Pathogen concentration method

Abstract

The invention relates to the technical field of biology, and discloses a pathogen concentration method which comprises the following steps: collecting pathogen microorganisms in air through liquid, adding a concentration agent into a solution containing pathogens to precipitate and concentrate the pathogens in the collected liquid to obtain a precipitate, resuspending the precipitate, and detecting whether the pathogens are positive or not and / or the content of the pathogens. The concentrating agent comprises one of inorganic salt, skim milk powder and diatomite, and one of PEG-6000 (Polyethylene Glycol) and PEG8000 (Polyethylene Glycol 8000). By optimizing the components of the concentrated solution, low-cost and high-recovery-rate detection of pathogens is realized, and the method is suitable for places such as common farms, hospitals and factories with high cleanliness requirements. Meanwhile, the invention further provides a sampling device applied to the method, and pathogens in the scene can be detected more effectively.

Description

Technical Field

[0001] This invention belongs to the field of biology, and more specifically, relates to a method for concentrating pathogens. Background Technology

[0002] The prior application for this project, publication number CN115369096A, entitled "A Method for Concentrating Porcine Viruses in Concentrated Water," discloses a method for concentrating, separating, and detecting viruses from concentrated water.

[0003] The plan mainly involves testing water bodies in places such as aquaculture farms.

[0004] As the project progresses further, different scenarios require relevant testing, such as the detection of airborne viruses in places like farms and hospitals, which is in high demand.

[0005] However, detecting viruses in the air has always been a challenging problem. For example, the local standard DB31 / T 1421-2023 requires the use of a high-flow bioaerosol sampler to sample and detect whether the pathogen is positive. The minimum market price for such a sampler is 10,000 yuan per unit. Furthermore, this equipment cannot support long-term sampling, which means that it cannot achieve high concentrations of virus enrichment. At the same time, it also requires relatively expensive experimental reagents.

[0006] Traditional methods for detecting airborne pathogens generally face two major challenges:

[0007] 1. The equipment is expensive;

[0008] 2. The low enrichment of pathogen particles necessitates higher requirements for subsequent nucleotide detection.

[0009] To overcome the above challenges, effectively improving the detectability of virus particles in sample solutions is an important and effective means of solving the problem. Summary of the Invention

[0010] The main objective of this invention is to collect air samples from a specific environment and provide a method for concentrating pathogens. By optimizing the composition of the concentrate, this invention achieves low-cost and high-recovery detection of pathogens, and is suitable for ordinary farms, hospitals, and factories with high cleanliness requirements.

[0011] According to a first aspect of the present invention, a method for concentrating pathogens is provided, comprising adding a concentrator to a solution containing pathogens to cause the pathogens in the collected solution to precipitate and concentrate, obtaining a precipitate, then resuspending the precipitate, and detecting whether the pathogen is positive and / or the content of the pathogen;

[0012] The concentrate includes one of inorganic salts, skim milk powder and diatomaceous earth, and one of PEG-6000 and PEG8000.

[0013] In the above method, the method for obtaining the solution containing pathogens is as follows: using one of physiological saline, PBS, or DMEM as an adsorbent, the adsorbent is sprayed into the air to adsorb pathogens in the air.

[0014] In the above method, the adsorbent is recycled during a single adsorption process, so that pathogens in the air are collected within a limited volume of adsorbent.

[0015] In the above method, the sampling device used to implement the method for obtaining the solution containing pathogens is a disposable device, and the sampling device is pre-sterilized to eliminate interference;

[0016] The sampling device has a nozzle for creating a miniature fountain and a circulation unit for continuously supplying liquid to the nozzle and collecting and circulating the liquid sprayed from the nozzle. The sampling device operates for at least 1 hour in the detection environment to enrich the liquid with pathogens.

[0017] In the above method, the inorganic salt is sodium chloride or potassium chloride, and the final concentration of the inorganic salt added to the solution is 0.9~3wt%.

[0018] In the above method, the concentration of the skim milk powder or diatomaceous earth in the solution is 0.005~0.02wt%.

[0019] In the above method, the concentration of PEG-6000 or PEG8000 in the solution is 5~10wt%.

[0020] In the above method, the resuspension solvent is one of physiological saline, PBS, and DMEM.

[0021] In the above method, the pathogen is a virus; the virus is porcine pseudorabies virus and / or porcine reproductive and respiratory syndrome virus.

[0022] In the above methods, if only porcine pseudorabies virus needs to be detected, the concentrate is Formula 1; if only porcine reproductive and respiratory syndrome virus (PRRSV) needs to be detected, the concentrate is Formula 2; if both porcine pseudorabies virus and PRRSV need to be detected simultaneously, the concentrate is Formula 1, Formula 2, or Formula 3.

[0023] Formula 1 is: 10wt% PEG-8000, 1.463wt% NaCl, and 0.01wt% skim milk powder are added to the solution;

[0024] Formula 2 is: 10wt% PEG-6000, 2.925% NaCl, and 0.01wt% skim milk powder are added to the solution;

[0025] Formula 3 is: 10wt% PEG-6000, 2.925% NaCl, and 0.01wt% diatomaceous earth are added to the solution.

[0026] One of the above-described technical solutions of the present invention has at least one of the following advantages or beneficial effects:

[0027] 1. In a preferred embodiment of the present invention, the enrichment of airborne pathogens is carried out by a circulating spraying method, which can enrich the pathogens for a longer period of time, overcoming the difficulties of traditional methods in terms of long-term sampling and high-concentration enrichment.

[0028] 2. By using a combination of skim milk powder / diatomaceous earth and PEG6000 / 8000, this invention can significantly improve the enrichment and precipitation efficiency of pathogens in solutions containing trace pathogens, thereby improving detection accuracy and enabling effective detection of trace pathogens.

[0029] The combination of the above methods can enable the accurate detection of pathogens in specific environments, such as farms, pharmaceutical factories, and hospitals. Attached Figure Description

[0030] The present invention will be further described below with reference to the accompanying drawings and embodiments;

[0031] Figure 1 This is a structural diagram of the pathogen sampling device without the placement seat in use according to Embodiment 2 of the present invention;

[0032] Figure 2 This is Embodiment 2 of the present invention. Figure 1 Enlarged diagram of A in the middle;

[0033] Figure 3 This is a structural diagram of the pathogen sampling device of Embodiment 2 of the present invention during use;

[0034] Figure 4 This is a cross-sectional view of the liquid receiving body, sampling container, and placement seat of the pathogen sampling device according to Embodiment 2 of the present invention when they are assembled.

[0035] Figure 5 This is a cross-sectional view of the nozzle, liquid receiving body, and hose of the pathogen sampling device according to Embodiment 2 of the present invention when they are in combination;

[0036] Figure 6 This is a structural diagram of the sampling container of the pathogen sampling device in Embodiment 2 of the present invention. Detailed Implementation

[0037] Embodiments of the present invention 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 are only used to explain the present invention, and should not be construed as limiting the present invention.

[0038] Example 1

[0039] In the applicant's prior application, it has been verified that PEG8000 has a certain concentration effect. Therefore, this project started with PEG8000 as the necessary verification object to conduct virus concentration experiments, while PEG6000 was used for parallel comparison.

[0040] The specific experimental method includes the following steps:

[0041] We purchased porcine pseudorabies live vaccine (trade name: Keweining) batch number 20240654 from Wuhan Keqian Biotechnology Co., Ltd., and porcine reproductive and respiratory syndrome live vaccine (trade name: Lanfulai) batch number FS2025009 from Boehringer Ingelheim Animal Health (Shanghai) Co., Ltd.

[0042] Nucleic acid was extracted from the viral fluid using an automated nucleic acid extractor (Shenzhen Xinsiwei Singu32, MTQ064V). Primers and probes were synthesized according to the national standard "Fluorescent PCR Detection Method for Pseudorabies Virus" (GB / T35911-2018) to detect the PRV-gH gene. Primers and probes were synthesized according to the national standard "Fluorescent RT-PCR Detection Method for Porcine Reproductive and Respiratory Syndrome Virus" (GB / T 35912-2018) to detect the ORF6-M gene. The PRV quantification standard was based on the method in the existing invention CN 115369096 A. The PRRSV quantification standard was the method established by the laboratory of the College of Veterinary Medicine, Huazhong Agricultural University: Y = -3.41 X + 38.52.

[0043] Step 1: Preparation of virus solution: In order to maintain the consistency of the total amount of virus seeding in each batch of experiments, PRV (pseudorabies virus of pigs) and PRRSV (porcine reproductive and respiratory syndrome virus) vaccine dry powder were dissolved and mixed in advance, and then the virus mixture was diluted by a certain factor and dispensed into centrifuge tubes, stored at -80℃ and left to stand overnight.

[0044] Step 2: Add skim milk powder or diatomaceous earth, PEG6000 or PEG8000 to the virus solution and adjust the concentration of inorganic salts in the virus solution. Refer to Table 1 for the amount of each substance. Use the virus solution without any of the substances mentioned in Step 2 as a blank control.

[0045] Step 3: Centrifuge the virus solution at 5000 rpm for 20 minutes and discard the supernatant;

[0046] Step 4: Resuspend the precipitate obtained in Step 3 in 400 μL of physiological saline, extract nucleic acids, and repeat the extraction in the concentrated group as a parallel control.

[0047] Step 5: Perform PRV / PRRSV quantitative PCR / quantitative RT-PCR amplification according to the method described above.

[0048] The amplification of PRV should be performed according to Section 2.1 of CN115369096A, which outlines the establishment of the PRV-qPCR method.

[0049] The amplification procedure for PRRSV is as follows: GB / T 35912-2018.

[0050] Table 1 Experimental Parameters and Results

[0051] Group PEG6000 PEG8000 Salt concentration Concentrate PRV-CT value PRV nucleic acid concentration (copies / μL) PRRSV-CT value PRRSV nucleic acid concentration (copies / μL) 1 5% 5% NaCl / 1.463% / 36.332 166 32.293 67 2 10% / NaCl / 1.463% / 34.574 521 29.926 331 3 / 10% NaCl / 1.463% / 37.895 60 31.863 90 4 / 10% NaCl / 0.9% / 37.449 80 31.801 93 5 / 10% NaCl / 1.463% / 36.957 111 32.254 69 6 / 10% NaCl / 2.925% / 31.348 4241 31.754 96 7 / 10% NaCl / 1.463% / 36.785 124 32.145 74 8 / 10% KCl / 1.863% / 34.332 610 32.582 55 9 / 10% <![CDATA[CaCl2 / 2.775%]]> / 38.473 41 No CT value 0 10 / 10% NaCl / 1.463% 0.01% diatomaceous earth 33.785 882 31.66 103 11 / 10% NaCl / 1.463% / 36.340 168 32.004 81 12 / 10% NaCl / 1.463% 0.01% skim milk powder 28.113 35168 31.215 139 13 10% / NaCl / 5% / 32.676 1812 30.269 263 14 10% / NaCl / 2.925% / 31.004 5372 30.820 181 15 10% / NaCl / 2.925% 0.01% skim milk powder 32.395 2176 28.258 1022 16 10% / NaCl / 2.925% 0.01% diatomaceous earth 31.906 2989 29.7345 377 Blank control 0 0 0 0 37.535 77 33.985 67

[0052] Note: Unless otherwise specified, % represents weight percentage.

[0053] As can be seen from the data in Table 1, PEG6000 showed a more prominent effect in detecting both PRV and PRRSV in groups 1 to 3.

[0054] In groups 4 to 6, the concentration of NaCl increased and the number of detected copies increased;

[0055] In groups 7 to 9, KCl showed the most significant effect;

[0056] In groups 10 to 12, the addition of diatomaceous earth or skim milk powder to PEG-8000 contributed to the detection of PRRSV (but the contribution was limited, with the copy number increasing from 21 in the blank group to 100 to 140). Diatomaceous earth made a substantial contribution to the detection of PRRSV.

[0057] The experiments in groups 1 to 12 show that increasing the concentration of NaCl significantly improves the detection rate of PRV. Furthermore, using skim milk powder at relatively low NaCl concentrations yields even more significant results. PEG6000 exhibits the most pronounced effect in PRRSV detection.

[0058] In groups 13 and 14, the relationship between PEG6000 and inorganic salts was verified. Combined with the experiments in group 2 and the blank group, it can be seen that increasing the amount of NaCl slightly improves the detection effect of PRV, but the concentration effect will decrease after exceeding a certain concentration, such as 2.925% NaCl having a better concentration effect than 5% NaCl.

[0059] In groups 15 and 16, although the detection of PRV was not as significant as that of PEG-8000 when using PEG-6000 and different concentrates, the use of skim milk powder had a very significant improvement in the detection of PRRSV.

[0060] Based on the above results, this embodiment offers the following recommendations for virus detection:

[0061] 1. If only PRV and DNA virus need to be tested, the formula is: 10wt% PEG8000 + 1.463wt% NaCl + 0.01wt% skim milk powder;

[0062] 2. If only PRRSV and RNA viruses need to be detected, the formula is: 10wt% PEG6000 + 2.925% NaCl + 0.01wt% skim milk powder;

[0063] 3. If simultaneous detection of PRV and PRRSV is required, the formulation is: 10wt% PEG6000 + 2.925% NaCl + 0.01wt% diatomaceous earth, or 10wt% PEG-8000 + 1.463wt% NaCl + 0.01wt% skim milk powder, or 10wt% PEG6000 + 2.925% NaCl + 0.01wt% skim milk powder; the optimal choice is: 10wt% PEG6000 + 2.925% NaCl + 0.01wt% diatomaceous earth.

[0064] Example 2

[0065] Reference Figures 1 to 6 As shown, a pathogen sampling device includes a receiving component 1, a sampling container 2, a tubing 3, a nozzle 4, and a peristaltic pump 5. The receiving component 1 has a receiving tank 101. The sampling container 2 has a receiving cavity 201 for storing sampling liquid. One end of the tubing 3 along its length is connected to the sampling container 2, and the other end is connected to the nozzle 4 to connect the receiving cavity 201 and the nozzle 4. The pump head of the peristaltic pump 5 is connected to the tubing 3, and the peristaltic pump 5 is used to squeeze the tubing 3 so that the tubing 3 delivers the sampling liquid in the receiving cavity 201 to the nozzle 4. The nozzle 4 is used to spray the sampling liquid into the air, and the receiving tank 101 is used to receive the sampling liquid sprayed from the nozzle 4.

[0066] In this embodiment of the pathogen sampling device, during actual use, the sampling container 2's receiving cavity 201 stores sampling liquid. The two ends of the flexible tube 3 along its length are connected to the sampling container 2 and the nozzle 4, respectively, and the flexible tube 3 is connected to the pump head of the peristaltic pump 5. The receiving tank 101 of the receiving component 1 is used to receive the sampling liquid sprayed from the nozzle 4. Based on this, when the peristaltic pump 5 is working, the sampling liquid in the receiving cavity 201 is sprayed into the air through the flexible tube 3 from the nozzle. During this process, the sampling liquid captures pathogens in the air and concentrates them in the receiving tank 101. Thus, the pathogen sampling device can successfully collect pathogen samples from the air. Furthermore, the pathogen sampling device, through the receiving component... 1. The sampling container 2, hose 3, nozzle 4, and peristaltic pump 5 can be used to collect pathogens from the air. It has a simple structure, small size, and is easy to carry and use in different environments. In addition, since the pathogen sampling device of this application only combines the liquid receiving part 1, sampling container 2, hose 3, nozzle 4, and peristaltic pump 5 during actual use, the liquid receiving part 1, sampling container 2, hose 3, and nozzle 4 can all be disposable consumables. Therefore, when used in different environments, there is no problem of pathogens from the previous environment mixing into the sampling liquid used to collect pathogens from the current environment, avoiding inaccurate sampling results and ensuring high accuracy of subsequent pathogen component analysis results.

[0067] In this invention, all components that come into contact with liquids are disposable sterile components, such as liquid receiving component 1, sampling container 2, tubing 3, and nozzle 4; most of these components are obtained through injection molding, which is low in cost.

[0068] In this invention, only the peristaltic pump is relatively expensive, with a single peristaltic pump having a liquid supply capacity of about 400 ml / min costing approximately 3,000 yuan. Commonly used peristaltic pumps in laboratories can be used as a substitute. Compared to commercially available high-flow bioaerosol samplers, it is not only much cheaper but also has a simpler structure and very low consumable costs.

[0069] In production, it is recommended to aseptically seal and store the saline solution directly in the sampling container 2. Users can then directly open the lid and connect the receiving part 1 for use.

[0070] In this embodiment, the nozzle 4 is detachably connected to the liquid receiving component 1, thereby ensuring that the nozzle 4 can be stably positioned when spraying the sampling liquid, thus ensuring the smooth progress of pathogen collection.

[0071] It should be noted that the nozzle 4 has multiple spray holes at the spray end, and the nozzle 4 sprays out the sampling liquid in a divergent manner. The advantage of this method is that the sampling liquid can come into full contact with the air, thereby collecting as many pathogen samples as possible.

[0072] In this embodiment, during actual use, the liquid receiving component 1 is placed horizontally, with the opening of the liquid receiving tank 101 facing upwards. The liquid receiving component 1 has a vertically penetrating connecting hole 102 located on the bottom surface of the liquid receiving tank 101. The nozzle 4 is inserted into the connecting hole 102, and the flexible hose 3 is used to connect to one end of the nozzle 4 inserted into the connecting hole 102. In practical applications, the nozzle 4 sprays the sampling liquid upwards. Thus, the sampling liquid sprayed by the nozzle 4 can fall directly back into the liquid receiving tank 101 after contacting the air, which can reduce the loss of the sampling liquid and ensure the smooth progress of pathogen collection.

[0073] It should be noted that, in this embodiment, the vertical direction refers to... Figure 1 The view shown is from top to bottom.

[0074] For example, the nozzle 4 is connected to the wall of the connecting hole 102 by a threaded connection. In other embodiments, the nozzle 4 can also be connected to the liquid receiving component 1 by an interference fit into the connecting hole 102. This application does not limit this.

[0075] In this embodiment, the liquid receiving component 1 includes a liquid receiving body 11 and a connecting column 12. The liquid receiving body 11 has a liquid receiving groove 101 and a connecting hole 102. The connecting column 12 is connected to the bottom surface of the liquid receiving body 11, and the axis of the connecting column 12 extends vertically. The connecting column 12 has a return hole 103 that passes through its axis and communicates with the liquid receiving groove 101. The sampling container 2 includes a container body 21, a first connecting tube 22, and a connecting cap 23. The container body 21 has a receiving cavity 201. One end of the first connecting tube 22 along its length is connected to the container body 21, and the inner hole of the first connecting tube 22 communicates with the receiving cavity 201. The other end of the first connecting tube 22 along its length is detachably connected to the connecting cap 23, and the connecting cap 23 seals the inner hole of the first connecting tube 22. The first connecting tube 22 is used to connect with the connecting column 12. When the first connecting tube 22 is connected to the connecting column 12, The reflux hole 103 connects the receiving cavity 201 and the liquid receiving tank 101 through the inner hole of the first connecting pipe 22. Specifically, in actual use, the pathogen sampling device first removes the connecting cover 23 from the first connecting pipe 22, and then connects the first connecting pipe 22 to the connecting column 12. At this time, the liquid receiving tank 101, the reflux hole 103, the inner hole of the first connecting pipe 22, and the receiving cavity 201 are connected. The advantage of this structure is that the sampling liquid sprayed by the nozzle 4 falls back into the liquid receiving tank 101 and then flows back into the receiving cavity 201 through the reflux hole 103 and the inner hole of the first connecting pipe 22. Thus, the continuous operation of the peristaltic pump 5 allows the sampling liquid to continuously undergo the above process until the peristaltic pump 5 stops working. Based on this, the sampling liquid can repeatedly collect pathogens in the air. By controlling the working time of the peristaltic pump 5, a sufficient concentration of pathogens can be collected, which is beneficial for subsequent pathogen component analysis.

[0076] Furthermore, the bottom surface of the liquid receiving tank 101 is an annular slope 1011 surrounding the return hole 103. The annular slope 1011 serves as a guide, enabling the sampling liquid in the liquid receiving tank 101 to flow smoothly back into the receiving cavity 201 of the sampling container 2.

[0077] In this embodiment, the outer wall surface of the connecting column 12 is provided with external threads, and the hole wall surface of the first connecting pipe 22 is provided with internal threads. The external threads are used to connect with the internal threads. That is, the connection method between the connecting column 12 and the first connecting pipe 22 is a threaded connection, which has a simple structure, is easy to install, and has high connection stability.

[0078] In this embodiment, the sampling container 2 further includes a second connecting tube 24 and a sealing component 25. One end of the second connecting tube 24 along its length is connected to the container body 21, and the inner hole of the second connecting tube 24 communicates with the receiving cavity 201. The other end of the second connecting tube 24 along its length is connected to the sealing component 25, and the sealing component 25 seals the inner hole of the second connecting tube 24. The sealing component 25 ensures that the sampling container 2 remains sealed when not in use, preventing leakage. In actual use, the hose 3 is connected to the second connecting tube 24, and can communicate with the receiving cavity 201 through the inner hole of the second connecting tube 24. The structure is simple and easy to use.

[0079] In some embodiments, the sealing component 25 can be detachably connected to the second connecting pipe 24. Thus, when the hose 3 is connected to the second connecting pipe 24, the sealing component 25 is removed first and then the hose 3 is connected to the second connecting pipe 24.

[0080] In this embodiment, the sealing assembly 25 includes a sealing gasket 251 and a fixing cap 252. The sealing gasket 251 is connected to the end face of the second connecting tube 24 facing away from the container body 21 and covers the inner hole of the second connecting tube 24. The fixing cap 252 is connected to the second connecting tube 24 to fix the sealing gasket 251. When the hose 3 is connected to the second connecting tube 24, the hose 3 passes through the fixing cap 252 and the sealing gasket 251. The sealing gasket 251 abuts against the outer peripheral wall of the hose 3. At this time, the sealing gasket 251 plays a sealing role and can prevent the collection liquid from leaking from the second connecting tube 24. The advantage of this structure is that the structure of the sampling container 2 is simple, the hose 3 is easy to connect to the second connecting tube 24, and since the collection liquid will not leak from the second connecting tube 24 after the hose 3 is connected to the second connecting tube 24, the second connecting tube 24 can be set at the lower part of the container body 21. For example, the second connecting tube 24 is set at the lower end of the container body 21 and located on the peripheral side wall of the container body 21, which is beneficial to the output of the sampling liquid.

[0081] In this embodiment, the hose 3 includes a tube body 31, a first straight connector 32, and a second straight connector 33. The first straight connector 32 and the second straight connector 33 are respectively connected to the two ends of the tube body 31 along its length. In actual use, the pump head of the peristaltic pump 5 is connected to the tube body 31, the first straight connector 32 is connected to the nozzle 4, and the second straight connector 33 is connected to the second connecting pipe 24. The second straight connector 33 is provided with a fixing cap 252 and a sealing gasket 251. The sealing gasket 251 abuts against the outer peripheral wall of the second straight connector 33. Specifically, the nozzle 4 and the tube body 31 are connected through the first straight connector 32, which has a simple structure, is easy to connect, and has high stability. The second straight connector 33 is used to connect the second connecting pipe 24 and the tube body 31, which allows the second straight connector 33 to easily penetrate the sealing gasket 251, resulting in a simple structure, easy connection, and high stability.

[0082] In this embodiment, the pathogen sampling device further includes a placement seat 6, which has a first mounting cavity 601, a second mounting cavity 602, and a limiting groove 603. The limiting groove 603 is disposed on the top surface of the placement seat 6. The first mounting cavity 601 and the second mounting cavity 602 are spaced apart within the placement seat 6, and the first mounting cavity 601 is disposed below the limiting groove 603. The bottom surface of the limiting groove 603 is provided with a first clearance hole 604 and a second clearance hole 605 penetrating vertically. 4. The second clearance holes 605 are all connected to the first mounting cavity 601. In actual use, the liquid receiving body 11 is placed in the limiting groove 603, and the connecting post 12 passes through the first clearance hole 604. Based on the setting of the limiting groove 603, the liquid receiving body 11 can be placed stably, which can prevent the liquid receiving body 11 from tipping over, which is conducive to the smooth progress of pathogen sampling. The first mounting cavity 601 has an opening 6011 extending to the front side of the placement seat 6. In actual use, the sampling container 2 is accommodated in the first mounting cavity 601. Inside cavity 601, the first connecting tube 22 is connected to the connecting post 12 passing through the first clearance hole 604. The first straight connector 32 of the hose 3 is housed in the first mounting cavity 601 and passes through the second clearance hole 605 to connect with the nozzle 4. Specifically, the first mounting cavity 601 can protect the sampling container 2 and ensure that pathogen sampling can be carried out smoothly. The opening 6011 facilitates the connection between the sampling container 2 and the connecting post 12, and facilitates the connection between the hose 3 and the nozzle 4 and the sampling container 2. The cavity wall of the second mounting cavity 602 is provided with a third clearance hole 606 that extends to the front side of the placement seat 6. The peristaltic pump 5 is connected in the second mounting cavity 602, and at least part of the pump head of the peristaltic pump 5 is located in the third clearance hole 606. This facilitates the connection of the tube body 31 to the pump head. Specifically, based on the setting of the placement seat 6, when pathogen sampling is carried out, the liquid receiving part 1, the sampling container 2, the hose 3, the nozzle 4 and the peristaltic pump 5 can all be stably placed, which can ensure that pathogen sampling is carried out smoothly.

[0083] In practical applications, the above sampling device was used to sample three air outlets in a pig farm. The peristaltic pump of the sampling device had a liquid supply capacity of 400 ml / min; the sampling time was 2 hours; and the volume of the sampling container was 1000 ml, containing 800 ml of physiological saline.

[0084] The concentrate was processed according to the method described in Example 1, with two replicates for each treatment.

[0085] Table 2 Sampling Results

[0086] The above experiments show that, compared with the blank group, the concentration groups 1 to 3 of the present invention can significantly increase the number of detected copies. This further illustrates that the sampling device and concentration detection method of the present invention can be applied to more demanding detection environments, without the need for secondary concentration, and the detection results can be obtained directly.

[0087] Furthermore, Table 2 above illustrates that the method of the present invention has good repeatability.

[0088] Although embodiments of the invention have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims

1. A method of concentrating pathogens, characterized by, Adding a concentrated agent to a solution containing a pathogen to precipitate and concentrate the pathogen in the collection solution, obtaining a precipitate, and then resuspending the precipitate to detect whether the pathogen is positive and / or the content of the pathogen; The concentrated agent includes one of inorganic salt, skimmed milk powder, and diatomite, one of PEG-6000 and PEG8000.

2. The method of claim 1, wherein, The method for obtaining the solution containing the pathogen is to use one of physiological saline, PBS, and DMEM as an adsorbent, and to spray the adsorbent into the air in a spraying manner to adsorb the pathogen in the air.

3. The method of claim 2, wherein, The adsorbent is recycled in a single adsorption process to enrich the pathogen in the air with a limited volume of adsorbent.

4. The method of claim 2, wherein, The sampling device for implementing the method for obtaining the solution containing the pathogen is a disposable device, and the sampling device is sterilized in advance to eliminate interference. The sampling device has a nozzle for manufacturing a micro-fountain, and a circulation unit for continuously supplying liquid to the nozzle and collecting and circulating the liquid sprayed by the nozzle, and the sampling device works in a detection environment for at least 1 h to enrich the pathogen in the liquid.

5. The method of claim 1, wherein, The inorganic salt is sodium chloride or potassium chloride, and the final concentration of the inorganic salt added to the solution is 0.9-3 wt%.

6. The method of claim 1, wherein, The concentration of the skimmed milk powder or diatomite in the solution is 0.005-0.02 wt%.

7. The method of claim 1, wherein, The concentration of the PEG6000 or PEG8000 in the solution is 5-10 wt%.

8. The method of claim 1, wherein, The resuspension solvent is one of physiological saline, PBS, and DMEM.

9. The method according to any one of claims 1 to 8, characterized in that Experiments are performed by taking porcine pseudorabies virus as a DNA virus model and porcine reproductive and respiratory syndrome virus (hereinafter referred to as blue ear virus) as an RNA virus model, and different types of viruses can be concentrated.

10. The method of claim 9, wherein, If only DNA viruses such as porcine pseudorabies virus need to be detected, the concentrated agent is formula 1; if only RNA viruses such as porcine blue ear virus need to be detected, the concentrated agent is formula 2; if DNA and RNA viruses need to be detected simultaneously, the concentrated agent is formula 1, formula 2, or formula 3. The formula 1 is to add 10 wt% PEG-8000, 1.463 wt% NaCl, and 0.01 wt% skimmed milk powder to the solution; The formula 2 is to add 10 wt% PEG-6000, 2.925% NaCl, and 0.01 wt% skimmed milk powder to the solution; The formula 3 is to add 10 wt% PEG-6000, 2.925% NaCl, and 0.01 wt% diatomite to the solution.

Images