Virus qPCR (quantitative polymerase chain reaction) detection method and application

Abstract

The invention discloses a virus qPCR (quantitative polymerase chain reaction) detection method and application, belongs to the technical field of biology, discloses a primer group, a primer / probe group and a product of a water-borne pathogenic virus, and further provides an amplification method, a detection method and application of a water-borne pathogenic virus sequence. The invention also provides a library for detecting a water-borne pathogenic virus.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a virus qPCR detection method and application. BACKGROUND

[0002] Water-borne diseases refer to diseases transmitted through drinking or contacting water contaminated by pathogens. Such diseases usually include the following types of pathogens: bacteria, such as Salmonella typhi, Salmonella paratyphi, Vibrio cholerae, and Shigella dysenteriae; viruses, such as hepatitis A virus, poliovirus, coxsackievirus, and adenovirus; and protozoa, such as Giardia lamblia, Entamoeba histolytica, and Schistosoma. These pathogens mainly come from human feces, domestic sewage, hospital waste, and waste water from livestock slaughtering, leather, and food industries. The epidemic characteristics of water-borne diseases include outbreak, consistent distribution of cases with water supply range, and rapid control of the epidemic after strengthening water purification and disinfection. The key to preventing water-borne diseases lies in ensuring the safety and hygiene of drinking water, avoiding drinking untreated or unknown source water, and seeking medical treatment in time when relevant symptoms occur. For high-risk areas, regular water quality testing and disinfection are necessary preventive measures. Therefore, pathogen detection for different water sources is extremely important. SUMMARY

[0003] To solve the technical problems in the prior art, the application provides the following technical solutions.

[0004] The application provides a primer set for detecting water-borne pathogenic viruses, and the sequence of the primer set is at least one of the following primer combinations: 1) primers SEQ ID NO: 1, 2, 4, and 5, 2) primers SEQ ID NO: 7, 8, 10, and 11, 3) primers SEQ ID NO: 13, 14, 16, and 17, 4) primers SEQ ID NO: 19, 20, 22, and 23, 5) primers SEQ ID NO: 25, 26, 28, and 29, 6) primers SEQ ID NO: 31, 32, 34, and 35, 7) primers SEQ ID NO: 37, 38, 40, 41, 43, and 44, 8) primers SEQ ID NO: 46, 47, 48, 49, 52, and 53, 9) primers SEQ ID NO: 55 and 56, and / or 10) primers SEQ ID NO: 58 and 59.

[0005] Further, the water-borne pathogenic viruses are enterovirus, hepatitis A virus, enterovirus A71, norovirus GⅠ, norovirus GⅡ, astrovirus, poliovirus, echovirus, hepatitis E virus, Sali virus, Aichi virus, coxsackievirus A16, coxsackievirus A6, rotavirus, Sapporo virus, bacteriophage MS2, bocavirus, and adenovirus.

[0006] Furthermore, the primer set includes degenerate positions.

[0007] Furthermore, the degenerate position includes R, W, M, Y, K, B, D, H, N, S, or V, wherein R includes A or G, W includes A or T, M includes A or C, Y includes C or T, K includes G or T, B includes A, G, or T, D includes A, G, or T, H includes A, C, or T, N includes A, G, C, or T, S includes G or C, and V includes A, C, or G.

[0008] In this invention, primer sets and primers are interchangeable. Primers refer to short nucleic acid molecules, and the primers disclosed in this invention are between 15 and 40 nucleotides in length. A hybrid can be formed between the primer and the target nucleic acid strand through nucleic acid hybridization and annealing with a complementary target nucleic acid molecule. The primer can be extended along the target nucleic acid molecule using polymerase. Therefore, primers can be used to amplify target nucleic acid molecules, wherein the primer sequence is specific to the target nucleic acid molecule.

[0009] In this invention, degenerate bases include, but are not limited to, nucleotide bases that do not follow the Watson-Crick base pairing rule but can combine with at least two, but not all four, of the four canonical bases A, T / U, C, and G. Degenerate bases may also be referred to as wobbly bases; these terms are used interchangeably in this invention.

[0010] In this invention, the modified nucleotides include, but are not limited to, 2'-modified nucleotides or 5-methylcytosine. Specifically, the 2'-modified nucleotides include, but are not limited to, 2'-O-methyl modified nucleotides and 2'-fluorine modified oligonucleotides; the 5-methylcytosine includes, but is not limited to, 5-methyl-deoxycytosine; and the 5-methyl-deoxycytosine includes, but is not limited to, 5-Me-dC and 5-methyl-2'-deoxycytosine. In some embodiments, the primers may include two or more modified nucleotides. The two or more modified nucleotides may have the same or different modifications. In some embodiments, the primer set may include one or more 5-methylcytosines. The primer set may have 0, 1, 2, 3, 4, 5, 6, 7, 8 or more 2'-O-methyl modified nucleotides, 2'-fluorine modified oligonucleotides, 5-methylcytosine, or combinations thereof.

[0011] This invention provides a primer / probe set for detecting waterborne pathogenic viruses. The sequence of the primer / probe set is at least one of the following primer / probe combinations: 1) Primers SEQ ID NO: 1, 2, 4, 5, probes SEQ ID NO: 3, 6; 2) Primers SEQ ID NO: 7, 8, 10, 11, probes SEQ ID NO: 9, 12; 3) Primers SEQ ID NO: 13, 14, 16, 17, probes SEQ ID NO: 15, 18; 4) Primers SEQ ID NO: 19, 20, 22, 23, probes SEQ ID NO: 21, 24; 5) Primers SEQ ID NO: 25, 26, 28, 29, probes SEQ ID NO: 27, 30; 6) Primers SEQ ID NO: 31, 32, 34, 35, probes SEQ ID NO: 33, 36; 7) Primers SEQ ID NO: 37, 38, 40, 41, 43, 44, probes SEQ ID NO: 36, 47, 4 ...9, 40 NO:39, 42, 45; 8) Primers SEQ ID NO:46, 47, 48, 49, 52, 53, probes SEQ ID NO:50, 51, 54; 9) Primers SEQ ID NO:55, 56, probes SEQ ID NO:57; and / or 10) Primers SEQ ID NO:58, 59, probes SEQ ID NO:60.

[0012] Furthermore, the waterborne pathogenic viruses mentioned are enteroviruses, hepatitis A viruses, enterovirus A71, norovirus G1, norovirus G2, astroviruses, polioviruses, echoviruses, hepatitis E viruses, Saliviruses, Aichi viruses, Cusackie viruses, Coxsackievirus A16, Coxsackievirus A6, rotaviruses, Sapporo viruses, bacteriophage MS2, bocaviruses, and adenoviruses.

[0013] Furthermore, the probe is labeled with a fluorescent dye.

[0014] Furthermore, the fluorescent dye labels include FAM, HEX, TAMRA, ROX, TET, JOE, Cy3, Cy5, VIC, Cy5.5, Texas Red, NED, FAM-dT, TAMRA-dT, CY3-dT, CY5-dT, HEX-dT, and FITC.

[0015] Furthermore, the probe also includes a quenching group.

[0016] Furthermore, the quenching groups include BHQ1, TAMRA, BHQ2, Rhodamine 6G, Rhodamine B, BHQ3, Dabcyl, Eclipse, MGB, BHQ1-dT, and BHQ2-dT.

[0017] Furthermore, the probe is labeled with a modifying group.

[0018] Furthermore, the modifying groups include amino groups, phosphate groups, biotin, biotin-TEG, and C3-spacer.

[0019] Furthermore, the primer set includes degenerate positions.

[0020] Furthermore, the degenerate position includes R, W, M, Y, K, B, D, H, N, S, or V, wherein R includes A or G, W includes A or T, M includes A or C, Y includes C or T, K includes G or T, B includes A, G, or T, D includes A, G, or T, H includes A, C, or T, N includes A, G, C, or T, S includes G or C, and V includes A, C, or G.

[0021] In the primers of this invention labeled with a labeling substance, the labeling substance can bind directly to the primer or bind via a linker. As the linker, any linker commonly used in this field is acceptable; specifically, for example, a nucleic acid of 1 to 3 bases is preferred, DNA of 1 to 3 bases is more preferred, DNA of 2 bases is even more preferred, and adenine (A)-adenine (A) is particularly preferred.

[0022] This invention provides a product for detecting waterborne pathogenic viruses, the product comprising the primer set described above, or the primer / probe set described above.

[0023] Furthermore, the products include nucleic acid membrane strips, reagent kits, lyophilized microspheres, and chips.

[0024] Furthermore, the kit also includes PCR amplification buffer and amplification enzyme.

[0025] Furthermore, the kit also includes reagents for multiplex PCR.

[0026] Furthermore, the amplification enzyme includes DNA polymerase and / or RNA polymerase.

[0027] Furthermore, the kit also includes a reverse transcription reaction system.

[0028] Furthermore, the reverse transcription reaction system includes primers.

[0029] Furthermore, the primers for the reverse transcription reaction system are random primers.

[0030] Furthermore, the kit also includes a fluorescent dye.

[0031] Furthermore, the kit also includes instructions.

[0032] In some embodiments, primers or probes are present or provided in one or more containers at a suitable dose, or immobilized on a matrix, wherein the primers or probes may be provided as a suspension in an aqueous solution, or, for example, as a lyophilized or freeze-dried powder. On the other hand, the container carrying the primers or probes may be any conventional container capable of containing the provided form, such as a microcentrifuge tube, ampoule, or bottle.

[0033] In this invention, various known fluorescent dyes can be used. Examples include methods using intercalators with marking functions, and methods using probes that bind fluorescent substances to nucleotides that specifically hybridize to relatively amplified DNA sequences. Examples of intercalators include ethidium bromide and SYBR Green I as unsaturated fluorescent dyes, and Resolight (manufactured by Roche) and EvaGreen (manufactured by Biotim) as saturated fluorescent dyes. Preferred intercalators are SYBR Green I as an unsaturated fluorescent dye, and EvaGreen and Resolight as saturated fluorescent dyes; more preferably, EvaGreen and Resolight are saturated fluorescent dyes. The dosage is determined according to the recommendations of the manufacturer or distributor of the fluorescent dye used.

[0034] In some embodiments, the kit also includes instructions for obtaining and processing samples. In some embodiments, the kit includes genomic DNA of a pathogen as a positive control for PCR and sterile water as a negative control.

[0035] Furthermore, the freeze-dried microspheres use 10% (w / v) trehalose, 5% (w / v) mannitol, 4% (w / v) cyclodextrin, and 0.5% (w / v) BSA as freeze-drying protectants.

[0036] In some embodiments, the freeze-dried microspheres are used for room-temperature storage and transportation of chips. During the freeze-drying process, attention must be paid to the drying process. In some implementations, the main purpose of freeze-drying is to remove free water from the material and some adsorbed water adsorbed in the interstices of the solid lattice in a low-temperature and vacuum environment. Conventional freeze-drying processes generally consist of three steps: pre-freezing, sublimation drying, and desorption drying. Unless otherwise specified, the freeze-drying operations performed in this invention are standard procedures for those skilled in the art.

[0037] Furthermore, the chip is a high-throughput qPCR detection chip.

[0038] Furthermore, the chip is a nucleic acid extraction structure and a nucleic acid detection structure.

[0039] Furthermore, the nucleic acid detection structure includes a PCR amplification chamber.

[0040] Furthermore, the nucleic acid detection structure incorporates a built-in lyophilized reagent formulation.

[0041] This invention provides a method for amplifying the sequence of a waterborne pathogenic virus, wherein the method uses the primer set, the primer / probe set, or the product described above to perform the amplification reaction.

[0042] Furthermore, the amplification reaction is performed using methods including LCR, NASBA, SDA, TMA, bDNA, and PCR.

[0043] Furthermore, the amplification reaction is performed using PCR.

[0044] Furthermore, the PCR includes real-time PCR and multiplex PCR.

[0045] Furthermore, the PCR is selected from multiplex PCR.

[0046] Furthermore, the amplification reaction is carried out under similar amplification conditions.

[0047] In this invention, amplification refers to the generation of multiple copies of a polynucleotide or a portion of a polynucleotide, typically starting from a small number of polynucleotides (e.g., a single polynucleotide molecule). The resulting amplification product is also called an amplicon, and the amplification product or amplicon is usually detectable. Polynucleotide amplification involves a variety of chemical and enzymatic processes.

[0048] The Embodiments section of this invention provides exemplary embodiments of amplification conditions. However, as used herein, the term "amplification conditions" refers to a temperature and / or incubation time suitable for obtaining a detectable amount of the target. Therefore, the term "similar amplification conditions" means that, if desired, targets can be assayed at similar temperatures. The term "similar amplification conditions" also means that, if desired, targets can be assayed at similar incubation times. In some cases, the term "similar amplification conditions" also refers to the number of amplification cycles. However, it is well known in the art that the number of cycles is not always strict. For example, some samples may be removed or left to undergo additional amplification cycles before other samples. In other cases, the term "similar amplification conditions" also refers to the nature of the buffer and amplification reagents (enzymes, nucleotides, salts, etc.) used. The term "similar amplification conditions" also means that conditions (e.g., time, buffer, number of cycles, temperature, etc.) may be slightly varied or may be the same.

[0049] This invention provides a method for detecting waterborne pathogenic viruses in samples, the method comprising amplifying using the primer set, primer / probe set, or product described above, obtaining amplification products, and sequencing using the amplification products.

[0050] Furthermore, the samples include human body samples and environmental samples, wherein the human body samples are ex vivo human body samples.

[0051] Furthermore, the human samples include blood, saliva, and urine.

[0052] Furthermore, the environmental samples include water sources.

[0053] Furthermore, the water source includes sewage.

[0054] Furthermore, the wastewater includes wastewater from wastewater treatment plants, rivers, and sewer systems.

[0055] Furthermore, the method described is not for diagnostic purposes.

[0056] This invention provides a library for detecting waterborne pathogenic viruses, the library comprising the primer set or the primer / probe set described above.

[0057] This invention provides the aforementioned primer set and its application in the preparation of products for amplifying or detecting waterborne pathogenic viruses.

[0058] This invention provides the application of the aforementioned primer set, the aforementioned primer / probe set, or the aforementioned product in the detection of waterborne pathogenic viruses for non-diagnostic purposes. Attached Figure Description

[0059] Figure 1 This is a graph showing the dimer detection results during the qPCR detection process of primer and probe sets for 18 waterborne pathogenic viruses after pairing.

[0060] Figure 2 This image shows the dimer detection results during qPCR testing of 18 waterborne pathogenic viruses after their combinations were confirmed. Note: ①: Multiple viral primer probes (without template); ②: Positive control of viral combinations; ③④⑤: Single viral primer probes (without template).

[0061] Figure 3 The graph shows the enterovirus qPCR amplification curve (left) and standard curve (right).

[0062] Figure 4 The graph shows the qPCR amplification curve (left) and standard curve (right) of enterovirus 71.

[0063] Figure 5 The graph shows the amplification curve (left) and standard curve (right) of norovirus GⅠ type qPCR.

[0064] Figure 6 The graph shows the amplification curve (left) and standard curve (right) of norovirus GII qPCR.

[0065] Figure 7 The graph shows the qPCR amplification curve (left) and standard curve (right) of poliovirus.

[0066] Figure 8 The graph shows the echovirus qPCR amplification curve (left) and standard curve (right).

[0067] Figure 9 The graph shows the hepatitis A virus qPCR amplification curve (left) and standard curve (right).

[0068] Figure 10 The graph shows the hepatitis E virus qPCR amplification curve (left) and standard curve (right).

[0069] Figure 11 The graph shows the Aichi virus qPCR amplification curve (left) and standard curve (right).

[0070] Figure 12 The graph shows the qPCR amplification curve (left) and standard curve (right) for the Cushn virus.

[0071] Figure 13 The graph shows the amplification curve (left) and standard curve (right) of astrovirus qPCR.

[0072] Figure 14 The graph shows the Sapporo virus qPCR amplification curve (left) and standard curve (right).

[0073] Figure 15 The graph shows the Sali virus qPCR amplification curve (left) and standard curve (right).

[0074] Figure 16 The graph shows the rotavirus qPCR amplification curve (left) and the standard curve (right).

[0075] Figure 17 The graph shows the qPCR amplification curve (left) and standard curve (right) for Coxsackievirus A16.

[0076] Figure 18 The graph shows the amplification curve (left) and standard curve (right) of Coxsackievirus A6 qPCR.

[0077] Figure 19 The graph shows the adenovirus qPCR amplification curve (left) and the standard curve (right).

[0078] Figure 20 The graph shows the amplification curve (left) and standard curve (right) of Bocavirus qPCR.

[0079] Figure 21 This is a diagram showing the state of the qPCR reaction system after lyophilization.

[0080] Figure 22 This is a graph showing the inhibitory effect of the lyophilization protectant formulation on the qPCR reaction. Note: *p<0.05, compared with before lyophilization.

[0081] Figure 23 This is a schematic diagram of the preparation of freeze-dried microspheres using a microsphere forming apparatus.

[0082] Figure 24 This is a picture of the finished freeze-dried microspheres.

[0083] Figure 25 This is a comparison of the virus detection capabilities of lyophilized microspheres and liquid systems using qPCR. Note: *p<0.05, compared to the non-lyophilized system.

[0084] Figure 26 This is a graph showing the effect of storage temperature of freeze-dried microspheres on virus detection capability. Note: *p<0.05, comparison of freeze-dried microspheres stored at 4℃ and room temperature; #p<0.05, comparison of freeze-dried microspheres stored at 4℃ and 37℃.

[0085] Figure 27 This is a schematic diagram of the main specifications of the microfluidic chip. Detailed Implementation

[0086] The invention is further illustrated below with reference to specific embodiments. It should be understood that the specific embodiments described herein are by way of example and are not intended to limit the invention. The main features of the invention can be used in various embodiments without departing from the scope of the invention.

[0087] Example 1: Screening of primers and probes for qPCR detection of waterborne pathogenic viruses

[0088] Gene sequences (VP1 / VP2) of 18 waterborne pathogenic viruses were retrieved from GenBank. Primers and probes were designed for each virus's conserved sequence using Oligo 7.0, and homology analysis was performed for comparison. Specific sequences are shown in Table 1. The primer target sequences were cloned into the pUC57 vector, and plasmid standards were synthesized. All primers, probes, and plasmid standards were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0089] Table 1 Primer and probe sequences for qPCR detection of 18 waterborne viruses

[0090]

[0091]

[0092]

[0093] Example 2: Establishment of a multiplex qPCR detection system for waterborne pathogenic viruses

[0094] To achieve high-throughput detection of waterborne pathogenic viruses, this invention employs multiplex quantitative PCR (qPCR) technology based on quantitative real-time PCR. This technology combines several different fluorescent groups and utilizes the instrument's ability to detect fluorescence in multiple channels to achieve real-time quantitative detection of multiple targets. However, when multiple primer pairs, probes, and templates are added to the same reaction tube, the number of nucleotide chains within the tube increases with the detection multiplex, significantly increasing the likelihood of dimers or even multimers forming between these nucleotide chains. To prevent the formation of dimers between multiple primers and probes, this invention utilizes... Figure 1 The experiment comparisons shown are as follows.

[0095] Finally, multiple primer-probe combinations for RNA viruses were performed, and no significant dimeric bands were found in the PCR agarose gel electrophoresis results for the following eight viral primer-probe combinations: enterovirus with hepatitis A virus, enterovirus A71 with norovirus G1, norovirus G2 with astrovirus, poliovirus with echovirus, hepatitis E virus with Salivirus, Aichi virus with Cuxay virus, Coxsackievirus A16, Coxsackievirus A6 with rotavirus, and Sapporo virus with the positive control. Figure 2 The fluorescent and quenching groups of some probes (FAM, CY5, and ROX probes) were replaced, as detailed in Table 2. The Ct values ​​of single qPCR and multiplex qPCR for detecting various viruses were compared. The results showed no significant difference in Ct values ​​between single and multiplex qPCR, indicating that the multiplex qPCR reaction system can perform high-throughput qPCR detection of waterborne viruses.

[0096] Table 2 Comparison of Ct values ​​for single / multiplex qPCR detection of 18 waterborne pathogenic viruses

[0097]

[0098] Example 3: Sensitivity Evaluation of qPCR Detection of Waterborne Pathogenic Viruses

[0099] The pUC57 plasmids of various RNA viruses were transcribed in vitro using the T7 RNA in vitro transcription kit, and the RNA was purified to obtain RNA standards, which were then serially diluted 10-fold (10... -1 -10 -11 Using a template and a negative control, amplification was performed using a real-time PCR instrument to construct a standard curve.

[0100] The results showed that as the copy number of the positive plasmid decreased, the peak time of the amplification curve gradually lengthened, but significant amplification was still observed, and the gradient was uniform, with a detection limit of 10. 2 The limits of detection (LODs) for enteroviruses, enterovirus A71, norovirus G1, norovirus G2, poliovirus, echovirus, hepatitis A virus, hepatitis E virus, Aichi virus, Cuxay virus, astrovirus, Sapporo virus, Sali virus, rotavirus, Coxsackievirus A16, Coxsackievirus A6, adenovirus, and bocavirus were 55.89 copies / μL, 12.81 copies / μL, 134.14 copies / μL, 116.34 copies / μL, 148.07 copies / μL, 64.05 copies / μL, 65.53 copies / μL, 16.29 copies / μL, 43.85 copies / μL, 109.63 copies / μL, 52.30 copies / μL, 877.48 copies / μL, 89.78 copies / μL, and 131.05 copies / µL, respectively. The results showed that the selected qPCR primers and probes had high sensitivity and could detect low-abundance viruses in the samples. The qPCR amplification curves and standard curves for 18 viruses are shown below. Figures 3-20 .

[0101] Example 4: Specificity Evaluation of qPCR Detection of Waterborne Pathogenic Viruses

[0102] After mixing all the constructed pUC57 plasmids, the selected primers and probes were used for detection. It was found that each viral primer and probe only recognized the corresponding template, indicating that each primer and probe had good single qPCR specificity. The results are shown in Table 3.

[0103] Table 3

[0104]

[0105]

[0106] Example 5: Repeatability Evaluation of qPCR Detection of Waterborne Pathogenic Viruses

[0107] The repeatability of the established multiplex qPCR detection method was evaluated, and the viral RNA labeling stock solution (10) was tested. 12 Perform 10-fold serial dilutions (copies / μL), selecting 3 dilution gradients (10 copies / μL).-3 10 -4 10 -5 Using viral RNA standards as templates, and with a negative control, three replicates were set up for each gradient, and the experiment was repeated three times. The coefficient of variation within and between groups was calculated. The formula is: coefficient of variation (CV) = standard deviation / mean × 100%.

[0108] Table 4 shows the repeatability test results. The coefficients of variation for intra-group qPCR reactions of each viral standard at different concentration gradients ranged from 0.002% to 1.99%, and the coefficients of variation between groups ranged from 0.32% to 4.97%, all less than 5%, indicating that the multiplex qPCR detection method has good repeatability and stability.

[0109] Table 4. Repeatability evaluation of multiplex qPCR detection of viruses in water at different concentrations

[0110]

[0111]

[0112] Example 6: Establishment and Evaluation of a High-Throughput Virus Microfluidic Detection System

[0113] 1. Screening of freeze-drying protectants

[0114] The room-temperature storage and transportation of microfluidic chips, as well as the design of ultramultiplexer detection, necessitate the crucial step of lyophilizing the qPCR reaction system. To facilitate the drying process, the lyophilized product solution is typically prepared as a dilute solution containing 4%–15% solids. In this solution, water primarily exists in molecular form as free water, with a small amount adsorbed in the interstitial spaces of the solid lattice or bound to some polar groups via hydrogen bonds. Additionally, some water is fixed in organisms and cells, representing bound water that is difficult to remove after freezing. The main purpose of lyophilization is to remove free water from the material and some adsorbed water from the interstitial spaces of the solid lattice under low temperature and vacuum conditions. Therefore, selecting an appropriate lyophilization protectant formulation is a key step in preparing stable and effective microfluidic qPCR lyophilized microspheres.

[0115] Based on the survey, five lyophilization protectant formulations (Table 5) were selected for lyophilizing qPCR reaction systems containing norovirus G1 type virus primers and probes. The lyophilized systems were then tested. Formulations 1 and 5 showed no significant difference compared to the liquid reaction systems after lyophilization. However, Formulation 1 showed a 19% inhibition rate in qPCR detection. Therefore, Formulation 5 was ultimately selected for subsequent lyophilization, and the results are as follows... Figure 21 , Figure 22 As shown.

[0116] Table 5. Formulation of freeze-drying protectant

[0117]

[0118] 2. Preparation of freeze-dried microspheres for RNA virus multiplex qPCR detection system

[0119] The freeze-drying process generally involves three steps: pre-freezing, sublimation drying, and desorption drying. Considering factors such as the appearance, color, reconstitution, and bioactivity of the freeze-dried pellets, it is necessary to screen the protective agent components and adjust the freeze-drying sequence based on the properties of relevant substances in the freeze-dried components.

[0120] First, pre-freeze the microspheres using a microsphere molding machine (MS-1R, Boyikang Instrument Co., Ltd.), such as Figure 23 As shown in Table 6, a liquid reaction system (excluding the RNA template) for multiplex qPCR detection of 16 RNA viruses was prepared on-site as the sample loading stock solution. This solution was used to debug the microsphere forming instrument, ensuring that each droplet was 20 μL. After the instrument was debugged successfully, a thermos was filled with liquid nitrogen. Droplets were added to the liquid nitrogen and rapidly frozen into spheres, which then settled to the bottom of the thermos, thus achieving pre-freeze-drying of the reaction system.

[0121] Table 6. Preparation system of multiplex qPCR lyophilized microspheres

[0122]

[0123] Microspheres were removed from liquid nitrogen and transferred to a freeze dryer (Pilot 2-4M, Boyikang Instruments Co., Ltd.) pre-cooled to -50°C for freeze drying according to the program in Table 7. After the program was completed, the microspheres were stoppered in the chamber using the stoppering system and then backfilled to atmospheric pressure to maintain negative pressure and dryness inside the vial. The freeze-dried microspheres were then stored at different temperatures. Figure 24 ).

[0124] Table 7. Lyophilization Procedure for Multiplex qPCR Microspheres

[0125]

[0126] The prepared lyophilized microspheres were reconstituted and then subjected to qPCR to test their detection activity. The results showed that the lyophilized microspheres had good solubility. Compared with the liquid system, there were no significant changes in Ct values ​​for rotavirus, enterovirus, astrovirus, hepatitis A virus, norovirus G1, norovirus G2, Salivirus, Coxsackievirus, and Sapporo virus; no amplification was observed in the NTC group. However, there were differences in Ct values ​​for Aichi virus, Cuxvirus, enterovirus A71, hepatitis E virus, echovirus, poliovirus, and Coxsackievirus A6, but the differences were all <2, indicating that the lyophilized system was stable. Figure 25 ), and based on 10 -3 ~10 -7Five dilution gradients were used to establish standard curves for qPCR detection of lyophilized microspheres, as shown in Table 8. The lyophilized microspheres maintained their detection activity after six months of storage at both 4℃ and room temperature (10℃~30℃). Compared to lyophilized microspheres stored at 4℃, those stored at room temperature became harder, but showed no significant differences in detection results for rotavirus, enterovirus A71, astrovirus, hepatitis E virus, echovirus, norovirus G1, poliovirus, Salivirus, Coxsackievirus A6, Coxsackievirus A16, and Sapporo virus. The Ct values ​​of lyophilized microspheres for other viruses differed by <2, indicating stable detection activity. After six months of storage at 37℃, the lyophilized microspheres showed no significant difference in quantitative PCR detection results only for enterovirus A71, astrovirus, hepatitis E virus, and poliovirus, but lost their detection function for other viruses. Figure 26 The above results indicate that lyophilized microspheres can be stored at room temperature (10℃~30℃) for a long time and maintain their detection activity, but cannot be stored at 37℃ for a long time.

[0127] Table 8 Standard curve for qPCR of RNA virus lyophilized microspheres

[0128]

[0129]

[0130] 3. Microfluidic chip design for high-throughput qPCR detection of viruses

[0131] The chip's structural design is based on the sample processing flow and reagent types of the kit, mainly divided into nucleic acid extraction structures (involving sample lysis and sample distribution functions) and nucleic acid detection structures (including PCR amplification chambers). The chip's internal space, serving as the site for sample processing and reagent reactions, must have corresponding reagent storage and reaction chamber structures. The shape of the chambers depends on the type and volume of the reagents; the volume of the reaction chamber for mixing the lysis buffer and sample should be larger than the total volume of the sample and lysis buffer mixture. The nucleic acid detection structure requires built-in lyophilized reagent formulations and is designed as multiple parallel chambers based on the composition of the lyophilized reagent formulations and the principles of the instrument's optical detection mechanism. These chambers are located within the optical path of the optical detection structure.

[0132] Due to the limited space of the chip, it is necessary to integrate the nucleic acid sample processing and detection processes therein. The volumes of the sample and reagents are usually at the μL level (<20 μL), and the liquid follows the continuous flow law of microfluidics.外加驱动力,如气泵压力和离心力,通过芯片内部结构对液体进行控制。芯片的设计还包括液体阀和泵浦结构、液体运输的管道网络设计以及密闭气路联通网络。这些结构通过外加驱动力和芯片内部结构对液体流动进行调控,实现芯片不同功能模块的联动和功能划分,最终在短时间内完成样本制备和检测。考虑到产品设计分析,微流控芯片需满足内置试剂、功能架构,实现检测流程,并适应仪器设计的反馈。基于内置试剂优化后的设计结果,芯片的扇形样式设计规格如 Figure 27 所示。

[0133] The maximum radius (R) of the chip is 120 mm, the angular radian of the fan-shaped area is 56°, the overall thickness < 12 mm (to avoid excessive thickness of the chip causing flow in the vertical direction), and there is a horizontal cutting space with a radial height of about 23.3 mm on the inside. The chip is divided into three temperature zones: the inner temperature zone (R < 71 mm), the middle zone (R 77 ~ 91 mm), and the outer temperature zone (R 97 ~ 120 mm). The PCR reaction chambers are located on the arc of R 115 mm, with a total of 8, and the interval between each chamber is 3°. The 4th chamber counted from left to right is located on the symmetry axis of the chip.

[0134] After the chip is processed, it is packaged, which mainly includes the encapsulation of freeze-dried microspheres, the encapsulation of the built-in components of the chip, the encapsulation of the chip bottom plate, the assembly of the sealing structure of the sample loading port, and the outer packaging of the chip. The chip packaging must meet the airtightness requirements for storage within the chip's expiration date to ensure that the internal reagents do not volatilize or deteriorate.

[0135] 4. Evaluation of the Detection Performance of the Microfluidic Chip

[0136] The freeze-dried microspheres containing the qPCR reaction system are encapsulated into the reaction chambers of the microfluidic chip, and the positive standard product is serially diluted to 10 -9The positive standards were tested using a microfluidic chip. The test results were stable and no amplification was observed in the negative control (Table 9). At this point, the detectable viral concentrations for each primer probe were as follows: Enterovirus (Enterovirus) 447.34 copies / μL, Hepatitis A Virus (Hepatitis A) 524.47 copies / μL, Aichi Virus (Aichi Virus) 350.99 copies / μL, Cushn virus (Cushn) 877.48 copies / μL, Poliovirus (Poliovirus) 1185.16 copies / μL, Echovirus (Echovirus) 512.68 copies / μL, Enterovirus 71 (Enterovirus 71) 1025.37 copies / μL, Norovirus GI (Norovirus GI) 1073.62 copies / μL, and Astrovirus (Astrovirus) 418.61 copies / μL. The concentrations of norovirus GII (abbreviated as Norovirus GII) and Sapporo virus (abbreviated as Sapporo) were 931.20 copies / μL. These results demonstrate that the virus detection microfluidic chip fabricated based on lyophilized microspheres and microfluidic technology can achieve high-throughput detection of viruses in samples.

[0137] Table 9

[0138]

[0139]

Claims

1. A primer set for detecting waterborne pathogenic viruses, wherein the sequence of the primer set comprises at least one of the following primer combinations: 1) Primers SEQ ID NO: 1, 2, 4, 5, 2) Primers SEQ ID NO: 7, 8, 10, 11, 3) Primers SEQ ID NO: 13, 14, 16, 17, 4) Primers SEQ ID NO: 19, 20, 22, 23, 5) Primers SEQ ID NO: 25, 26, 28, 29, 6) Primers SEQ ID NO: 31, 32, 34, 35, 7) Primers SEQ ID NO: 37, 38, 40, 41, 43, 44, 8) Primers SEQ ID NO: 46, 47, 48, 49, 52, 53, 9) Primers SEQ ID NO: 55, 56, and / or, 10) Primers SEQ ID NO:58, 59.

2. The primer set as described in claim 1, wherein the waterborne pathogenic virus is enterovirus, hepatitis A virus, enterovirus A71, norovirus G1, norovirus G2, astrovirus, poliovirus, echovirus, hepatitis E virus, Salivirus, Aichi virus, Cusackie virus, Coxsackie virus A16, Coxsackie virus A6, rotavirus, Sapporo virus, bacteriophage MS2, bocavirus, or adenovirus; Preferably, the primer set includes degenerate positions; Preferably, the degenerate position includes R, W, M, Y, K, B, D, H, N, S, or V, wherein, R includes A or G, W includes A or T, M includes A or C, Y includes C or T, K includes G or T, B includes A, G or T, D includes A, G or T, H includes A, C or T, N includes A, G, C or T, S includes G or C, and V includes A, C or G.

3. A primer / probe set for detecting waterborne pathogenic viruses, wherein the sequence of the primer / probe set is at least one of the following primer / probe combinations: 1) Primers SEQ ID NO:1, 2, 4, 5; Probes SEQ ID NO:3, 6; 2) Primers SEQ ID NO:7, 8, 10, 11; probes SEQ ID NO:9, 12; 3) Primers SEQ ID NO: 13, 14, 16, 17; probes SEQ ID NO: 15, 18; 4) Primers SEQ ID NO: 19, 20, 22, 23; probes SEQ ID NO: 21, 24; 5) Primers SEQ ID NO:25, 26, 28, 29; Probes SEQ ID NO:27, 30; 6) Primers SEQ ID NO: 31, 32, 34, 35; probes SEQ ID NO: 33, 36; 7) Primers SEQ ID NO: 37, 38, 40, 41, 43, 44; Probes SEQ ID NO: 39, 42, 45; 8) Primers SEQ ID NO: 46, 47, 48, 49, 52, 53; Probes SEQ ID NO: 50, 51, 54; 9) Primers SEQ ID NO:55, 56, probe SEQ ID NO:57; and / or 10) Primers SEQ ID NO:58, 59, probe SEQ ID NO:

60.

4. The primer / probe set as described in claim 3, wherein the waterborne pathogenic virus is enterovirus, hepatitis A virus, enterovirus A71, norovirus G1, norovirus G2, astrovirus, poliovirus, echovirus, hepatitis E virus, Salivirus, Aichi virus, Cusackie virus, Coxsackie virus A16, Coxsackie virus A6, rotavirus, Sapporo virus, bacteriophage MS2, bocavirus, or adenovirus; Preferably, the probe is labeled with a fluorescent dye; Preferably, the fluorescent dye label includes FAM, HEX, TAMRA, ROX, TET, JOE, Cy3, Cy5, VIC, Cy5.5, Texas Red, NED, FAM-dT, TAMRA-dT, CY3-dT, CY5-dT, HEX-dT, and FITC; Preferably, the probe further includes a quenching group; Preferably, the quenching groups include BHQ1, TAMRA, BHQ2, Rhodamine 6G, Rhodamine B, BHQ3, Dabcyl, Eclipse, MGB, BHQ1-dT, and BHQ2-dT; Preferably, the probe is labeled with a modifying group; Preferably, the modifying groups include amino groups, phosphate groups, biotin, biotin-TEG, and C3-spacer; Preferably, the primer set includes degenerate positions; Preferably, the degenerate position includes R, W, M, Y, K, B, D, H, N, S, or V, wherein, R includes A or G, W includes A or T, M includes A or C, Y includes C or T, K includes G or T, B includes A, G or T, D includes A, G or T, H includes A, C or T, N includes A, G, C or T, S includes G or C, and V includes A, C or G.

5. A product for detecting waterborne pathogenic viruses, the product comprising the primer set as described in claim 1 or 2, or the primer / probe set as described in claim 3 or 4; Preferably, the product includes nucleic acid membrane strips, reagent kits, lyophilized microspheres, and chips; Preferably, the kit further includes PCR amplification buffer and amplification enzyme; Preferably, the amplification enzyme includes DNA polymerase and / or RNA polymerase; Preferably, the kit further includes a reverse transcription reaction system; Preferably, the reverse transcription reaction system includes primers; Preferably, the primers for the reverse transcription reaction system are random primers; Preferably, the kit further includes a fluorescent dye; Preferably, the kit also includes instructions; Preferably, the freeze-dried microspheres use 10% (w / v) trehalose, 5% (w / v) mannitol, 4% (w / v) cyclodextrin, and 0.5% (w / v) BSA as freeze-drying protectants. Preferably, the chip is a high-throughput qPCR detection chip; Preferably, the chip is a nucleic acid extraction structure or a nucleic acid detection structure; Preferably, the nucleic acid detection structure includes a PCR amplification chamber; Preferably, the nucleic acid detection structure incorporates a lyophilized reagent formulation.

6. A method for amplifying a waterborne pathogenic virus sequence, wherein the method uses the primer set of claim 1 or 2, the primer / probe set of claim 3 or 4, or the product of claim 5 to perform the amplification reaction; Preferably, the amplification reaction is performed using methods including LCR, NASBA, SDA, TMA, bDNA, and PCR. Preferably, the amplification reaction is performed by PCR. Preferably, the PCR includes real-time PCR and multiplex PCR; Preferably, the PCR is selected from multiplex PCR; Preferably, the amplification reaction is carried out under similar amplification conditions.

7. A method for detecting waterborne pathogenic viruses in a sample, the method comprising amplifying using the primer set of claim 1 or 2, the primer / probe set of claim 3 or 4, or the product of claim 5, obtaining an amplification product, and sequencing using the amplification product; Preferably, the samples include human body samples and environmental samples, wherein the human body samples are ex vivo human body samples; Preferably, the human sample includes blood, saliva, and urine; Preferably, the environmental sample includes a water source; Preferably, the water source includes sewage; Preferably, the wastewater includes wastewater from wastewater treatment plants, rivers, and sewer systems; Preferably, the method is a non-diagnostic method.

8. A library for detecting waterborne pathogenic viruses, said library comprising the primer set of claim 1 or 2, or the primer / probe set of claim 3 or 4.

9. The use of the primer set of claim 1 or 2, or the primer / probe set of claim 3 or 4, in the preparation of products for amplifying or detecting waterborne pathogenic viruses.

10. The use of the primer set of claim 1 or 2, the primer / probe set of claim 3 or 4, or the product of claim 5 in the detection of waterborne pathogenic viruses for non-diagnostic purposes.

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