteritis virus fluorescent RT-PCR using the genotyping detection system for sewage diarrhea viruses of this invention;

[0071] Figure 12 This is a graph showing the repeatability detection results of adenovirus fluorescent PCR in the genotyping detection system for sewage diarrhea viruses of the present invention. Detailed Implementation

[0072] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0073] Example

[0074] Reference Figures 1-12As an embodiment of the present invention, a typing and detection system for sewage diarrhea viruses is provided. This typing and detection system for sewage diarrhea viruses includes:

[0075] Pathogen enrichment unit: A modified polyethylene glycol (PEG) precipitation method is used to enrich pathogens in wastewater samples. This modified PEG precipitation method includes the following steps:

[0076] Adjust the pH of the wastewater sample to neutral to ensure the stability of the pathogens during the subsequent enrichment process;

[0077] A certain concentration of polyethylene glycol solution with a molecular weight of 6,000-20,000 was added to the sewage sample, and the amount added was 5%-15% of the sewage sample volume, in order to promote the aggregation of pathogens.

[0078] At a specific temperature, such as 4℃-25℃, slowly stir for a certain period of time, such as 1-4 hours, to allow the pathogens to fully combine with polyethylene glycol to form a precipitate;

[0079] After centrifugation, the precipitate is collected to remove most impurities from the wastewater, improving the efficiency of subsequent nucleic acid extraction. This modified polyethylene glycol precipitation method effectively enriches pathogens from complex wastewater samples, increasing virus enrichment efficiency and providing high-quality samples for subsequent nucleic acid testing.

[0080] Nucleic acid extraction unit: Extracts nucleic acids from the enriched sample using a magnetic bead extraction method, which includes the following steps:

[0081] The enriched sample is mixed with magnetic beads containing specific binding groups. The magnetic beads can specifically bind to viral nucleic acids. Under the action of an external magnetic field, the magnetic beads are separated from the liquid, thereby separating the nucleic acid from the sample.

[0082] The magnetic beads bound to nucleic acids were washed multiple times with a washing solution to remove residual impurities and further improve the purity of the nucleic acids.

[0083] Finally, the nucleic acid was eluted from the magnetic beads using elution buffer to obtain high-purity viral nucleic acid, providing a high-quality template for subsequent multiplex fluorescent RT-PCR detection.

[0084] Primer and probe design unit: Primers and probes are designed for the conserved sequences of nucleic acids of seven viruses: norovirus G1, norovirus G2, rotavirus, adenovirus, astrovirus, human paraenteritis virus and zarrovirus. The design of the primers and probes has undergone rigorous gene-specific alignment analysis and gene variation region analysis to ensure that they can specifically recognize the nucleic acid sequences of the target viruses and have no cross-reaction with non-target sequences.

[0085] Internal standard virus addition unit: Adds chili pepper mottle virus as an ideal internal standard virus for the detection of pathogens in sewage. The chili pepper mottle virus is abundant in human feces and has high stability, which can effectively monitor the reliability of experimental operations. By detecting the recovery rate and amplification curve of the internal standard virus, it is possible to determine whether there are errors or contamination in the entire detection process, thereby ensuring the accuracy and reliability of the detection results.

[0086] Multiplex Detection System Establishment Unit: Based on the designed primers and probes, a multiplex detection system is established, comprising three PCR amplification reaction systems (A, B, and C), each targeting a specific virus type. Wherein:

[0087] Tube A detects the following virus types: Norovirus G1, Norovirus G2, and Rotavirus.

[0088] B tube detects virus types: human paraenterovirus, adenovirus, astrovirus;

[0089] C-tube detection of virus types: Zaru virus, Capsicum mild mottle virus;

[0090] Fluorescent PCR Cycling Conditions Setting Unit: Sets the cycling conditions for the fluorescent PCR instrument, specifically:

[0091] One cycle: 50℃, 10min;

[0092] One cycle: 97℃, 1 min;

[0093] 45 cycles: 97℃, 5 sec; 58℃, 30 sec, with fluorescence collected at 58℃;

[0094] Threshold setting and quality control standard unit: The threshold is set to be just above the highest point of fluorescence signal of normal negative control, or adjusted according to the instrument noise. At the same time, a quality control standard is established, that is, the blank control and negative control have no amplification curve, and the positive control has an S-shaped amplification curve in the detection channel. The experiment is valid, otherwise the experimental results are invalid.

[0095] Primer pool design unit: Based on the gene characteristics of seven pathogens, and through gene-specific comparison analysis, gene variation region analysis, and pathogen subtype characteristic analysis, highly conserved regions of each pathogen were selected as target genes. A pair of primers was designed for each target gene, with amplicon length ranging from 1000-500 bp, ultimately forming a primer set, namely the "diarrhea pathogen primer pool," consisting of 14 pairs of primers. The primers in the primer pool have been optimized to ensure that no cross-reaction or primer dimer formation occurs between primers during multiplex PCR amplification, thereby improving amplification efficiency and specificity.

[0096] Targeted sequencing cDNA synthesis unit: Using wastewater samples that tested positive by multiplex fluorescent RT-PCR as templates, single-stranded cDNA is synthesized using reverse transcriptase and random primers. The reverse transcriptase possesses efficient and stable reverse transcription activity, ensuring the synthesis of high-quality cDNA from RNA virus samples and providing sufficient template for subsequent PCR amplification.

[0097] Targeted sequencing multiplex PCR amplification unit: Using synthesized single-stranded cDNA as a template, amplification is performed using a "diarrhea pathogen primer pool". The PCR reaction system includes specific concentrations of dNTPs, Mg2+, Taq DNA polymerase, and other components to ensure smooth amplification. The amplification program is optimized, including pre-denaturation, denaturation, annealing, and extension steps to ensure sufficient amplification of each target gene.

[0098] Product purification unit: After the PCR reaction, magnetic beads are used for product purification. These magnetic beads have specific binding properties and can specifically bind to the PCR product. An external magnetic field is used to separate the product from impurities, thereby obtaining high-purity amplified products. The magnetic bead kit used is the MGIEAsyDNA purification magnetic bead kit, which offers efficient and rapid purification, improving the success rate of sequencing library construction.

[0099] Sequencing library construction and sequencing unit: The NGS library was prepared and sequenced using BGI Genomics' single-end sequencing (S100). The sequencing library construction process included steps such as end repair, adapter addition, and library screening of purified PCR products to prepare libraries that met sequencing requirements. The sequencing process used advanced high-throughput sequencing technology, which can quickly and accurately obtain a large amount of sequencing data. By analyzing the sequencing data, it is possible to detect the typing of diarrhea viruses in sewage, providing detailed genetic information for virus tracing and epidemiological research.

[0100] Sequencing library construction and sequencing units also include the workflow for analyzing sequencing data, as detailed below:

[0101] Data preprocessing: The raw sequencing data after sequencing is subjected to quality assessment, and impurities such as low-quality sequences and adapter sequences are removed to obtain high-quality clean data;

[0102] Sequence alignment analysis: The clean data is compared with known diarrhea virus gene databases. Bioinformatics tools such as BWA and Bowtie are used to perform sequence alignment to determine the types of diarrhea viruses contained in the sewage samples.

[0103] Virus typing: Based on the comparison results and combined with the genetic characteristics of different viruses, the detected viruses are accurately typed to determine the specific type of the virus;

[0104] Virulence gene detection: Further analysis of virulence-related gene fragments in the viral gene sequence to assess the potential harm of the virus.

[0105] The sequencing library construction and sequencing steps are as follows:

[0106] Sequencing library construction: The purified amplification products are processed by end repair and barcode adapter addition to construct a library suitable for high-throughput sequencing platforms. End repair makes the DNA fragment ends flush, which facilitates subsequent adapter ligation. Adding barcode adapters gives each sample a unique identifier, which makes it easier to distinguish data from different samples after sequencing.

[0107] Sequencing: The NGS library was prepared and sequenced using BGI Genomics' single-end sequencing (S100). During the sequencing process, sequencing depth and data quality were controlled to ensure that sufficient effective data could be obtained for accurate detection and typing of the virus.

[0108] After sequencing is completed, the sequencing data is monitored and preliminarily analyzed in real time to promptly identify and address any potential data quality issues, ensuring the accuracy and reliability of the sequencing results and providing strong data support for the monitoring and early warning of diarrhea viruses in sewage.

[0109] Post-sequencing result verification involves using commercially available reagents to retest positive samples detected by sequencing, ensuring the accuracy of the results. By comparing the test results of the self-developed kit with those of commercially available reagents, the targeted sequencing results are verified and calibrated, improving the reliability of the detection system. Simultaneously, multiple comparisons and analyses of the sequencing results are performed, combined with clinical sample information and results from other testing methods, to further confirm the infection status and epidemic trend of the virus, providing a scientific basis for public health decision-making.

[0110] 1. Method Establishment

[0111] 1.1 Preparation of the reaction system

[0112] To comprehensively detect the various gastrointestinal viruses covered by the kit, each sample should undergo three PCR amplification processes: A, B, and C. After dissolving, mixing, and centrifuging the reaction solutions for A, B, and C, aliquot 10 μL into PCR tubes suitable for a fluorescence PCR instrument at each well. Add 10 μL of the nucleic acid to be tested to each of the three reaction systems (A, B, and C), for a total reaction volume of 20 μL. Add 10 μL of the corresponding template to the positive control reaction tube.

[0113] 1.2 Setting up Cyclic Conditions for Fluorescent PCR

[0114]

[0115] Detection settings: "reporterDye" is set to FAM, VIC, ROX, and CY5 respectively. See Result Interpretation for details on the corresponding channels for each target. "QuencherDye" is set to None. For ABI series instruments, ensure "PassiveReference" is set to None.

[0116] 1.3 Threshold Setting

[0117] The threshold setting principle is to use the highest point of fluorescence signal that just exceeds that of the normal negative control as the threshold line, or to adjust it according to the instrument noise level.

[0118] 1.4 Quality Control Standards

[0119] The experiment is considered valid if the blank control and negative control show no amplification curves, and the positive control shows an S-shaped amplification curve in the detection channel. Otherwise, the experimental results are considered invalid.

[0120] 1.5 Results Analysis and Judgment

[0121] 1.5.1 Under normal instrument conditions and when the test results of positive and negative controls meet the quality control standards, analyze and interpret the test results of the sample to be tested.

[0122] 1.5.2 A sample is considered positive if its amplification curve exhibits a typical S-shaped curve and meets the following conditions:

[0123]

[0124]

[0125] 1.5.3 Experimental gray area:

[0126] If the detection channel shows a clear S-shaped amplification curve and 38 < Ct value ≤ 40, the result is considered to be in the experimental gray zone and is determined to be an uncertain sample. Nucleic acid needs to be extracted again for testing. If the retest result still shows a clear S-shaped amplification curve and Ct value ≤ 40, the result is considered positive. If there is no clear S-shaped amplification curve, the result is considered negative.

[0127] 2. Method Validation

[0128] 2.1 Sensitivity test: The three positive plasmids were diluted to 1×10⁸, 1×10⁷, 1×10⁶, 1×10⁵, 1×10⁴, 1×10³, and 500 copies / ml, respectively, and detected using a kit.

[0129] 2.2 Specificity tests: Specificity tests were performed on RNA-positive samples of zarrovirus, astrovirus, adenovirus, norovirus, rotavirus, and human paraenteritis virus that had been preserved in the laboratory.

[0130] 2.3 Repeatability test: Plasmid standards with concentrations of 10⁷ and 10⁴ copies / ml were tested, and each concentration sample was tested twice.

[0131] 2.4 Accuracy testing: Positive samples detected by this kit were compared with commercially available reagents.

[0132] 3 Results

[0133] 3.1 Sensitivity Experiment

[0134] The three positive plasmids were diluted to 1×10⁸, 1×10⁷, 1×10⁶, 1×10⁵, 1×10⁴, 1×10³, and 500 copies / ml, respectively, and detected using a kit. The results are as follows: the limit of detection (LOD) for rotavirus, norovirus GI, GII, and pepper mottle virus was 500 copies / ml; the LOD for adenovirus, human paraenterovirus, and astrovirus was 1×10³ copies / ml; and the LOD for zarrovirus was 1×10⁴ copies / ml.

[0135] 3.2 Specificity Testing: Positive samples of RNA from pre-existing strains of zajugvirus, hepatitis A virus, astrovirus, adenovirus, norovirus, rotavirus, and human paraenteritis virus were subjected to specificity testing (see reference). Figure 4 , 5 (and 6). Each probe can only detect its own specific pathogen, and so far there has been no cross-reaction with other pathogens.

[0136] 3.3 Repeatability experiments were conducted using plasmid standards at concentrations of 10⁷ and 10⁴ copies / ml, with each concentration sample tested twice (reference). Figure 7-12 ).

[0137] 3.4. Accuracy Verification. Positive samples were verified and compared using commercially available reagents.

[0138]

[0139]

[0140] 3. Targeted sequencing

[0141] 3.1. Primer Design for Multiplex PCR Amplification

[0142] Based on pathogen gene characteristics, and through gene-specific comparison analysis, gene variation region analysis, and pathogen subtype characteristic analysis, highly conserved regions were selected as target genes for each of the seven pathogens (norovirus G1, norovirus G2, rotavirus, adenovirus, astrovirus, human paraenteritis virus, and zarozygovirus). Through multiple design, analysis, and comparison processes, one pair of primers was designed for each target gene, with amplicon length ranging from 1000-500 bp. This resulted in a primer set – the "Diarrhea Pathogen Primer Pool" – comprising 14 primer pairs, as shown in the table. Specific target genes are shown in the figure.

[0143] 3.2 Targeted sequencing workflow:

[0144] Wastewater samples that tested positive for diarrhea virus nucleic acid by multiplex fluorescent RT-PCR were used as templates to synthesize single-stranded cDNA using reverse transcriptase and random primers.

[0145] Using cDNA single-stranded DNA as a template, the target region in the sample was amplified using the constructed "diarrhea pathogen primer pool". The kit used was the ATOPlex DNA multiplex PCR universal library preparation module. The specific reaction system and reaction procedure are shown in the table. After the PCR reaction, the product was purified using magnetic beads. The kit used was the MGIEAsyDNA purification magnetic bead kit.

[0146] 3.3 Sequencing Library Construction and Sequencing

[0147] The preparation and sequencing of the NGS library were performed using BGI Genomics single-end sequencing (S100).

[0148] This invention provides a method for simultaneous detection and typing of seven possible diarrheal pathogens in wastewater using targeted sequencing combined with multi-pathogen detection of diarrheal viruses. The primer combination provided by this invention simplifies manual operation and improves pathogen detection efficiency without interference between primers. The primer combination provided by this invention has strong specificity and sensitivity, and can accurately capture target genes from pathogen samples. Therefore, the pathogen infection status can be judged based on the detection of target genes in the sequencing results, realizing the simultaneous and rapid subtype classification and virulence gene detection of multiple diarrheal pathogens.

[0149] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A typing and detection system for sewage diarrhea viruses, characterized in that, include: Pathogen enrichment unit: Pathogens were enriched in wastewater samples using a modified polyethylene glycol precipitation method; Nucleic acid extraction unit: Extracts nucleic acids from enriched samples using magnetic bead extraction method; Primer and probe design unit: Primers and probes were designed for the conserved sequences of nucleic acids of seven viruses: norovirus G1, norovirus G2, rotavirus, adenovirus, astrovirus, human paraenteritis virus and zarovirus. Internal control virus addition unit: Adds chili pepper mottle virus as an ideal internal control virus for wastewater pathogen detection; Multiplex Detection System Establishment Unit: Based on the designed primers and probes, establish a multiplex detection system; Fluorescent PCR cycling condition setting unit; Threshold setting and quality control standard unit: Set the threshold to be just above the highest point of fluorescence signal of the normal negative control, or adjust it according to the instrument noise level; Primer pool design unit: Based on the gene characteristics of seven pathogens, gene-specific comparison analysis, gene variation region analysis, and pathogen subtype characteristic analysis were performed. Targeted sequencing cDNA synthesis unit: Using wastewater samples that are positive for multiplex fluorescent RT-PCR as templates, single strands of cDNA are synthesized using reverse transcriptase and random primers; Targeted sequencing multiplex PCR amplification unit: using synthesized cDNA single strands as templates, amplification is performed using the "diarrhea pathogen primer pool"; Product purification unit: After the PCR reaction, magnetic beads are used to purify the product; Sequencing library construction and sequencing unit: The NGS library was prepared and sequenced using BGI single-end sequencing (S100).

2. The typing and detection system for sewage diarrhea virus according to claim 1, characterized in that: The improved polyethylene glycol precipitation method includes the following steps: Adjust the pH of the wastewater sample to neutral to ensure the stability of the pathogens during the subsequent enrichment process; A certain concentration of polyethylene glycol solution with a molecular weight of 6,000-20,000 was added to the sewage sample, and the amount added was 5%-15% of the sewage sample volume, in order to promote the aggregation of pathogens. At a specific temperature, such as 4℃-25℃, slowly stir for a certain period of time, such as 1-4 hours, to allow the pathogens to fully combine with polyethylene glycol to form a precipitate; After centrifugation, the precipitate is collected to remove most of the impurities from the wastewater and improve the efficiency of subsequent nucleic acid extraction.

3. The typing and detection system for sewage diarrhea virus according to claim 1, characterized in that: The magnetic bead extraction method includes the following steps: The enriched sample is mixed with magnetic beads containing specific binding groups. The magnetic beads can specifically bind to viral nucleic acids. Under the action of an external magnetic field, the magnetic beads are separated from the liquid, thereby separating the nucleic acid from the sample. The magnetic beads bound to nucleic acids were washed multiple times with a washing solution to remove residual impurities and further improve the purity of the nucleic acids. Finally, the nucleic acid was eluted from the magnetic beads using elution buffer to obtain high-purity viral nucleic acid, providing a high-quality template for subsequent multiplex fluorescent RT-PCR detection.

4. The typing and detection system for sewage diarrhea virus according to claim 3, characterized in that: The multiplex detection system includes three PCR amplification reaction systems, A, B, and C, each targeting a specific virus type. Tube A detects the following virus types: Norovirus G1, Norovirus G2, and Rotavirus. B tube detects virus types: human paraenterovirus, adenovirus, astrovirus; C-tube detection of virus types: Zaruzin virus, chili pepper mottled virus.

5. The typing and detection system for sewage diarrhea virus according to claim 4, characterized in that: The fluorescence PCR cycling condition setting unit sets the cycling conditions of the fluorescence PCR instrument, specifically as follows: One cycle: 50℃, 10min; One cycle: 97℃, 1 min; 45 cycles: 97℃, 5 sec; 58℃, 30 sec, with fluorescence collected at 58℃.

6. The typing and detection system for sewage diarrhea virus according to claim 5, characterized in that: The primer pool design unit selects highly conserved regions of each pathogen as target genes, designs a pair of primers for each target gene, and the amplicon length ranges from 1000-500 bp, ultimately forming a primer set, namely the "diarrhea pathogen primer pool", with a total of 14 pairs of primers. The primers in the primer pool are optimized to ensure that no cross-reaction or primer dimer occurs between primers during multiplex PCR amplification, thereby improving amplification efficiency and specificity.

7. The typing and detection system for sewage diarrhea virus according to claim 6, characterized in that: The PCR reaction system includes specific concentrations of dNTPs, Mg2+, Taq DNA polymerase, and other components to ensure the smooth progress of the amplification reaction. The amplification program has been optimized and includes steps such as pre-denaturation, denaturation, annealing, and extension to ensure that each target gene is fully amplified.

8. The typing and detection system for sewage diarrhea virus according to claim 7, characterized in that: The sequencing library construction and sequencing unit also includes a sequencing data analysis process, as detailed below: Data preprocessing: The raw sequencing data after sequencing is subjected to quality assessment, and impurities such as low-quality sequences and adapter sequences are removed to obtain high-quality clean data; Sequence alignment analysis: The clean data is compared with known diarrhea virus gene databases. Bioinformatics tools such as BWA and Bowtie are used to perform sequence alignment to determine the types of diarrhea viruses contained in the sewage samples. Virus typing: Based on the comparison results and combined with the genetic characteristics of different viruses, the detected viruses are accurately typed to determine the specific type of the virus; Virulence gene detection: Further analysis of virulence-related gene fragments in the viral gene sequence to assess the potential harm of the virus.

9. The typing and detection system for sewage diarrhea virus according to claim 8, characterized in that: The targeted sequencing process is as follows: Using sewage samples that tested positive for diarrhea virus nucleic acid by multiplex fluorescent RT-PCR as templates, cDNA single strands were synthesized using reverse transcriptase and random primers; Using cDNA single strands as templates, the target region in the sample was amplified using the constructed "diarrhea pathogen primer pool". The kit used was the ATOPlex DNA multiplex PCR universal library preparation module. After the PCR reaction, magnetic beads were used to purify the product. The kit used was the MGIEAsyDNA Purification Magnetic Bead Kit. The magnetic beads specifically bind to the product, and impurities are separated under the action of an external magnetic field. After washing and elution steps, the purified amplification product is obtained, which improves the accuracy and reliability of sequencing results.

10. The typing and detection system for sewage diarrhea virus according to claim 9, characterized in that: The sequencing library construction and sequencing steps are as follows: Sequencing library construction: The purified amplification products are processed by end repair and barcode adapter addition to construct a library suitable for high-throughput sequencing platforms. End repair makes the DNA fragment ends flush, which facilitates subsequent adapter ligation. Adding barcode adapters gives each sample a unique identifier, which makes it easier to distinguish data from different samples after sequencing. Sequencing: The NGS library was prepared and sequenced using BGI Genomics' single-end sequencing (S100). During the sequencing process, sequencing depth and data quality were controlled to ensure that sufficient effective data could be obtained for accurate detection and typing of the virus. After sequencing is completed, the sequencing data is monitored and preliminarily analyzed in real time to ensure the accuracy and reliability of the sequencing results, providing strong data support for the monitoring and early warning of diarrhea viruses in sewage. For post-sequencing result verification, commercial reagents were used to retest the positive samples detected by sequencing to ensure the accuracy of the test results.

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