Method for detecting live viruses in live virus wastewater in high-grade biosafety laboratory

Abstract

The invention discloses a method for detecting live viruses in live virus wastewater in a high-grade biosafety laboratory, which comprises the following steps of: performing preliminary impurity removal treatment on the wastewater, and intercepting large particles and solid wastes at the front end; the pathogenic microorganisms are enriched on the membrane through a reverse osmosis membrane in a physical enrichment mode; the enriched pathogenic microorganisms are eluted from the membrane in a high-frequency vibration and backflushing mode and stored in a collection bottle; adding the collected active toxic wastewater into cells, culturing, centrifuging, and taking a precipitate; purifying the precipitate, and extracting nucleic acid; detecting the extracted nucleic acid concentration by adopting a PCR (Polymerase Chain Reaction) method; the treatment time of the whole active toxic wastewater can be shortened, whether pathogenic microorganisms in the wastewater are completely inactivated or not can be judged within 2 hours through sampling, and the volumes of a water storage tank and a sterilization tank of a wastewater treatment system are reduced. The problem that whether the treated wastewater contains live pathogenic microorganisms cannot be judged is solved, the treatment time is prolonged, and the treatment process is scientifically accelerated.

Description

Technical Field

[0001] The invention relates to the technical field of virus treatment in live virus wastewater, in particular to a method for detecting live viruses in live virus wastewater in a high-level biosafety laboratory. Background Art

[0002] During the experimental activities, high-level biosafety laboratories will produce wastewater containing live pathogenic microorganisms due to activities such as animal breeding and experimental operations. This type of live toxic wastewater contains live pathogenic microorganisms. If it is not effectively treated, it will cause great harm to humans and the environment. At present, there are two ways to treat live toxic wastewater in high-level biosafety laboratories: 1) Chemical treatment: The live toxic wastewater is mixed with disinfectant and discharged after a certain period of time. 2) High temperature and high pressure treatment: After high temperature and high pressure treatment, it is discharged after a certain period of time. Generally, the procedure is 121 degrees for 30 minutes (except for prions).

[0003] Whether it is chemical treatment or high temperature and high pressure treatment, there are two important problems. 1) It is impossible to monitor whether the target pathogenic microorganisms are effectively inactivated. At this stage, verification is carried out through indirect verification. For example: high temperature and high pressure treatment, using moist heat Bacillus as a biological indicator, after one operation cycle, sampling and culture are carried out to determine whether it is completely inactivated. The culture is 48 hours, and the inactivation effect of the wastewater treatment system is evaluated indirectly. 2) Another problem is the control of time. In order to reduce risks, chemical disinfection generally sets the disinfection time at 4 hours to allow the disinfectant to fully react with the wastewater. The overall operation cycle of high temperature and high pressure treatment is about 8 hours. Both methods have greatly improved the action time and action conditions in order to set a higher safety margin.

[0004] Therefore, we proposed a method for detecting live viruses in live virus wastewater from high-level biosafety laboratories. Summary of the invention

[0005] The object of the present invention is to provide a method for detecting live viruses in live virus wastewater of a high-level biosafety laboratory to solve the problems raised in the above-mentioned background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A method for detecting live viruses in live virus wastewater in a high-level biosafety laboratory, comprising:

[0008] Step 1: Enrichment and collection of active poison wastewater;

[0009] Step 2: Use rapid culture methods to determine whether pathogenic microorganisms are active.

[0010] As a further solution of the present invention: the enrichment and collection of active virus wastewater in step 1 includes preliminary impurity removal treatment of the wastewater, a large-pore filter screen is placed in front to intercept large particles and solid waste at the front end; since the enrichment of pathogenic microorganisms cannot be affected by chemical reagents, the pathogenic microorganisms are enriched on the membrane by physical enrichment through a reverse osmosis membrane; the enriched pathogenic microorganisms are eluted from the membrane by high-frequency vibration and backwashing and stored in a collection bottle.

[0011] As a further solution of the present invention: since the enrichment of pathogenic microorganisms cannot be affected by chemical reagents, the pathogenic microorganisms are enriched on the membrane by physical enrichment through a reverse osmosis membrane, the sampling time is 5 minutes, the amount of water collected is as representative as possible, and the processing volume is 100L.

[0012] As a further solution of the present invention: the enriched pathogenic microorganisms are eluted from the membrane by high-frequency vibration and backwashing and stored in a collection bottle, and the recovered sample volume is less than 300 ml.

[0013] As a further solution of the present invention: the step 2 adopts a rapid culture method to determine whether the pathogenic microorganism is active, and the specific process includes: adding the collected active poison wastewater into cells for culture, centrifugation, and taking the precipitate; purifying the precipitate and extracting nucleic acid; using the PCR method to detect the concentration of the nucleic acid.

[0014] As a further solution of the present invention: the collected live virus wastewater is added into cells for cultivation, centrifuged, and the precipitate is taken. The collected live virus wastewater is added into specific susceptible cells for cultivation for 2 hours.

[0015] As a further solution of the present invention: the PCR method for detecting the proposed nucleic acid concentration includes a PCR detection system, and the PCR detection system includes an amplification module, a real-time fluorescence detection module, a droplet transfer module and a droplet reading and analysis module.

[0016] As a further scheme of the present invention: the amplification module is used to perform thermal cycling treatment on the sample to be detected so as to amplify the amount of nucleic acid in the sample to be detected; the real-time fluorescence detection module is used to perform fluorescence quantitative detection and analysis on the sample to be detected in the thermal cycle when the sample to be detected is to be subjected to real-time fluorescence quantitative PCR detection, so as to obtain the nucleic acid content in the sample to be detected; the droplet transfer device is used to transfer a plurality of droplets formed by the dispersion of the mixture in the sample to be detected to the droplet reading and analysis module when the sample to be detected is to be subjected to digital PCR detection; the droplet reading and analysis device is used to perform droplet reading analysis on the droplets to obtain the nucleic acid content in the sample to be detected.

[0017] As a further scheme of the present invention: the method for detecting the nucleic acid content by the PCR detection system includes: performing thermal cycle treatment on the sample to be detected through the amplification module to amplify the amount of nucleic acid in the sample to be detected; performing fluorescence quantitative detection and analysis on the sample to be detected in the thermal cycle by the real-time fluorescence quantitative detection module to obtain the nucleic acid content of the sample to be detected; when the sample to be detected is to be subjected to digital PCR detection, transferring multiple droplets formed by the dispersion of the mixture in the sample to be detected to the droplet reading and analysis module through the droplet transfer module; performing droplet reading analysis on the droplets by the droplet reading and analysis module to obtain the nucleic acid content in the sample to be detected.

[0018] As a further solution of the present invention: the amplification module performs thermal cycle treatment on the sample to be detected, including: in the amplification module, the sample to be detected is subjected to thermal cycle treatment by a semiconductor refrigeration plate that performs a temperature rise and fall cycle.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The method for detecting live viruses in live virus wastewater in a high-level biosafety laboratory is to enrich and collect live virus wastewater, and then use a rapid culture method to determine whether the pathogenic microorganisms are active; the wastewater is preliminarily treated for impurities, and a large-aperture filter is placed in front to intercept large particles and solid waste at the front end; since the enrichment of pathogenic microorganisms cannot be affected by chemical reagents, the pathogenic microorganisms are enriched on the membrane by physical enrichment through a reverse osmosis membrane; the enriched pathogenic microorganisms are eluted from the membrane by high-frequency vibration and backwashing and stored in a collection bottle; the collected live virus wastewater is added to cells for culture, centrifuged, and precipitated; the precipitate is purified and nucleic acid is extracted; the PCR method is used to detect the proposed nucleic acid concentration; the processing time of the entire live virus wastewater can be accelerated, and after sampling, it can be determined within 2 hours whether the pathogenic microorganisms in the wastewater are completely inactivated, the wastewater treatment efficiency is improved, and the volume of the water storage tank and sterilization tank of the wastewater treatment system is reduced. The problem of not being able to determine whether the treated wastewater contains live pathogenic microorganisms and the processing time is increased, and the processing process is scientifically accelerated. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The present invention is a schematic flow chart of a method for detecting live viruses in live virus wastewater in a high-level biosafety laboratory.

[0022] Figure 2 This is the dengue virus type 2 infection 0min detection result in the present invention.

[0023] Figure 3 This is the 5-minute detection result of dengue virus type 2 infection in the present invention.

[0024] Figure 4This is the detection result of dengue virus type 2 infection for 15 minutes in the present invention.

[0025] Figure 5 This is the 30-min detection result of dengue virus type 2 infection in the present invention.

[0026] Figure 6 This is the 60-min detection result of dengue virus type 2 infection in the present invention.

[0027] Figure 7 This is the 90-min detection result of dengue virus type 2 infection in the present invention.

[0028] Figure 8 This is the detection result of dengue virus type 2 infection for 120 minutes in the present invention. DETAILED DESCRIPTION

[0029] In one embodiment, Figure 1 As shown, a method for detecting live viruses in live virus wastewater in a high-level biosafety laboratory comprises the following steps:

[0030] Step 1: Enrichment and collection of active poison wastewater;

[0031] Step 2: Use rapid culture method to determine whether the pathogenic microorganism is active;

[0032] Step 1: The enrichment and collection of active virus wastewater includes preliminary impurity removal treatment of the wastewater, and a large-aperture filter is placed in front to intercept large particles and solid waste at the front end; since the enrichment of pathogenic microorganisms cannot be affected by chemical reagents, the pathogenic microorganisms are enriched on the membrane by physical enrichment through a reverse osmosis membrane; the enriched pathogenic microorganisms are eluted from the membrane by high-frequency vibration and backwashing and stored in a collection bottle;

[0033] Since the enrichment of pathogenic microorganisms cannot be affected by chemical reagents, the pathogenic microorganisms are enriched on the membrane by physical enrichment through reverse osmosis membrane. The sampling time is 5 minutes and the processing volume is 100L.

[0034] The enriched pathogenic microorganisms are eluted from the membrane by high-frequency vibration and backwashing and stored in a collection bottle. The recovered sample volume is less than 300 ml.

[0035] Step 2 uses a rapid culture method to determine whether the pathogenic microorganism is active. The specific process includes: adding the collected live poison wastewater to cells for culture, centrifugation, and taking the precipitate; purifying the precipitate and extracting nucleic acid; using the PCR method to detect the concentration of the nucleic acid;

[0036] The collected live poison wastewater was added to the cells for cultivation, centrifuged, and the precipitate was taken. The collected live poison wastewater was added to the cells for cultivation for 2 hours;

[0037] The enrichment efficiency was determined in the following manner.

[0038] 1) The virus was simulated by nanospheres, and the enrichment efficiency was calculated by weighing. The following is the enrichment efficiency experimental data, as shown in Table 1.

[0039] Table 1 Enrichment efficiency experimental data

[0040]

[0041] 2) Take 50ul of H3N2 virus culture, dilute it to 500mL with pure water, shake the stock solution thoroughly, take 1mL to extract nucleic acid, dilute the remaining water sample to 50L, filter it through a reverse osmosis membrane, and recover 300mL of sample. Take the filtered wastewater and 1mL of each sample to extract nucleic acid, and perform digital PCR detection with the H3N2 nucleic acid detection kit. The test results are shown in Table 2:

[0042] Table 2 Fluorescence PCR test results of stock solution, wastewater and pure water

[0043] Copies Original solution 1618.4 sample 1079.0 Wastewater 0

[0044] No H3N2 nucleic acid fragments were detected in the wastewater at the rear end of the membrane, and the surface H3N2 virus culture and its nucleic acid fragments were enriched on the reverse osmosis membrane, with an enrichment efficiency of 100% in theory.

[0045] The PCR method for detecting the proposed nucleic acid concentration includes a PCR detection system, wherein the PCR detection system includes an amplification module, a real-time fluorescence detection module, a droplet transfer module, and a droplet reading and analysis module;

[0046] The amplification module is used to perform thermal cycling treatment on the sample to be detected to amplify the amount of nucleic acid in the sample to be detected; the real-time fluorescence detection module is used to perform fluorescence quantitative detection and analysis on the sample to be detected in the thermal cycle when the sample to be detected is to be subjected to real-time fluorescence quantitative PCR detection, so as to obtain the nucleic acid content in the sample to be detected; the droplet transfer device is used to transfer a plurality of droplets dispersed from the mixture in the sample to be detected to the droplet reading and analysis module when the sample to be detected is to be subjected to digital PCR detection; the droplet reading and analysis device is used to perform droplet reading and analysis on the droplets to obtain the nucleic acid content in the sample to be detected;

[0047] The method for detecting the nucleic acid content by the PCR detection system includes: performing a thermal cycle treatment on the sample to be detected by an amplification module to amplify the amount of nucleic acid in the sample to be detected; performing a fluorescence quantitative detection analysis on the sample to be detected in the thermal cycle by a real-time fluorescence quantitative detection module to obtain the nucleic acid content of the sample to be detected; when the sample to be detected is to be subjected to digital PCR detection, a plurality of droplets formed by the dispersion of the mixture in the sample to be detected are transferred to a droplet reading and analysis module by a droplet transfer module; and performing a droplet reading and analysis on the droplets by the droplet reading and analysis module to obtain the nucleic acid content in the sample to be detected;

[0048] The amplification module performs thermal cycle treatment on the sample to be detected, including: in the amplification module, the sample to be detected is thermally cycled by a semiconductor refrigeration piece that performs a temperature rise and fall cycle;

[0049] Through the amplification module in the PCR detection system, the sample to be detected is subjected to thermal cycle treatment to amplify the nucleic acid content in the reaction unit, so that it is easier to detect the nucleic acid content in the sample to be detected; when the sample to be detected is to be subjected to real-time fluorescence quantitative PCR detection, the real-time fluorescence quantitative detection module in the PCR detection system performs fluorescence quantitative detection and analysis on the sample to be detected in the thermal cycle to obtain the nucleic acid content in the sample to be detected, thereby realizing real-time fluorescence quantitative PCR detection; when the sample to be detected is to be subjected to digital PCR detection, through the droplet transfer module in the PCR detection system, multiple droplets formed by the dispersion of the mixture in the reaction unit are transferred to the droplet reading and analysis module in the PCR detection system, and the droplets are read and analyzed by the droplet reading and analysis module to obtain the nucleic acid content in the sample to be detected, thereby realizing digital PCR detection. Thus, digital PCR detection and / or fluorescence quantitative PCR detection can be realized according to the detection requirements, giving play to the advantages of the two PCR detection technologies, providing a more comprehensive means for nucleic acid detection, expanding the application scope of PCR detection, improving the efficiency, accuracy and flexibility of nucleic acid detection, and reducing economic and space costs.

[0050] Specific primer fragments were designed for Dengue Virus (type 2) (Table 3). Infection time was determined on specific susceptible cells to verify that the method for rapid culture of live virus within 2 hours was feasible.

[0051] Table 3 Dengue Virus (type 2) primer design

[0052]

[0053]

[0054] Follow the steps below to operate.

[0055] Experimental materials: Dengue virus type 2: 2.37×10 5 TCID50 / ml (wastewater containing viruses after enrichment)

[0056] Experimental steps:

[0057] 1) Inoculate the cells into a 24-well plate one day in advance, so that the cells can grow to more than 90% after attaching to the plate the next day;

[0058] 2) Discard the original cell culture medium and add appropriate amount of PBS to wash the cell surface once;

[0059] 3) Infect cells with 200 μL virus solution / well, add 200 μL culture medium to the control group, shake well after inoculation, and let stand for a while;

[0060] 4) Set different time points to collect samples, which can be 0h, 5min, 15min, 30min, 60min, 90min, 120min, with 3 replicates for each group;

[0061] 5) Take the cell culture plate at the corresponding time point and discard the culture supernatant;

[0062] 6) Add appropriate amount of sterile PBS to the infected cells for washing, wash three times, and aspirate the PBS thoroughly when discarding it for the last time;

[0063] 7) Add 300 μL TRIzol to the infected cells to lyse the cells. After pipetting and mixing, transfer the cell lysate to a new 1.5 mL eppendorf tube and mark it.

[0064] 8) Extract nucleic acid and perform qPCR detection using designed primers.

[0065] Table 4 Dengue virus type 2 detection results

[0066]

[0067]

[0068] Reference Figure 2-Figure 8 The experimental results showed that within 2 hours of cell infection, the virus began to replicate from 5 minutes onwards, and the PCR method could identify the obvious virus proliferation process from the cell extract, indicating that this method can distinguish whether the virus in the water sample is active.

[0069] The present invention enriches and collects the live poison wastewater, and then uses a rapid culture method to determine whether the pathogenic microorganisms are active; performs preliminary impurity removal treatment on the wastewater, and places a large-aperture filter in front to intercept large particles and solid waste at the front end; because the enrichment of pathogenic microorganisms cannot be affected by chemical reagents, the pathogenic microorganisms are enriched on the membrane by physical enrichment through a reverse osmosis membrane; the enriched pathogenic microorganisms are eluted from the membrane by high-frequency vibration and backwashing and stored in a collection bottle; the collected live poison wastewater is added to cells for culture, centrifuged, and precipitates are taken; the precipitates are purified and nucleic acids are extracted; the PCR method is used to detect the proposed nucleic acid concentration; the processing time of the entire live poison wastewater can be accelerated, and after sampling, it can be determined within 2 hours whether the pathogenic microorganisms in the wastewater are completely inactivated, the treatment efficiency of the wastewater is improved, and the volume of the water storage tank and the sterilization tank of the wastewater treatment system is reduced. The problem of being unable to determine whether the treated wastewater contains live pathogenic microorganisms and the processing time is increased, and the processing process is scientifically accelerated.

[0070] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for detecting live viruses in live virus wastewater in a high-level biosafety laboratory, characterized in that: The following steps are involved: Step 1: Enrichment and collection of active poison wastewater; Step 2: Use rapid culture methods to determine whether pathogenic microorganisms are active.

2. The method for detecting live viruses in live virus wastewater of a high-level biosafety laboratory according to claim 1, characterized in that: The step 1 of enriching and collecting the active virus wastewater includes preliminary impurity removal treatment of the wastewater, a large-aperture filter screen in front, and intercepting large particles and solid waste at the front end; since the enrichment of pathogenic microorganisms cannot be affected by chemical reagents, the pathogenic microorganisms are enriched on the membrane by physical enrichment through a reverse osmosis membrane; the enriched pathogenic microorganisms are eluted from the membrane by high-frequency vibration and backwashing and stored in a collection bottle.

3. The method for detecting live viruses in live virus wastewater of a high-level biosafety laboratory according to claim 2, characterized in that: Since the enrichment of pathogenic microorganisms cannot be affected by chemical reagents, the pathogenic microorganisms are enriched on the membrane by physical enrichment through a reverse osmosis membrane. The sampling time is 5 minutes and the processing volume is 100L.

4. The method for detecting live viruses in live virus wastewater of a high-level biosafety laboratory according to claim 2, characterized in that: The enriched pathogenic microorganisms are eluted from the membrane by high-frequency vibration and backwashing and stored in a collection bottle. The recovered sample volume is less than 300 ml.

5. The method for detecting live viruses in live virus wastewater of a high-level biosafety laboratory according to claim 1, characterized in that: The second step adopts a rapid culture method to determine whether the pathogenic microorganism is active. The specific process includes: adding the collected active poison wastewater into cells for culture, centrifugation, and taking the precipitate; purifying the precipitate and extracting nucleic acid; and using the PCR method to detect the concentration of the nucleic acid.

6. The method for detecting live viruses in live virus wastewater of a high-level biosafety laboratory according to claim 5, characterized in that: The collected active poison wastewater was added to the cells for cultivation, centrifuged, and the precipitate was taken. The collected active poison wastewater was added to the cells for cultivation for 2 hours.

7. The method for detecting live viruses in live virus wastewater of a high-level biosafety laboratory according to claim 5, characterized in that: The PCR method for detecting the proposed nucleic acid concentration includes a PCR detection system, which includes an amplification module, a real-time fluorescence detection module, a droplet transfer module, and a droplet reading and analysis module.

8. The method for detecting live viruses in live virus wastewater of a high-level biosafety laboratory according to claim 7, characterized in that: The amplification module is used to perform thermal cycling treatment on the sample to be detected to amplify the amount of nucleic acid in the sample to be detected; the real-time fluorescence detection module is used to perform fluorescence quantitative detection and analysis on the sample to be detected in the thermal cycle when the sample to be detected is to be subjected to real-time fluorescence quantitative PCR detection, so as to obtain the nucleic acid content in the sample to be detected; the droplet transfer device is used to transfer a plurality of droplets dispersed from the mixture in the sample to be detected to the droplet reading and analysis module when the sample to be detected is to be subjected to digital PCR detection; the droplet reading and analysis device is used to perform droplet reading analysis on the droplets to obtain the nucleic acid content in the sample to be detected.

9. The method for detecting live viruses in live virus wastewater of a high-level biosafety laboratory according to claim 8, characterized in that: The method for detecting the nucleic acid content of the PCR detection system includes: performing a thermal cycle treatment on the sample to be detected by an amplification module to amplify the amount of nucleic acid in the sample to be detected; performing a fluorescence quantitative detection analysis on the sample to be detected in the thermal cycle by a real-time fluorescence quantitative detection module to obtain the nucleic acid content of the sample to be detected; when the sample to be detected is to be subjected to digital PCR detection, transferring a plurality of droplets formed by the dispersion of a mixture in the sample to be detected to a droplet reading and analysis module by a droplet transfer module; performing a droplet reading and analysis on the droplets by the droplet reading and analysis module to obtain the nucleic acid content in the sample to be detected.

10. The method for detecting live viruses in live virus wastewater of a high-level biosafety laboratory according to claim 9, characterized in that: The amplification module performs thermal cycle processing on the sample to be detected, including: in the amplification module, the sample to be detected is subjected to thermal cycle processing by a semiconductor refrigeration piece that performs a temperature rise and fall cycle.

Images