Method for enriching foodborne pathogenic microorganisms in agricultural water and application thereof

Abstract

The application provides an enrichment method and application of foodborne pathogenic microorganisms in agricultural water, and belongs to the technical field of foodborne pathogenic microorganism detection. The enrichment method is that the pretreated agricultural water is filtered by a filter membrane; the filter membrane with microorganisms is eluted, the obtained eluate is separated, the obtained solid phase is a membrane bacterial component, and the obtained liquid phase is a solution containing a membrane virus component; and the filter membrane filtrate is treated by a tangential flow filter to obtain a tangential flow filtration virus component. The method can simultaneously enrich and separate various pathogenic bacteria and foodborne viruses in the water sample, shorten the enrichment treatment time, and the recovery rate of the foodborne viruses can be up to 62.60%, and the recovery rate of the pathogenic bacteria can be up to 94.24%, so that various common pathogenic bacteria, foodborne viruses and coronaviruses can be effectively enriched, and the method is applied to rapid detection of foodborne pathogenic microorganisms in agricultural water in cooperation with high-throughput sequencing technology or real-time fluorescent quantitative PCR detection technology.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of detection of foodborne pathogenic microorganisms, and particularly relates to a method for enriching foodborne pathogenic microorganisms in agricultural water and application thereof. BACKGROUND

[0002] Foodborne pathogenic microorganisms are the main biological hazards that contaminate food and agricultural products, seriously threatening human health and causing huge economic losses to the food and agricultural product industry. Fresh fruits and vegetables are contaminated by a variety of foodborne pathogenic microorganisms. The production environment is the main source of contamination of foodborne pathogenic microorganisms in fruits and vegetables. Fresh fruits and vegetables are easily contaminated by pathogenic microorganisms such as norovirus and diarrheal E. coli during production, and because fresh fruits and vegetables need cold chain transportation due to their short shelf life, pathogenic microorganisms can survive for a long time in the supply chain (and foodborne viruses are not easily completely eliminated), and there is a great risk of safety when processing or eating raw.

[0003] The content of pathogenic microorganisms in agricultural water is low, and the types are various, so synchronous, comprehensive and effective enrichment of pathogenic microorganisms in agricultural water is a key link for synchronous detection of pathogenic bacteria and foodborne viruses. At present, the commonly used method for enriching pathogenic microorganisms is mainly for pathogenic bacteria or foodborne viruses, and there are certain limitations for all common pathogenic microorganisms (pathogenic bacteria and foodborne viruses) in agricultural water.

[0004] The existing enrichment methods for microorganisms in agricultural water mainly include filter membrane method for bacteria and glass wool, graphene and other material enrichment method for virus enrichment, and pressure filter membrane method, and there is no method for simultaneous enrichment of bacteria and viruses in agricultural water. SUMMARY

[0005] Therefore, the purpose of the present application is to provide a method for enriching foodborne pathogenic microorganisms in agricultural water and application thereof, which realizes the simultaneous enrichment of pathogenic bacteria and foodborne viruses.

[0006] The present application provides a method for enriching foodborne pathogenic microorganisms in agricultural water, comprising the following steps:

[0007] 1) The pretreated agricultural water is filtered through a filter membrane to obtain a filter membrane with microorganisms trapped and a filter membrane filtrate;

[0008] 2) The filter membrane with microorganisms trapped in step 1) is eluted, and the eluate is subjected to solid-liquid separation, the solid phase is a membrane bacterial component, and the liquid phase is a membrane viral component;

[0009] 3) The filter membrane filtrate in step 1) is subjected to tangential flow filtration to obtain a tangential flow filtered viral component;

[0010] There is no time sequence limitation between step 2) and step 3).

[0011] Preferably, the pore size of the filter membrane in step 1) is 0.2-0.8 μm.

[0012] Preferably, the filtration of the filter membrane in step 1) comprises sequentially using a large-pore filter membrane and a small-pore filter membrane.

[0013] The large-pore filter membrane is a filter membrane with a pore size of 0.45-0.8 μm; and the small-pore filter membrane includes a filter membrane with a pore size of 0.2 μm.

[0014] Preferably, the elution solution in step 2) is a first elution buffer.

[0015] The first elution buffer includes the following components at the following concentrations: beef powder 10-15 g / L, Tris-Base 0.1-0.2 mol / L, and glycine 0.05-0.1 mol / L.

[0016] The separation in step 2) is a centrifugation method, the centrifugation speed is 3000-6000 x g, and the centrifugation time is 5-10 min.

[0017] Preferably, the tangential flow filtration treatment in step 3) comprises tangential flow filtration concentration and elution sequentially.

[0018] Preferably, the tangential flow filtration concentration time is 30-60 min.

[0019] Preferably, the elution solution is a second elution buffer; and the second elution buffer includes the following components at the following concentrations: beef powder 20-30 g / L, Tris-Base 0.2-0.4 mol / L, and glycine 0.1-0.2 mol / L.

[0020] Preferably, the method further comprises the following steps: performing virus precipitation on the virus-containing solution in step 2) and / or the tangential flow filtration virus component in step 3) to obtain a virus solution containing foodborne viruses and / or coronaviruses.

[0021] Preferably, the virus precipitation method comprises PEG precipitation reagent treatment and solid-liquid separation sequentially.

[0022] Preferably, the PEG precipitation reagent includes an aqueous solution with the following concentrations: PEG-8000 8-200 g / L, and sodium chloride 17.5-37.4 g / L.

[0023] The PEG precipitation reagent treatment time is 1-12 h.

[0024] The PEG precipitation reagent treatment temperature is 1-5 °C.

[0025] The solid-liquid separation comprises centrifugation; the centrifugation speed is 4500-8000 rpm, and the centrifugation time is 30-120 min.

[0026] Preferably, the pathogenic bacteria comprise one or more of the following bacteria: Listeria monocytogenes, Staphylococcus aureus, Escherichia coli and Salmonella;

[0027] The foodborne viruses comprise one or more of the following viruses: Norovirus, Hepatitis A virus and Rotavirus;

[0028] The coronavirus comprises Porcine epidemic diarrhea virus.

[0029] The enrichment method provided by the present application is combined with real-time fluorescent quantitative PCR detection technology or high-throughput sequencing technology to rapidly detect foodborne pathogenic microorganisms in agricultural water.

[0030] The enrichment method for foodborne pathogenic microorganisms in agricultural water provided by the present application realizes the primary separation of microorganisms by using a pressurized filter membrane method on pretreated agricultural water, so that bacteria and other microorganisms are intercepted on the filter membrane, and part of the viruses can pass through the filter membrane and exist in the filter membrane filtrate; the microorganisms intercepted on the filter membrane are eluted and subjected to solid-liquid separation, the obtained solid phase is a membrane bacterial component, and the obtained liquid phase is a virus component-containing solution; and the filter membrane filtrate is subjected to tangential flow filtration treatment to obtain a tangential flow filtration virus component. The enrichment method provided by the present application can simultaneously enrich and separate various pathogenic bacteria and foodborne viruses in a water sample, shorten the enrichment time, and the recovery rate of foodborne viruses can be up to 62.60%, and the recovery rate of pathogenic bacteria can be up to 94.24%, so that various common pathogenic bacteria (Listeria monocytogenes, Staphylococcus aureus, Salmonella, Escherichia coli), foodborne viruses (human norovirus (GI and GII type), hepatitis A virus, rotavirus) and coronavirus (porcine epidemic diarrhea virus) can be effectively enriched, and the high-throughput sequencing technology can be used for rapid detection of foodborne pathogenic microorganisms in agricultural water. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 The figure is a schematic diagram of a filter membrane suction filtration device involved in the enrichment process of the present application.

[0032] Figure 2 The figure is a schematic diagram of a tangential flow filtration concentration device involved in the enrichment process of the present application. DETAILED DESCRIPTION

[0033] The present application provides an enrichment method for foodborne pathogenic microorganisms in agricultural water, comprising the following steps:

[0034] 1) Pretreated agricultural water is subjected to filter membrane suction filtration to obtain a filter membrane with intercepted microorganisms and a filter membrane filtrate;

[0035] 2) eluting the filter membrane retaining microorganism in step 1), and performing solid-liquid separation on the obtained eluate, wherein the obtained solid phase is a membrane bacterial component, and the obtained liquid phase is a membrane virus component;

[0036] 3) performing tangential flow filtration treatment on the filter membrane filtrate in step 1), to obtain a tangential flow filtration virus component.

[0037] The present application filters the pretreated agricultural water through a filter membrane, to obtain a filter membrane retaining microorganism and a filter membrane filtrate.

[0038] In the present application, the agricultural water preferably includes agricultural irrigation water and / or agricultural breeding water. The pretreatment method preferably removes weeds, algae and other impurities in the agricultural water. The impurity removal method is preferably completed by using a filter screen with a pore size of 1-2 mm. In the embodiments of the present application, the impurity removal method uses 4-6 layers of sterilized gauze filtration. The present application does not have special restrictions on the source of the agricultural water, and any source of agricultural water known in the art can be used. In the embodiments of the present application, the agricultural water is from natural samples of agricultural irrigation water and breeding water in Nanjing City, Jurong City and Yancheng City.

[0039] In the present application, the pore size of the filter membrane preferably includes 0.2-0.8 μm, and specifically 0.2, 0.45, 0.6, 0.8 μm. When the concentration of microorganisms contained in the agricultural water is high, the filter membrane filtration preferably includes sequentially using a large-pore filter membrane to obtain a large-pore filter membrane retaining microorganism and a filtrate; then replacing the filter device with a small-pore filter membrane, and filtering the obtained filtrate through the small-pore filter membrane; and obtaining a filter membrane filtrate and a small-pore filter membrane retaining microorganism after the small-pore filter membrane filtration. The large-pore filter membrane is preferably a filter membrane with a pore size of 0.45-0.8 μm; and the small-pore filter membrane preferably includes a filter membrane with a pore size of 0.2 μm. The filter membrane filtration is preferably completed by using the filter membrane filtration device shown in the figure. The pressure of the filter membrane filtration is preferably negative pressure, specifically 50-70 KPa, and more preferably 60 KPa. Figure 1

[0040] After obtaining the filter membrane retaining microorganism, the present application elutes the filter membrane retaining microorganism, and performs solid-liquid separation on the obtained eluate, wherein the obtained solid phase is a membrane bacterial component, and the obtained liquid phase is a membrane virus component solution.

[0041] ​In this invention, the preferred elution method involves immersing the filter membrane (including macroporous and microporous membranes) containing microorganisms with an elution solution using agitation. The immersion time is preferably 30-60 minutes, most preferably 60 minutes. The agitation speed is preferably 100-300 rpm, most preferably 200 rpm. The elution solution is a first elution buffer. The first elution buffer preferably comprises the following components at the following concentrations: beef meal 10-15 g / L, Tris-Base 0.1-0.2 mol / L, and glycine 0.05-0.1 mol / L. The first elution buffer can elute most of the microorganisms retained on the filter membrane. The preferred solid-liquid separation method is centrifugation. The centrifugation speed is preferably 3000-6000 × g, most preferably 6000 × g. The centrifugation time is preferably 5-10 minutes, most preferably 10 minutes. The resulting precipitate is a membrane bacterial component, and the supernatant is a membrane viral component. The membrane bacterial components mainly consist of most of the pathogenic bacteria enriched in agricultural water.

[0042] After obtaining the membrane filtrate, the present invention performs tangential flow filtration on the membrane filtrate to obtain tangential flow filtered viral components.

[0043] In this invention, because virus particles are small, most viruses pass through the filter membrane and enter the filtrate during membrane filtration. A tangential flow filtration method is used to enrich most viruses in the water. The filtration process preferably employs... Figure 2 The tangential flow filtration process is carried out in the tangential flow filtration device shown. The tangential flow filtration process preferably includes tangential flow filtration concentration and elution sequentially. In this embodiment of the invention, the tangential flow filtration process specifically involves placing the inlet and outlet pipes of the filtration device into the filtrate, turning on the peristaltic pump to a negative pressure state in the filtration device; transferring the concentrated filtrate entirely to a sterile centrifuge tube through the outlet pipe; then introducing an equal volume of elution solution into the filtration device through the inlet pipe for cyclic elution, and transferring the eluted solution to a sterile centrifuge tube through the outlet. This component is the virus-enriched component from the tangential flow filtration. The negative pressure is preferably 1.5–2.0 MPa. The tangential flow filtration concentration time is preferably 30–60 min, most preferably 60 min. The cyclic elution time is preferably 10–20 minutes, and the pressure is preferably 0.5–1.0 MPa. The elution solution is preferably a second elution buffer; the second elution buffer preferably comprises the following components at the following concentrations: beef powder 20-30 g / L, Tris-Base 0.2-0.4 mol / L, and glycine 0.1-0.2 mol / L. This invention does not impose any special restrictions on the source of the tangential flow filtration and concentration device; any tangential flow filtration and concentration device well-known in the art can be used.

[0044] In the present application, it is preferred to further comprise a solution containing viral components on the membrane and / or the tangential flow filtration viral components to carry out virus precipitation to obtain a virus liquid containing foodborne viruses and / or coronaviruses. The method of virus precipitation preferably comprises PEG precipitation reagent treatment and solid-liquid separation in sequence. The PEG precipitation reagent preferably comprises an aqueous solution with the following final concentrations: PEG-8000 8-200 g / L, sodium chloride 17.5-37.4 g / L, and most preferably PEG-8000 200 g / L, sodium chloride 37.4 g / L. The treatment time of the PEG precipitation reagent is preferably 1-12 h, and most preferably 10 h. The treatment temperature of the PEG precipitation reagent is preferably 1-5°C, and most preferably 4°C. The solid-liquid separation preferably comprises centrifugation. The rotation speed of the centrifugation is preferably 4500-8000 rpm, and most preferably 8000 rpm. The centrifugation time is preferably 30-120 min, and most preferably 30 min.

[0045] In the present application, the enrichment method is suitable for the enrichment of all kinds of pathogenic bacteria, foodborne viruses and coronaviruses. The enriched bacterial components are preferably subjected to enrichment culture to ensure the detection of low-concentration pathogenic bacteria. The nucleic acids obtained from the enriched liquid containing the pathogenic bacteria, foodborne viruses and coronaviruses are detected by Taqman real-time fluorescent quantitative PCR detection method to detect the enriched bacteria, viruses.

[0046] The primer pairs and probes of murine norovirus are derived from Kitajima et al. (2010), the primer pairs and probes of human norovirus (GI and GII types) and hepatitis A virus are derived from international standard ISO / TS 15216-1 (2017), the primer pairs and probes of rotavirus are derived from Asmah, et. al. (2001), the primer pairs and probes of coronavirus (porcine epidemic diarrhea virus) are derived from Fan et al. (2019). The primer pairs and probes of pathogenic bacteria are derived from national standard SNT 1870-2016.

[0047] In the embodiments of the present application, common pathogenic bacteria (Listeria monocytogenes, Staphylococcus aureus, Salmonella and Escherichia coli O157), foodborne viruses (GI and GII norovirus, rotavirus and hepatitis A virus) and coronavirus (porcine epidemic diarrhea virus) are detected in the natural samples of agricultural irrigation water and aquaculture water in Nanjing City, Jurong City and Yancheng City. The minimum detection limit of pathogenic bacteria is 1.61-146 CFU / ml, the minimum detection limit of foodborne viruses is 1.32-55 gene copies (GC) / ml, and the minimum detection limit of coronavirus (porcine epidemic diarrhea virus) is 103 gene copies (GC) / ml.

[0048] The application provides the application of the enrichment method in rapid detection of foodborne pathogenic microorganisms in agricultural water in combination with real-time fluorescent quantitative PCR detection technology or high-throughput sequencing technology.

[0049] The application will be described in detail below in combination with examples of an enrichment method of foodborne pathogenic microorganisms in agricultural water, but they should not be understood as limitations to the protection scope of the application.

[0050] Example 1

[0051] Enrichment method of pathogenic microorganisms in agricultural irrigation water samples artificially added with foodborne pathogenic microorganisms (Listeria monocytogenes, Staphylococcus aureus, Salmonella enteritidis, Norovirus, Rotavirus, murine Norovirus (quality control virus), hepatitis A virus and coronavirus)

[0052] 500ml of agricultural irrigation water is taken, and the mixed solution of the above viruses and bacteria is added, the virus mother liquor is of the order of 10 8 copies (GC) / ml, and the bacterial mother liquor is of the order of 10 8 CFU / ml, and the final concentrations of the viruses and bacteria in the water sample are 10 0 , 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 0 , 10 1 , 10 2 , 10 3 , 10 4 , 10 5 CFU / ml; the viruses are human Norovirus (GI and GII types), murine Norovirus or Rotavirus; coronavirus (porcine epidemic diarrhea virus); the bacteria include Listeria monocytogenes, Staphylococcus aureus or Salmonella, and different pore size filter membranes are sequentially used for suction filtration.

[0053] Large-pore-size filter membrane suction filtration: 0.45μm sterilized filter membrane is installed in a sterilized filter membrane suction filtration device ( Figure 1 ), a pressure pump is connected, the pressure pump is turned on to a negative pressure state of the filter membrane; the water sample added with viruses is added to the upper container of the filter membrane suction filtration device, suction filtration is performed through the filter membrane to the lower conical flask, and a large-pore-size filter membrane filtrate is obtained; the pressure pump is turned off, the filter membrane is removed, and is transferred to a sterile culture dish, soaked with an elution buffer, eluted on a shaking bed for 60 minutes at a speed of 200rpm, and the component is a large-pore-size filter membrane enriched component; after elution, the eluted component is centrifuged at a speed of 6000xg for 5 minutes, the precipitate is a large-pore-size membrane bacterial component, and the supernatant is a large-pore-size membrane virus component; the large-pore-size membrane bacterial component is dissolved with DPBS.

[0054] Small pore size filter filtration: 0.2 μm sterilization filter is installed in (another) sterilized filter filtration device ( Figure 1 ), connect the pressure pump, open the pressure pump to the filter negative pressure state; the filtrate of the large pore size filter is added to the upper container, filtered to the lower conical flask through the filter, and the 0.2 μm filter filtrate is obtained; the pressure pump is closed, the filter is removed and transferred to a sterile culture dish, soaked with elution buffer, eluted on a shaker for 60 minutes at a speed of 200 rpm, and the component is a 0.2 μm filter enriched component; after elution, the eluted component is centrifuged at a speed of 6000 x g for 5 minutes, the precipitate is a 0.2 μm membrane bacterial component, and the supernatant is a 0.2 μm membrane viral component; the 0.2 μm membrane bacterial component is dissolved with DPBS.

[0055] The 0.2 μm filter filtrate ( Figure 1 , lower conical flask) is enriched by tangential flow filtration ( Figure 2 ), the water inlet pipe and water outlet pipe of the filtration device are placed in the filtrate, the peristaltic pump is turned on to a negative pressure state of the filtration device, the pressure is 2.0 MPa, and the time is 60 minutes; the concentrated filtrate is completely transferred to a sterile centrifuge tube through the water outlet pipe, and an equal volume of eluent is introduced into the filtration device through the water inlet, and the cycle elution is 20 minutes, the pressure is 1.0 MPa, and it is transferred to a sterile centrifuge tube through the water outlet. The component is a tangential flow filtration viral component.

[0056] Virus precipitation: the membrane viral components and tangential flow filtration viral components obtained by large pore size and 0.2 μm filter filtration are subjected to virus precipitation, PEG-8000 and NaCl are added according to the volume of each component, the final concentration of PEG-8000 is 100 g / L, and the final concentration of the sodium chloride is 24 g / L; after mixing, precipitate at 4℃ for 4h; centrifuge at 8000 rpm for 30 minutes at 4℃; the precipitate of the membrane viral component or the tangential flow filtration viral component is dissolved with DPBS.

[0057] Foodborne viral nucleic acid extraction: the membrane viral components and tangential flow filtration viral components enriched by (large pore size and 0.2 μm) filter are extracted with TRIZOL-LS to extract RNA, and one-step RNA reverse transcription real-time fluorescent quantitative detection kit (TOYOBO, Japan; Qiagen, Germany) is used for Taqman probe real-time fluorescent quantitative detection of each virus. The primer sequence is shown in Table 1, and the reaction procedure is shown in Table 2.

[0058] Table 1 primer sequence table

[0059]

[0060]

[0061] Reaction procedure for virus detection

[0062]

[0063] The recovery rate of the virus was calculated according to Formula I.

[0064] Recovery rate (%) = (Enriched virus gene copy number / Added virus gene copy number) x 100% Formula I

[0065] The results are shown in Table 3. The recovery rates of human norovirus (GI and GII types) were 38.05% to 62.60% and 14.80% to 39.81%, respectively, and the minimum detection limits were 4.48 GC / ml and 11 GC / ml, respectively; the recovery rate of rotavirus was 17.41% to 57.35%, and the minimum detection limit was 16 GC / ml; the recovery rate of hepatitis A virus was 54.04% to 82.22%, and the minimum detection limit was 8.72 GC / ml; the recovery rate of coronavirus (porcine epidemic diarrhea virus) was 10.96% to 30.63%, and the minimum detection limit was 103 GC / ml; the recovery rate of murine norovirus (quality control virus) was 14.69% to 65.30%, and the minimum detection limit was 1.74 x 10 3 GC / ml.

[0066] Table 3 Recovery rate results of each virus recovered

[0067]

[0068]

[0069] Extraction of nucleic acids of common pathogenic bacteria: The bacterial components of the (large pore size and 0.2 pm) membrane were extracted for bacterial DNA using a bacterial DNA extraction kit, and each bacterium was subjected to Taqman probe real-time fluorescent quantitative detection using a real-time fluorescent quantitative detection kit (Vazyme, China). The primer sequences are shown in Table 1, and the reaction procedure is shown in Table 4.

[0070] Table 4 Reaction procedure for bacterial detection

[0071]

[0072] The recovery rate of the pathogenic bacteria was calculated using Formula II.

[0073] Recovery rate (%) = (Enriched bacterial number CFU / Added bacterial number CFU) x 100% Formula II

[0074] The results are shown in Table 5. The recovery rate of Listeria monocytogenes was 22.32% to 46.44%, the minimum detection limit was 0.175 CFU / ml, the recovery rate of Staphylococcus aureus was 13.68% to 35.48%, the minimum detection limit was 0.0293 CFU / ml, and the recovery rate of Salmonella enteritidis was 45.61% to 94.24%, the minimum detection limit was 31.9 CFU / ml.

[0075] Table 5. Recovery rate of each bacterium

[0076]

[0077]

[0078] Example 2

[0079] Method for enriching pathogenic microorganisms in agricultural irrigation water and aquaculture water samples

[0080] 500 ml of agricultural irrigation water or aquaculture water was taken, a quality control virus MNV was added, and different pore size filter membranes were sequentially subjected to suction filtration.

[0081] Large-pore filter membrane suction filtration: a 0.8 μm sterilized filter membrane was installed in a sterilized filter membrane suction filtration device ( Figure 1 ), a pressure pump was connected, the pressure pump was turned on until the filter membrane was in a negative pressure state; the water sample to which the virus had been added was added to the upper container of the filter membrane suction filtration device, and suction filtration was performed through the filter membrane to the lower conical flask to obtain a large-pore filter membrane filtrate; the pressure pump was turned off, the filter membrane was removed, and was transferred to a sterile culture dish, soaked with an elution buffer, and eluted on a shaking bed for 60 minutes at a speed of 200 rpm. This component was a large-pore filter membrane enriched component; after elution, the eluted component was centrifuged at a speed of 6000 x g for 5 minutes, the precipitate was a large-pore membrane bacterial component, and the supernatant was a large-pore membrane viral component; the large-pore membrane bacterial component was dissolved with DPBS.

[0082] Small-pore filter membrane suction filtration: a 0.2 μm sterilized filter membrane was installed in another sterilized filter membrane suction filtration device ( Figure 1 ), a pressure pump was connected, the pressure pump was turned on until the filter membrane was in a negative pressure state; the large-pore filter membrane filtrate was added to the upper container, and suction filtration was performed through the filter membrane to the lower conical flask to obtain a 0.2 μm filter membrane filtrate; the pressure pump was turned off, the filter membrane was removed, and was transferred to a sterile culture dish, soaked with an elution buffer, and eluted on a shaking bed for 60 minutes at a speed of 200 rpm. This component was a 0.2 μm filter membrane enriched component; after elution, the eluted component was centrifuged at a speed of 6000 x g for 5 minutes, the precipitate was a 0.2 μm membrane bacterial component, and the supernatant was a 0.2 μm membrane viral component; the 0.2 μm membrane bacterial component was dissolved with DPBS.

[0083] The 0.2 μm filter membrane filtrate (Figure 1 , the lower layer conical flask) enriches the tangential flow filtration virus component by tangential flow filtration Figure 2 ), the water inlet pipe and the water outlet pipe of the filtration device are put into the filtrate, the peristaltic pump is turned on to the negative pressure state of the filtration device, the pressure is 2.0 MPa, and the time is 50 minutes; the concentrated filtrate is completely transferred to a sterile centrifuge tube through the water outlet pipe, and an equal volume of eluent is introduced into the filtration device through the water inlet, and the cycle elution is 15 minutes, the pressure is 1.0 Mpa, and it is transferred to a sterile centrifuge tube through the water outlet. The component is a tangential flow filtration virus component.

[0084] Virus precipitation: the membrane virus component enriched by the large pore size and 0.2 μm filter membrane and the tangential flow filtration virus component need to be subjected to virus precipitation, PEG-8000 and NaCl are added according to the volume of each component, the amount of PEG-8000 is 200 g / L, and the amount of sodium chloride is 37.4 g / L; after mixing, precipitate at 4°C for 10h; centrifuge at 4°C, 8000 rpm for 30 minutes; the precipitate of the membrane virus component enriched by the large pore size and 0.2 μm filter membrane or the tangential flow filtration virus component is dissolved with DPBS.

[0085] Extraction of foodborne virus nucleic acid: the viruses in the membrane virus component enriched by the large pore size and 0.2 μm filter membrane or the tangential flow filtration virus component are extracted by TRIZOL-LS to extract RNA, and a one-step RNA reverse transcription real-time fluorescent quantitative detection kit is used to detect human norovirus (GI and GII), hepatitis A virus, rotavirus, porcine epidemic diarrhea virus and quality control virus MNV by Taqman probe real-time fluorescent quantitative detection.

[0086] The detection results are as follows: in 500 ml agricultural irrigation water in Nanjing, GI norovirus (5, 2.39-798.63 GC / ml), GII norovirus (6, 0.731-7.22 GC / ml), hepatitis A virus (1, 233 GC / ml), rotavirus (1, 366 GC / ml) and porcine epidemic diarrhea virus (1, 57.6 GC / ml) were detected; in 500 ml agricultural irrigation water in Jurong City, GI norovirus (2, 7.45-18.78 GC / ml) and GII norovirus (6, 0.365-369 GC / ml) were detected; in 500 ml agricultural irrigation water and breeding water in Yancheng City, GI norovirus (3, 29.2-167 GC / ml) and GII norovirus (3, 1.3-19.5 GC / ml) were detected. The recovery rate of quality control virus MNV is between 4.24% and 62.29%, all greater than 1%.

[0087] Common pathogenic bacteria nucleic acid extraction: the membrane bacterial components enriched by large aperture and 0.2 μm filter membrane were extracted by bacterial DNA extraction kit, and the Staphylococcus aureus, Escherichia coli O157, Salmonella and Listeria monocytogenes were detected by Taqman probe real-time fluorescent quantitative detection.

[0088] The detection results are as follows: Staphylococcus aureus (1, 2.42×10 -2 CFU / ml), Salmonella (2, 69.5-201 CFU / ml) and Listeria monocytogenes (1, 1.89 CFU / ml) were detected in 500 ml agricultural irrigation water in Nanjing; Staphylococcus aureus (2, 6.27×10 -2 ~1.26×10 -2 CFU / ml), Salmonella (5, 62.5-2260 CFU / ml) and Listeria monocytogenes (3, 0.59-1.77 CFU / ml) were detected in 500 ml agricultural irrigation water in Jurong; Salmonella (2, 7.74-66.4 CFU / ml) and Listeria monocytogenes (1, 3.2 CFU / ml) were detected in 500 ml agricultural irrigation water in Yancheng; and no Escherichia coli O157 was detected in the above water samples.

[0089] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered within the protection scope of the present application. SEQUENCE LISTING <110> Jiangsu Academy of Agricultural Sciences <120> A method for enriching foodborne pathogenic microorganisms in agricultural water and application <160> 24 <170> SIPOSequenceListing 1.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence (Artificial Sequence) <400> 1 cgctggatgc gnttccat 18 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence (Artificial Sequence) <400> 2 ccttagacgc catcatcatt tac 23 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 tggacaggag aycgcratct 20 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <400> 4 atgttcagrt ggatgagrtt ctcwga 26 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <400> 5 tcgacgccat cttcattcac a 21 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <400> 6 agcacgtggg agggcgatcg 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <400> 7 gtctgaaaag ccaatcattc 20 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <400> 8 ttgcctctgt tgttactc 18 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <400> 9 ctgttgttgc cattgccacg a 21 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <400> 10 ccgcaggaac gctcagcag 19 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <400> 11 ggytgaatgg ggacggcctg 20 <210> 12 <211> 15 <212> DNA <213> Artificial Sequence <400> 12 atgagtgatg gcgca 15 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <400> 13 accatctaca catgaccctc 20 <210> 14 <211> 16 <212> DNA <213> Artificial Sequence <400> 14 ggtcacataa cgcccc 16 <210> 15 <211> 33 <212> DNA <213> Artificial Sequence <400> 15 atgagcacaa tagttaaaag ctaacactgt caa 33 <210> 16 <211> 19 <212> DNA <213> Artificial Sequence <400> 16 gcggcgttgg agagtgata 19 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <400> 17 agcaatggaa aaagcaggat g 21 <210> 18 <211> 28 <212> DNA <213> Artificial Sequence <400> 18 catttcttaa acggcggtgt ctttccct 28 <210> 19 <211> 25 <212> DNA <213> Artificial Sequence <400> 19 ttcttcacga ctaaataaac gctca 25 <210> 20 <211> 28 <212> DNA <213> Artificial Sequence <400> 20 ggtactacta aagattatca agacggct 28 <210> 21 <211> 27 <212> DNA <213> Artificial Sequence <400> 21 cagaacacaa tgtttccgat gcaacgt 27 <210> 22 <211> 23 <212> DNA <213> Artificial Sequence <400> 22 ctgaatctca agcaaaacct ggt 23 <210> 23 <211> 18 <212> DNA <213> Artificial Sequence <400> 23 cgcgaccgaa gccaacta 18 <210> 24 <211> 28 <212> DNA <213> Artificial Sequence <400> 24 atacgataac atccacggct ctggctgg 28

Claims

1. A method for enriching food-borne pathogenic microorganisms in agricultural water, characterized by, The method comprises the following steps: 1) filtering the pretreated agricultural water through a filter membrane to obtain a filter membrane retaining microorganisms and a filter membrane filtrate; the filter membrane filtration comprises sequentially filtering through a large-pore filter membrane and a small-pore filter membrane; the large-pore filter membrane is a filter membrane with a pore size of 0.45-0.8 μm; the small-pore filter membrane is a filter membrane with a pore size of 0.2 μm; 2) eluting the filter membrane retaining microorganisms in step 1), and subjecting the eluate to solid-liquid separation, wherein the obtained solid phase is a membrane bacterial component, and the obtained liquid phase is a membrane viral component; the elution solution is a first elution buffer; the first elution buffer comprises the following components at the following concentrations: beef powder 10-15 g / L, Tris-Base 0.1-0.2 mol / L, and glycine 0.05-0.1 mol / L; 3) treating the filter membrane filtrate in step 1) through a tangential flow filter to obtain a tangential flow filtered viral component; the tangential flow filter treatment comprises tangential flow filter concentration and elution in sequence; the negative pressure in the tangential flow filter treatment is 1.5-2.0 MPa; the elution solution is a second elution buffer; the second elution buffer comprises the following components at the following concentrations: beef powder 20-30 g / L, Tris-Base 0.2-0.4 mol / L, and glycine 0.1-0.2 mol / L; subjecting the solution of the membrane viral component or the tangential flow filtered viral component to viral precipitation to obtain a foodborne virus; steps 2) and 3) are not limited in time sequence; the separation in step 2) is centrifugation, the centrifugation is performed at a speed of 3000-6000 x g for 5-10 min; the viral precipitation method in step 2) comprises PEG precipitation reagent treatment and solid-liquid separation in sequence; the PEG precipitation reagent comprises an aqueous solution of PEG-8000 8-200 g / L and sodium chloride 17.5-37.4 g / L; the PEG precipitation reagent is treated for 1-12 h; the treatment temperature of the PEG precipitation reagent is 3-5 ℃; the solid-liquid separation comprises centrifugation; the centrifugation is performed at a speed of 4500-8000 rpm for 30-120 min.

2. Application of the enrichment method of claim 1 in combination with real-time fluorescent quantitative PCR detection technology or high-throughput sequencing technology in detection of foodborne pathogenic microorganisms in agricultural water.

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