Norovirus gi and gii double genotype synchronous quantitative detection method, application and detection box

Abstract

The application belongs to the technical field of sewage detection, and provides a norovirus GI and GII double genotype synchronous quantitative detection method, which realizes synchronous detection and absolute quantification of norovirus GI and GII in the same reaction tube, does not need to rely on a standard curve, and avoids errors caused by batch differences of standard products; in terms of primer and probe design, the constructed detection system shows high specificity, and even under the coexistence condition of other viruses with close genetic relationship, no non-specific amplification occurs; through gradient dilution experiment verification, the digital PCR method shows good linear response in a wide dynamic range, excellent linear correlation, low repeat variation coefficient, and shows excellent detection consistency and stability; the droplet generation performance is stable, the effective droplet number is always maintained at more than 19500, and the reliability of the quantitative result is ensured.

Description

Technical Field

[0001] This invention relates to the field of wastewater detection technology, specifically to a method, application, and detection kit for simultaneous quantitative detection of norovirus genotypes GI and GII. Background Technology

[0002] Norovirus is a leading cause of acute gastroenteritis (AGE) worldwide. It is estimated that approximately 699 million people are infected with norovirus globally each year, resulting in about 212,000 deaths. This virus is characterized by low infectious doses, high transmissibility, and strong environmental resistance, making it a significant challenge in public health.

[0003] Wastewater, as a "hidden carrier" of viruses, can accurately reflect the prevalence of pathogens within a community. Wastewater surveillance technology can enable early warning and dynamic tracking of norovirus. Studies have shown a significant positive correlation between norovirus load in wastewater and the number of clinical cases (rs=0.51), especially during the peak winter and spring seasons. Quantitative detection of norovirus load in wastewater is of significant strategic importance for prevention and the optimal allocation of public health resources.

[0004] Currently, the quantitative detection of norovirus in wastewater mainly relies on molecular diagnostic techniques. In 2013, Brinkman et al. used real-time quantitative PCR (qPCR) combined with diatomaceous earth concentration to achieve the quantitative detection of norovirus and other enteroviruses in wastewater samples. In 2016, Fout's team further optimized the detection process by combining viral RNA extraction with reverse transcription real-time quantitative PCR (RT-qPCR). In 2024, Sun et al., based on the RPA-CRISPR / Cas13a platform, significantly improved the convenience of detecting norovirus GII in aquatic environments. Although the above methods have overcome the qualitative limitations of traditional RT-PCR, their quantitative results still require conversion based on standard curves, making absolute quantification difficult. Furthermore, they are significantly affected by sample matrix interference, impacting the accuracy and repeatability of the detection.

[0005] Digital PCR (dPCR) technology, with its absolute quantification capability, high specificity, high sensitivity, and good tolerance to inhibitors, has become a new path to overcome the aforementioned technical bottlenecks. However, current dPCR detection methods for norovirus in wastewater mostly focus on single genotype detection, and there is still a lack of a detection system that can simultaneously achieve accurate typing and absolute quantification of norovirus GI and GII genotypes in a single reaction system.

[0006] Therefore, developing a detection method suitable for complex wastewater matrices, capable of simultaneous quantitative detection of norovirus types GI and GII, and requiring no standard curve, is of great practical significance for improving the efficiency and reliability of wastewater monitoring and achieving early warning and precise prevention and control. Summary of the Invention

[0007] In view of this, considering the characteristics of urban sewage samples with complex matrix, low virus content and strong inhibitor interference, a simultaneous quantitative detection method, application and detection kit are established that is suitable for complex sewage matrix, can simultaneously and quantitatively detect norovirus GI and GII types, and does not require a standard curve.

[0008] To solve the above problems, this application adopts the following technical solution: In one aspect, this application provides a method for simultaneous quantitative detection of norovirus genotypes GI and GII, comprising the following steps: Extract viral nucleic acid from wastewater samples to be tested; A digital PCR reaction system was constructed, comprising a specific primer and probe set for norovirus GI gene, a specific primer and probe set for norovirus GII gene, reverse transcriptase, DNA polymerase, and nucleic acid template. The constructed reaction system was subjected to droplet generation and reverse transcription digital PCR amplification. Signals from the FAM and HEX fluorescence channels were acquired separately, with the FAM channel corresponding to norovirus GI type and the HEX channel corresponding to norovirus GII type. Based on the number of positive droplets and Poisson distribution, absolute quantitative results of norovirus types GI and GII in sewage were obtained simultaneously.

[0009] In some embodiments, the norovirus GI probe is labeled with FAM fluorescent dye, and the norovirus GII probe is labeled with HEX fluorescent dye.

[0010] In some embodiments, the digital PCR amplification program is as follows: reverse transcription at 50°C for 30 minutes; hot start at 95°C for 5 minutes; 40-50 cycles of denaturation at 96°C for 5 seconds and annealing / extension at 59°C for 30 seconds; and finally stabilization at 25°C for 1 minute.

[0011] In some embodiments, the final concentration of each primer pair and each probe in the digital PCR reaction system is 100–300 nM.

[0012] In some embodiments, when interpreting results, a positive threshold of ≥2 positive droplets is used, and the detection limit can reach 0.6 copies / μL.

[0013] In some embodiments, nucleic acid extraction of the wastewater sample is performed using a magnetic bead-based viral nucleic acid extraction kit, and includes a pre-centrifugation step.

[0014] Secondly, this application provides a digital PCR kit for simultaneous quantitative detection of norovirus types GI and GII in wastewater, comprising: Specific primer pairs and probes for norovirus GI type; Specific primer pairs and probes for norovirus GII; Digital PCR premix solution contains at least reverse transcriptase, DNA polymerase, and digital PCR-specific buffer. Positive and negative control samples; The kit is used to perform any of the methods described.

[0015] In some embodiments, the FAM fluorescence channel is used to detect norovirus GI and the HEX fluorescence channel is used to detect norovirus GII.

[0016] In some embodiments, the kit has a linear detection range of 1.5 to 15,000 copies / μL for norovirus types GI and GII, a coefficient of determination R² ≥ 0.999, and a relative standard deviation of ≤ 25% for repeated tests.

[0017] In some embodiments, the kit showed no nonspecific amplification signals for adenovirus, astrovirus, zaruzin virus, and rotavirus in wastewater samples.

[0018] Thirdly, this application provides the application of the method or the kit described herein in the preparation of products for wastewater public health monitoring, norovirus early warning, or community virus epidemic situation assessment.

[0019] Compared with the prior art, the present invention has the following significant advantages: This invention provides a method for simultaneous quantitative detection of norovirus genotypes GI and GII, enabling simultaneous detection and absolute quantification of norovirus GI and GII in the same reaction tube without relying on a standard curve, thus avoiding errors caused by batch-to-batch variations in standards. Regarding primer and probe design, the constructed detection system exhibits high specificity, showing no non-specific amplification even under coexistence conditions with other closely related viruses. Validation through gradient dilution experiments demonstrates good linearity over a wide dynamic range, with excellent linear correlation (R² = 0.999) and a low coefficient of variation (RSD = ±2%), exhibiting excellent detection consistency and stability. Droplet generation performance is stable, with the effective droplet count consistently maintained above 19,500, ensuring the reliability of the quantitative results. Sensitivity experiments show that the detection limit for norovirus can reach at least 0.6 copies / μL, demonstrating extremely high detection sensitivity.

[0020] In addition, this application also provides a digital PCR kit for simultaneous quantitative detection of norovirus GI and GII types, which provides a reliable, efficient and interference-resistant technical tool for monitoring norovirus in wastewater. It has important practical application value and promotion prospects in norovirus outbreak monitoring, transmission early warning and public health risk assessment. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the description of the embodiments of this application or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The flowchart illustrates the steps of the simultaneous quantitative detection method for norovirus genotypes GI and GII provided in this embodiment of the invention.

[0023] Figure 2 The digital PCR detection results provided in this embodiment of the invention include one-dimensional scatter plots of norovirus GI (A) and GII (B) before and after amplification, and norovirus GI (C) and GII (D).

[0024] Figure 3 The results show the specificity test results of the norovirus multiplex digital PCR system provided in Example 2 of this invention. The FAM fluorescence channel represents the norovirus GⅠ type signal (A), and the HEX fluorescence channel represents the norovirus GⅡ type signal (B).

[0025] Figure 4The results show the linear detection of norovirus multiplex digital PCR system provided in this embodiment of the invention. Linear one-dimensional scatter plot (A) and linear fit (B) of norovirus GⅠ type; linear one-dimensional scatter plot (C) and linear fit (D) of norovirus GⅡ type; each concentration gradient was repeated 3 times.

[0026] Figure 5 Scatter plot of repeatability test of norovirus GⅠ and GⅡ multiplex fluorescent quantitative PCR for embodiments of the present invention.

[0027] Figure 6 Scatter plots of norovirus multiplex digital PCR detection in 10 sewage samples provided in this embodiment of the invention, norovirus GⅠ type detection results (A) and norovirus GⅡ type detection results (B).

[0028] Figure 7 The images show the norovirus qPCR detection results of 10 sewage samples provided in this embodiment of the invention, including the norovirus GⅠ type detection result (A) and the norovirus GⅡ type detection result (B). Detailed Implementation

[0029] The embodiments of this application are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain this application, and should not be construed as limiting this application. In the description of this application, it should be understood that the terms "upper", "lower", "horizontal", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this application. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, "multiple" means two or more, unless otherwise explicitly specified.

[0030] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments.

[0031] Please see Figure 1 This application provides a flowchart of a method for simultaneous quantitative detection of norovirus genotypes GI and GII, comprising the following steps: Step S10: Extract viral nucleic acid from the wastewater sample to be tested.

[0032] Furthermore, the nucleic acid extraction of the wastewater sample was performed using a magnetic bead-based viral nucleic acid extraction kit, and included a pre-centrifugation step.

[0033] Furthermore, the viral RNA in the sample was extracted using the magnetic bead method.

[0034] Understandably, the magnetic bead method can effectively remove PCR inhibitors such as humic acid and heavy metals from sewage, thereby improving RNA purity; it is suitable for enriching samples with low viral load and improving detection sensitivity; and the magnetic bead method is easy to automate extraction, meeting the needs of batch sewage sample processing.

[0035] Furthermore, the sample can be a sewage sample, an environmental water sample, a food sample, or a clinical fecal sample; when the sample is a food sample, homogenization and proteinase K digestion steps are included before step S10; when the sample is a clinical fecal sample, dilution and centrifugation steps are included before step S10.

[0036] Step S20: Construct a digital PCR reaction system, wherein the reaction system comprises a specific primer and probe set for norovirus GI gene, a specific primer and probe set for norovirus GII gene, reverse transcriptase, DNA polymerase and nucleic acid template; Furthermore, the primers for the norovirus GⅠ type have sequences as shown in SEQ ID NO:1 and SEQ ID NO:2, the probe has a sequence as shown in SEQ ID NO:3, and the first label is FAM.

[0037] Furthermore, the primers for the norovirus GII type have sequences as shown in SEQ ID NO:4 and SEQ ID NO:5, the probe has a sequence as shown in SEQ ID NO:6, and the second label is HEX.

[0038] Table 1

[0039] Furthermore, the norovirus GI probe was labeled with FAM fluorescent dye, and the GII probe was labeled with HEX fluorescent dye.

[0040] It is understandable that during probe synthesis, primer synthesis companies (such as Bailige Biotechnology) label the 5' end of the norovirus GI type specific probe with a FAM fluorescent group and the 3' end with a quenching group; and the 5' end of the GII type probe with a HEX fluorescent group and the 3' end with a quenching group.

[0041] It is understandable that FAM and HEX are two commonly used non-overlapping fluorescence channels in digital PCR, offering high spectral separation and non-interference, ensuring independent interpretation of dual genotype signals. Experimental results showed that the signal-to-noise ratio (SNR) for genotype GI was 6.3, and for genotype GII it was 13.4, indicating good differentiation between positive and negative droplets (see...). Figure 2 C, 2D).

[0042] Step S30: The constructed reaction system is subjected to droplet generation and reverse transcription digital PCR amplification.

[0043] Furthermore, the digital PCR amplification program is as follows: reverse transcription at 50°C for 30 minutes; hot start at 95°C for 5 minutes; 40-50 cycles of denaturation at 96°C for 5 seconds and annealing / extension at 59°C for 30 seconds; and finally stabilization at 25°C for 1 minute.

[0044] Specifically, the above three-step amplification program was set up on a digital PCR instrument. Reverse transcription at 50℃ for 30 minutes ensured efficient transcription of RNA into cDNA; a hot start at 95℃ for 5 minutes activated the hot-start enzyme and inactivated the reverse transcriptase; 45 cycles of rapid denaturation and annealing extension (96℃ for 5 seconds, 59℃ for 30 seconds) ensured efficient amplification within the droplet; and finally, 25℃ for 1 minute stabilized the droplet for signal acquisition.

[0045] Furthermore, in the digital PCR reaction system, the final concentration of each primer pair and each probe is 100–300 nM.

[0046] Specifically, when preparing a 30 μL reaction system, the amounts of primer and probe mixtures added were optimized through concentration gradient pre-experiments, and the optimal final concentration was determined to be 200 nM (i.e., 200 nM for each primer pair and 200 nM for each probe). This concentration falls within the range of 100-300 nM.

[0047] Step S40: Collect signals from the FAM and HEX fluorescence channels respectively, where the FAM channel corresponds to norovirus GI and the HEX channel corresponds to norovirus GII.

[0048] It should be noted that norovirus G1 and G2 genomes share certain sequence homology, especially in the polymerase region or capsid region. Improper primer and probe design may lead to G1 primer amplification of the G2 template or probe cross-hybridization, resulting in typing errors. This application designs specific primers and TaqMan probes targeting conserved regions of norovirus G1 and G2. Both sets of primers and probes must undergo sequence alignment and experimental verification to ensure that only their respective targets are amplified in multiplex systems. The 5′ nuclease activity of the TaqMan probe requires a perfect match between the probe and the target to release fluorescence; even a single base mismatch can significantly reduce the signal, which inherently provides high specificity. Dual-channel independent detection further ensures the clarity of the signal source—only G1-positive droplets will show a signal in the first channel, and only G2-positive droplets will show a signal in the second channel, eliminating inter-channel crosstalk. The specificity of this method, screened for conserved regions and verified by dual fluorescence, can accurately distinguish between G1 and G2 norovirus, avoiding false-positive typing errors and providing reliable data for assessing the prevalence trends of different genotypes in community outbreaks. Step S50: Based on the number of positive droplets and Poisson distribution, the absolute quantitative results of norovirus GI and GII types in sewage are obtained simultaneously.

[0049] Specifically, using pure water as a template, 20 repeated tests were performed, and no positive droplets were detected (0), proving that the system was free of contamination or background signal. To avoid false positives caused by any possible random noise or extremely low concentration contamination, a positive threshold of ≥2 positive droplets was set. Samples with theoretical concentrations of 0.6 copies / μL and 0.3 copies / μL were prepared, with 20 replicates for each. The detection rate of the 0.6 copies / μL group was 100% (20 / 20), and the detection rate of the 0.3 copies / μL group was approximately 70%. Based on this, the limit of detection (LOD) was determined to be 0.6 copies / μL.

[0050] In this embodiment, the clearly defined positive threshold (≥2 droplets) eliminates the "gray zone" problem common in other molecular detection methods, ensuring objective result interpretation. The detection limit reaches 0.6 copies / μL, which is 5-10 times the sensitivity of traditional qPCR (qPCR typically has a LOD of 5-10 copies / reaction). This high sensitivity allows for the stable detection of extremely low viral loads in wastewater samples (such as in the early warning stage).

[0051] This invention provides a method for simultaneous quantitative detection of norovirus genotypes GI and GII, enabling simultaneous detection and absolute quantification of norovirus GI and GII in the same reaction tube without relying on a standard curve, thus avoiding errors caused by batch-to-batch variations in standards. Regarding primer and probe design, the constructed detection system exhibits high specificity, showing no non-specific amplification even under coexistence conditions with other closely related viruses. Validation through gradient dilution experiments demonstrates good linearity over a wide dynamic range, with excellent linear correlation (R² = 0.999) and a low coefficient of variation (RSD = ±2%), exhibiting excellent detection consistency and stability. Droplet generation performance is stable, with the effective droplet count consistently maintained above 19,500, ensuring the reliability of the quantitative results. Sensitivity experiments show that the detection limit for norovirus can reach at least 0.6 copies / μL, demonstrating extremely high detection sensitivity.

[0052] This application also provides a digital PCR kit for simultaneous quantitative detection of norovirus GI and GII types, comprising: specific primer pairs and probes for norovirus GI; specific primer pairs and probes for norovirus GII; digital PCR premix containing at least reverse transcriptase, DNA polymerase and digital PCR buffer; positive control and negative control; the kit is used to perform the method described.

[0053] Specifically, the kit components include: Primer-probe mixture: contains the GI-FAM probe, GII-HEX probe and corresponding upstream and downstream primers. Primer-probe sequences are shown in Table 1 (based on NCBI conserved region design).

[0054] Digital PCR premix: contains reverse transcriptase, hot-start DNA polymerase, dNTPs, MgCl2, and digital PCR-specific buffer.

[0055] Positive control samples: Norovirus GI and GII standard RNA (provided by Shanghai Institute of Metrology and Testing Technology), known concentration.

[0056] Negative control: Nuclease-free water.

[0057] It is understood that the integrated kit provided in this embodiment eliminates the tedious steps of users preparing and optimizing primers and probes themselves, reducing operational errors. Positive controls verify the validity of each batch of experiments, while negative controls monitor for contamination. The kit has been validated to be stable at -20°C for 6 months.

[0058] Furthermore, the FAM fluorescence channel is used to detect norovirus GI type, and the HEX fluorescence channel is used to detect norovirus GII type.

[0059] Specifically, in the signal acquisition settings of the digital PCR instrument, the gain parameter of the FAM channel is adjusted to a suitable detection range for the GI probe, and the HEX channel is set independently. During data analysis, the number of positive droplets from both channels is read separately to achieve simultaneous detection of two colors and two targets. Compared with single-channel single-target detection, the throughput is doubled, the reagent cost is reduced by about 40%, and sample handling errors caused by two separate detections are avoided.

[0060] Furthermore, the kit exhibits a linear detection range of 1.5–15,000 copies / μL for norovirus types GI and GII, with a coefficient of determination R² ≥ 0.999 and a relative standard deviation of ≤ 25% for repeated tests.

[0061] Specifically, the norovirus standard was serially diluted 10-fold to obtain five concentration points: 1.5, 15, 150, 1500, and 15000 copies / μL. Each concentration was repeated in triplicate. A linear regression was performed between the measured concentrations and the theoretical concentrations to calculate R².

[0062] Eight replicate tests were performed on 3000 copies / μL samples, and the relative standard deviation (RSD) of the quantitative concentration was calculated.

[0063] In this embodiment, R²=0.999 indicates excellent quantitative linearity over a four-order-of-magnitude range, eliminating the need for segmented calibration. The RSD of approximately 2% (far below the 25% quality control requirement) demonstrates excellent intra-batch repeatability and reliable results. This wide linear range allows the kit to detect both high-load samples during wastewater outbreaks and low-load samples during non-outbreak periods.

[0064] Furthermore, the kit showed no nonspecific amplification signals for adenovirus, astrovirus, zaruzin virus, and rotavirus in the wastewater samples.

[0065] In specificity assays, digital PCR detection was performed using nucleic acids from adenovirus (ADV), astrovirus (AstV), zaruvirus (SaV), and rotavirus (RV) as templates (template concentrations were comparable to the target concentrations). Negative controls and positive controls were also included. The results showed that no positive droplet signals were observed in either the FAM or HEX channels for the four viruses (see [link to kit]). Figure 3 ).

[0066] This embodiment demonstrates that the primer-probe design has high species specificity, avoiding cross-reactions with other enteroviruses coexisting in sewage and ensuring the accuracy of norovirus typing results. It reduces the risk of false positives and is particularly suitable for situations where multiple viruses coexist in environmental samples.

[0067] The digital PCR kit for simultaneous quantitative detection of norovirus GI and GII types provided in this application offers a reliable, efficient, and interference-resistant technical tool for monitoring norovirus in wastewater. It has significant practical application value and promising prospects in norovirus outbreak monitoring, transmission early warning, and public health risk assessment.

[0068] The above methods or kits are used in the preparation of products for wastewater public health monitoring, norovirus outbreak early warning, or community virus epidemic situation assessment.

[0069] For example, in assessing the community epidemic situation: Samples are regularly collected from the inlet of wastewater treatment plants (e.g., twice a week), and this method is used to detect norovirus GI / GII copy number concentrations. A correlation model between the positive detection rate and the community incidence rate is established by combining clinical case report data.

[0070] Epidemic warning: Set warning thresholds (such as two consecutive tests showing a concentration exceeding twice the historical baseline). When the sewage concentration exceeds the threshold, issue an early warning to the health department, prompting them to strengthen clinical monitoring and public education.

[0071] Public health resource allocation: During the peak season in winter and spring, medical resources (such as antiviral drugs and rehydration salts) are prioritized for high-load areas based on sewage testing results.

[0072] It is understandable that wastewater monitoring can detect viral circulation changes 3-7 days earlier than the onset of clinical symptoms, enabling early warning. Compared to monitoring based on clinical cases, wastewater monitoring is unbiased (covering asymptomatic infections), low-cost, and highly timely.

[0073] The absolute quantitative nature of this method allows for direct comparison of data from different times and locations, facilitating the establishment of a standardized monitoring network.

[0074] To more clearly illustrate the technical solution and expected effects of the present invention, the present invention will be described in detail below with reference to specific embodiments and experimental data. It should be understood that these embodiments are for illustrative purposes only and do not constitute any limitation on the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0075] Example Materials and reagents: Norovirus G1 and G2 RNA standards were purchased from Shanghai Institute of Metrology and Testing Technology. Digital PCR reverse transcription detection kits were purchased from Shanghai Little Turtle Technology Co., Ltd.; viral nucleic acid extraction kits were purchased from Suzhou Beaver Biomedical Engineering Co., Ltd.

[0076] Instruments and equipment: Centrifuge, Eppendorf GmbH, Germany; SCI DigitalPRO fully automated digital PCR machine, Shanghai Xiaohaigui Technology Co., Ltd.; Nucleic acid extractor (NPA-32P), Hangzhou Borui Technology Co., Ltd.; Real-time PCR instrument, Shanghai Hongshi Medical Technology Co., Ltd.

[0077] Primer and probe design Whole genome datasets of the species and closely related species were downloaded from the NCBI Genome database (https: / / www.ncbi.nlm.nih.gov / datasets / genome / ). Species-specific gene conservation analysis was performed using BLAST (Basic Local Alignment Search Tool) to screen for specific genes. Mega 11 was used for multiple sequence alignment of the target genes, and primers and probes were designed in conserved regions. This system is a multiplex digital PCR quantitative detection system for norovirus G1 and G2 types. The norovirus G1 probe was labeled with FAM fluorophore, and the norovirus G2 probe was labeled with HEX fluorophore. Primer and probe sequence information is shown in Table 1. Both primers and probes were synthesized by Bailige Biotechnology (Shanghai) Co., Ltd.

[0078] Wastewater sample enrichment and nucleic acid extraction: After pre-centrifugation of 15 mL of water sample, 8 mL of supernatant was transferred and nucleic acid was extracted using the Wastewater Nucleic Acid Extraction Kit (BeaverBeads® Wastewater Nucleic Acid Extraction Kit).

[0079] Real-time quantitative PCR detection: Norovirus GI / GII type premixed nucleic acid detection kit (fluorescent PCR method) was used for sample comparison testing. 5 μL each of the pre-treated test sample nucleic acid, positive control, and blank control were added to the PCR reaction tubes, for a final volume of 25 μL / tube. The PCR reaction program was set as follows: reverse transcription at 50℃ for 10 minutes; hot start at 95℃ for 1 minute; followed by 45 cycles of amplification reaction, including denaturation at 95℃ for 5 seconds and annealing / extension at 58℃ for 20 seconds.

[0080] Digital PCR detection conditions: The 30 μL digital PCR detection system contains 10 μL digital PCR buffer (dPCRBuffer), 3 μL DNA polymerase, 2 μL primers and 2 μL probe (final concentration 200 nM), and 5 μL template DNA, finally brought to 30 μL with nuclease-free water. Following the digital PCR instrument's operating instructions, prepare the necessary reagents and consumables and install the chip. The PCR reaction program is set as follows: reverse transcription at 50℃ for 30 minutes; hot start at 95℃ for 5 minutes; followed by 45 cycles of amplification, including denaturation at 96℃ for 5 seconds and annealing / extension at 59℃ for 30 seconds; finally, heat stabilization at 25℃ for 1 minute. Signal acquisition is performed using the FAM and HEX fluorescence channels. The entire digital PCR detection process is as follows: Figure 1 As shown, the process of generating droplets, PCR amplification, signal acquisition, and data analysis is completed automatically in sequence.

[0081] Specificity test: The nucleic acids of other viruses similar to norovirus, such as adenovirus (ADV), astrovirus (AstV), zarovirus (SaV), and rotavirus (RV), were selected as templates to verify the specificity of the developed multiplex norovirus detection digital PCR system. The relevant samples were obtained from samples preserved by the Shanghai Center for Disease Control and Prevention.

[0082] Linearity and repeatability tests: Norovirus standard, diluted to an initial concentration of 1.5 × 10⁻⁶. 4 Copies / µL, were serially diluted 10-fold using a diluent containing 20 ng / µL carrier RNA (to protect target nucleic acids and reduce loss during dilution). Five dilution gradients were set up (1.5 × 10⁻⁶). 4 Up to 1.5×10 0 (Copies / µL), with 3 replicates per gradient. 5 µL of diluted sample was used for testing. According to currently accepted EU quality control standards, points within the quantitative detection range should satisfy a linear fit to the standard curve, with a linear correlation coefficient >95%, and the relative standard deviation (RSD) of the test values ​​within all groups <25%.

[0083] Sensitivity testing: First, a blank limit test was performed using pure water samples, with 20 tests conducted. A blank detection standard and a positive / negative cutoff value were established to avoid laboratory contamination and interference from other fluorescent impurities. Starting with a sample at the lower limit of linear range (1.5 copies / µL of norovirus RNA), samples were diluted to prepare theoretical concentrations of 0.6 and 0.3 copies / µL for limit of detection (LOD) testing. Twenty replicates were set for each concentration gradient. A concentration was considered stably detectable when the detection rate was ≥95%.

[0084] Wastewater sample testing: Nucleic acid was extracted from wastewater samples at 10 different sites and dPCR was performed. The limit of detection (LOD) determined in section 1.3.7 was used as the threshold. A result greater than or equal to the LOD was considered positive, and a result less than the LOD was considered negative. Simultaneously, qPCR tests were performed on the 10 wastewater samples using a qPCR kit (Norovirus GI / GII Premixed Nucleic Acid Detection Kit, Jiangsu ShuoShi Biotechnology Co., Ltd.).

[0085] Results and Analysis Validation of primers, probes, and amplification conditions: The above detection procedure was used to detect norovirus GI and GII standards, and imaging analysis was performed on the results before and after PCR amplification. Imaging before PCR amplification helps identify signal anomalies that may be caused by high background fluorescence of the probe itself, thus avoiding false positives. Imaging after PCR amplification is used to confirm whether the number of droplets generated on the chip meets the requirements, and further analyzes the signal-to-noise ratio (SNR) of positive and negative droplets to assess whether it meets the quality standards of digital PCR experiments, ensuring that the results are reliable and usable for subsequent quantitative analysis. From the experimental results ( Figure 2 As shown in A and B), before PCR amplification, the fluorescence signals of norovirus GI and GII were very low. After amplification, the positive and negative droplets were well distinguished, with signal-to-noise ratios reaching 6.3 and 13.4, respectively. Figure 2 (C, D) It can also be seen that positive signals are very easy to distinguish from negative signals. The experimental results show that this digital PCR system and experimental conditions can be used to detect norovirus GI and GII.

[0086] Specificity test: A one-dimensional scatter plot of digital PCR data for specificity testing is shown below. Figure 3 As shown, the detection system can effectively amplify the target fragments of norovirus GⅠ (FAM channel) and GⅡ (HEX channel), and the signal clusters are clearly separated; no signal was detected in the negative control (NTC), confirming that the sample processing was uncontaminated; no positive signals were amplified in the two fluorescent channels for adenovirus (ADV), astrovirus (AstV), zaaruvirus (SaV), and rotavirus (RV) nucleic acid samples, indicating that the detection system has high specificity.

[0087] Linearity Experiment: Results of linearity validation experiment for norovirus multiplex digital PCR are as follows Figure 4 As shown. Both GⅠ and GⅡ types exhibit good signal discrimination and channel specificity, and the number of positive droplets decreases gradient with decreasing template concentration. Linear fitting results show that ( Figure 2(B, D) The standard curve determination coefficients (R²) for both G1 and G2 norovirus types reached 0.999, indicating that the method exhibits excellent linearity for the detection of both genotypes within a concentration range of 1.5–15000 copies / μL. Furthermore, the linearity and repeatability statistics for G1 and G2 types (Tables 2 and 3) show that the RSD for repeated detections at each concentration gradient was less than 25%, further demonstrating the good repeatability and stable, reliable quantitative results of this detection system.

[0088] Table 2. Linearity test results of the norovirus GⅠ type multiplex fluorescence quantitative system

[0089] Table 3. Linearity test results of the norovirus GII type multiplex fluorescence quantitative system

[0090] Repeatability Test: To further verify the repeatability and stability of the established multiplex digital PCR norovirus detection system, this study simultaneously performed quantitative detection and analysis of norovirus types G1 and G2. The sample concentration was diluted to 3000 copies / μL, and eight replicate tests were performed. The number of positive droplets and the quantitative concentration in each experiment were counted, and the results are shown in Table 6. The number of positive droplets in all eight replicate tests was higher than 19,500, and the relative standard deviation (RSD) of all concentration measurements was around 2%, indicating that the method has good repeatability. One-dimensional scatter plot of repeatability experiment (…) Figure 5 The results showed that the number of positive droplets in the FAM and HEX channels was highly consistent, further confirming the stability of the detection system.

[0091] Table 4. Results of Norovirus Multiplex Digital PCR Repeatability Test

[0092] Sensitivity testing: First, the system was examined using blank limit tests. No positive results were detected in any of the 20 blank tests, indicating that the entire experimental system was uncontaminated. To accommodate environmental differences in different laboratories and reduce the risk of false positives, a result of ≥2 positive droplets was considered positive. Subsequently, samples near the critical concentrations (0.6 copies / μL and 0.3 copies / μL) were analyzed, and the results are shown in Tables 5 and 6. At a concentration of 0.6 copies / μL, all 20 samples were detected; when the concentration was below 0.3 copies / μL, the detection rate was approximately 70%. Therefore, the detection limit of this method can reach at least 0.6 copies / μL.

[0093] Table 5. Sensitivity test results of the norovirus GⅠ type multiplex digital PCR detection system

[0094] Table 6. Sensitivity test results of the norovirus GII multiplex digital PCR detection system

[0095] Quantitative detection of wastewater samples: The results of a one-dimensional scatter plot of norovirus typing detection in 10 wastewater samples are shown below. Figure 6 As shown. The experimental quality control was good: no abnormal amplification was observed in NTC (template-free control), and the signal clustering in PTC (positive template control) was clear, indicating that the test results for this batch were valid. Of all samples, 9 were positive for norovirus GII, and sample number 9 was negative. Comparison with qPCR data showed that the digital PCR results were completely consistent (100% concordance rate). From... Figure 6 As shown in B, there are certain differences in the positive signal intensity among different samples. Of particular note is that while the copy number concentrations of samples 1 and 8 are relatively similar, their signal intensities differ significantly. Correspondingly, in the qPCR results, the Ct value of sample 8 is also significantly greater than that of sample 1.

[0096] This result indicates that inhibitors present in wastewater matrices significantly suppress qPCR amplification efficiency, leading to higher Ct values. In digital PCR, inhibitors also reduce amplification efficiency within individual droplets, manifested as weakened signal intensity; however, because digital PCR uses endpoint detection, the quantitative result is determined solely by the number of positive droplets, unaffected by signal intensity. This phenomenon further suggests that digital PCR has higher tolerance to inhibitors and is more suitable for the accurate quantitative detection of pathogens in complex matrices such as wastewater.

[0097] Table 7. Norovirus digital PCR and qPCR data from 10 sewage samples.

[0098]

[0099] This application provides a multiplex digital PCR (dPCR) quantitative detection method for norovirus in wastewater samples. This method can achieve accurate quantification of norovirus types GI and GII without relying on a standard curve, and has the advantages of simple operation, good repeatability and precision, significantly improving the efficiency and reliability of virus quantification detection in complex wastewater matrices.

[0100] Regarding primer and probe design, the constructed detection system exhibited high specificity, showing no non-specific amplification even under coexistence conditions with other closely related viruses. Validation through gradient dilution experiments demonstrated that this digital PCR method exhibited good linear response over a wide dynamic range, with excellent linear correlation (R² = 0.999) and low repeatability coefficient of variation (RSD = ±2%), showing excellent detection consistency and stability. Droplet generation performance was stable, with the effective droplet number consistently maintained above 19,500, ensuring the reliability of the quantitative results. Sensitivity experiments showed that the detection limit for norovirus reached at least 0.6 copies / μL, demonstrating extremely high detection sensitivity.

[0101] In actual wastewater sample validation, the results of 10 samples showed 100% consistency between digital PCR and qPCR, with no false negatives or missed detections observed in any samples. Notably, in wastewater samples with varying inhibitor concentrations, digital PCR demonstrated significantly better quantitative stability than qPCR, with less matrix interference. This indicates that the method is more tolerant to inhibitors in complex environmental samples and is suitable for accurate virus quantification in highly interfering samples such as wastewater.

[0102] The multiplex digital PCR detection kit developed in this study provides a reliable, efficient, and interference-resistant technical tool for monitoring norovirus in wastewater. It has significant practical application value and promising prospects in norovirus outbreak monitoring, transmission early warning, and public health risk assessment.

[0103] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0104] It is understood that the technical features of the above embodiments can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0105] The above are merely preferred embodiments of this application, and only specifically describe the technical principles of this application. These descriptions are only for explaining the principles of this application and should not be construed as limiting the scope of protection of this application in any way. Based on this explanation, any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application, as well as other specific embodiments of this application that can be conceived by those skilled in the art without creative effort, should be included within the scope of protection of this application.

Claims

1. A method for simultaneous quantitative detection of norovirus genotypes GI and GII, characterized in that, Includes the following steps: Extract viral nucleic acid from wastewater samples to be tested; A digital PCR reaction system was constructed, comprising a specific primer and probe set for norovirus GI gene, a specific primer and probe set for norovirus GII gene, reverse transcriptase, DNA polymerase, and nucleic acid template. The constructed reaction system was subjected to droplet generation and reverse transcription digital PCR amplification. Signals from the FAM and HEX fluorescence channels were acquired separately, with the FAM channel corresponding to norovirus GI type and the HEX channel corresponding to norovirus GII type. Based on the number of positive droplets and Poisson distribution, absolute quantitative results of norovirus types GI and GII in sewage were obtained simultaneously.

2. The method according to claim 1, characterized in that, The norovirus GI probe is labeled with FAM fluorescent pigment, and the norovirus GII probe is labeled with HEX fluorescent pigment.

3. The method according to claim 1, characterized in that, The digital PCR amplification program is as follows: reverse transcription at 50°C for 30 minutes; hot start at 95°C for 5 minutes; 40-50 cycles of denaturation at 96°C for 5 seconds and annealing / extension at 59°C for 30 seconds; and finally stabilization at 25°C for 1 minute.

4. The method according to claim 1, characterized in that, In the digital PCR reaction system, the final concentration of each primer pair and each probe is 100–300 nM.

5. The method according to claim 1, characterized in that, When interpreting the results, a positive threshold of ≥2 positive droplets was used, and the detection limit was 0.6 copies / μL.

6. The method according to claim 1, characterized in that, The nucleic acid extraction of the wastewater samples was performed using a magnetic bead-based viral nucleic acid extraction kit, which included a pre-centrifugation step.

7. A digital PCR kit for simultaneous quantitative detection of norovirus types GI and GII, characterized in that, Include: Specific primer pairs and probes for norovirus GI type; Specific primer pairs and probes for norovirus GII; Digital PCR premix solution contains at least reverse transcriptase, DNA polymerase, and digital PCR-specific buffer. Positive and negative control samples; The kit is used to perform the method according to any one of claims 1 to 6.

8. The reagent kit according to claim 7, characterized in that, The FAM fluorescence channel is used to detect norovirus GI, and the HEX fluorescence channel is used to detect norovirus GII.

9. The reagent kit according to claim 7, characterized in that, The kit exhibits a linear detection range of 1.5–15,000 copies / μL for norovirus types GI and GII, with a coefficient of determination R² ≥ 0.999 and a relative standard deviation of ≤ 25% for repeated tests.

10. The kit according to claim 7, characterized in that, The kit showed no nonspecific amplification signals for adenovirus, astrovirus, zarjor virus, and rotavirus in the wastewater samples.

11. The use of the method according to any one of claims 1 to 6 or the kit according to any one of claims 7 to 10 in the preparation of products for wastewater public health monitoring, norovirus outbreak early warning or community virus epidemic situation assessment.

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