A method for detecting trace amounts of enteroviruses in fruits and vegetables

Abstract

The invention discloses a method for detecting trace enterovirus in fruits and vegetables. The method comprises the following steps: mixing a fruit and vegetable sample to be tested with an elution buffer KNTG, washing the sample and centrifuging the sample, taking a supernatant and adjusting the pH value to 7.0; adding montmorillonite-sealed activated carbon to the supernatant after adjusting the pH value, shaking the supernatant and centrifuging the sample, collecting the supernatant, adding an aqueous solution of polyethylene glycol and NaCl, shaking the supernatant and centrifuging the supernatant, suspending the precipitate with PBS, and obtaining a suspension; extracting RNA, and detecting the content of enterovirus by RT-qPCR; the KNTG elution buffer provided by the invention is combined with BCAC to pre-treat the fruit and vegetable sample, so that enterovirus detection can be completed within 6 hours, and the average detection limit of enterovirus is reduced by 1-2 log10, and the method has the advantages of simple operation, short time consumption, rapidity, reliability, high sensitivity, and the like.

Description

(I) Technical field

[0001] The invention relates to a method for detecting trace enterovirus in fruits and vegetables. (II) Background technology

[0002] Human norovirus (HuNoV) belongs to the Caliciviridae family and is one of the pathogens of acute gastroenteritis. According to the amino acid sequence of the norovirus capsid protein (VP1), it can be divided into seven genomes: GⅠ, GⅡ, GⅢ, GIV, GⅤ, GⅥ and GⅦ. Among them, the fourth genotype (GⅡ.4) of the GⅡ gene family is the most common norovirus that causes human gastroenteritis. Norovirus (NoVs) is mainly transmitted through the fecal-oral route. Agricultural products contaminated by NoVs mainly include lettuce, strawberries, tomatoes, raspberries, carrots and bulb vegetables. The incubation period of patients infected with NoVs exceeds 10 hours. After the incubation period, symptoms such as vomiting, low-grade fever and diarrhea may occur. About 7 million people are infected with NoVs each year, resulting in more than 200,000 deaths and an economic burden of more than 64 million US dollars. At present, murine norovirus MNV-1 (murine norovirus) and Tulane virus TV (Tulanevirus) are the most common substitutes for human norovirus. MNV-1 is a culturable norovirus that has highly similar structure and genetic characteristics to HuNoV and is considered to be the most suitable alternative to HuNoV; the capsid of TV is very similar to that of HuNoV, especially the capsid region and its structure.

[0003] At present, the commonly used detection steps of NoVs mainly include virus elution and concentration, nucleic acid extraction and polymerase chain reaction (PCR) detection. However, due to the low virus content in fruits and vegetables and the difficulty in fully eluting, and the presence of PCR inhibitors and virus contamination to a certain extent, PCR cannot accurately detect foodborne viruses in fruits and vegetables. Based on the low-dose pathogenicity of enteroviruses in fruits and vegetables, how to fully elute and enrich enteroviruses in fruits and vegetables and improve the sensitivity of enterovirus detection methods has received increasing attention. (III) Summary of the invention

[0004] The present invention aims to provide a method for detecting trace amounts of enterovirus in fruits and vegetables, which can improve the recovery rate and detection sensitivity of enterovirus when the sample to be tested is further tested in the subsequent testing by washing with KNTG elution buffer.

[0005] The technical solution adopted by the present invention is:

[0006] The present invention provides a method for detecting trace amounts of enterovirus in fruits and vegetables, the method comprising the following steps: (1) mixing a fruit and vegetable sample to be tested with a KNTG elution buffer, washing and centrifuging, taking the supernatant and adjusting the pH to 7.0; the KNTG elution buffer comprises: 0.01-0.1 mM KH2PO4, 0.5-2 mM NaCl, and a volume concentration of 0.01-1.0% Triton X-100, 0.01-0.1 mM glycine (Glycine), the solvent is water, and the pH is 8-10; (2) adding montmorillonite-capped activated carbon (BCAC) to the supernatant after adjusting the pH value in step (1), shaking and centrifuging to remove RT-qPCR inhibitors in the fruit and vegetable samples, and collecting the supernatant; (3) adding an aqueous solution of polyethylene glycol (PEG) and NaCl to the supernatant in step (2), shaking and centrifuging, and suspending the precipitate with PBS to obtain a suspension; (4) extracting RNA from the suspension in step (3), and detecting the enterovirus content by RT-qPCR; the enterovirus includes murine norovirus MNV-1 (murine norovirus-1), Tulane virus TV (Tulane virus) or human norovirus HuNoV (human norovirus).

[0007] Preferably, the KNTG elution buffer in step (1) comprises: 0.05 mM KH2PO4, 1 mM NaCl, 0.1% Triton X-100, 0.05 mM glycine, the solvent is water, and the pH is 9.5.

[0008] Preferably, the fruit and vegetable samples to be tested in step (1) include lettuce, cherry tomatoes, blueberries, strawberries or spinach. The fresh fruit and vegetable samples are cut into pieces (preferably into small pieces of about 2.5 cm×2.5 cm×2.5 cm), the surface stains are cleaned, and the pieces are naturally air-dried to obtain the fruit and vegetable samples to be tested.

[0009] Preferably, in step (1), if the fruit and vegetable samples to be tested are vegetable samples, cellulase is added to the KNTG elution buffer, and the amount of the cellulase added is 1-10 U / mL (preferably 7.5 U / mL) based on the volume of the KNTG elution buffer; if the fruit and vegetable samples to be tested are fruit samples, pectinase is added to the KNTG elution buffer, and the amount of the pectinase added is 15-30 U / mL (preferably 28.5 U / mL) based on the volume of the KNTG elution buffer.

[0010] Preferably, the volume of KNTG elution buffer added in step (1) is 1-6 mL / g, preferably 1.6 mL / g, based on the mass of the fruit and vegetable sample to be tested.

[0011] Preferably, the post-wash centrifugation method in step (1) is: adding the fruit and vegetable sample to be tested to the KNTG elution buffer, shaking at 120 r / min for 20 min at room temperature, measuring the pH value every 10 min during the period to ensure the pH stability of the KNTG elution buffer; then centrifuging at 11000×g for 30 min at 4°C, transferring the supernatant to a clean 50 mL centrifuge tube, and adjusting the pH to 7.0 with 1 mM HCl.

[0012] Preferably, the montmorillonite-sealed activated carbon in step (2) is prepared as follows: (1) granular activated carbon with a particle size of 1.25-2 mm is taken, washed with deionized water until the supernatant is clarified, and dried at 60°C for later use; (2) 1 g of montmorillonite is fully mixed with 50 mL of deionized water, and then homogenized at 10000 r / min and 4°C for 3 min to ensure that the montmorillonite is fully mixed, and then centrifuged at 100×g and 4°C for 2 min, and the supernatant is taken as a sealing agent for later use; (3) The granular activated carbon in step (1) is immersed in the sealing agent of step (2), and sealed at 120 r / min for 3 h in a shaker at 37°C, and then the supernatant is discarded, and after drying at 60°C, the montmorillonite-sealed activated carbon is obtained.

[0013] Preferably, the amount of montmorillonite-sealed activated carbon added in step (2) is 0.001-0.1 g / mL, preferably (0.025 g / mL), based on the volume of the KNTG elution buffer in step (1); after the montmorillonite-sealed activated carbon is added, the mixture is shaken at 120 r / min for 15 min and centrifuged at 100×g for 2 min.

[0014] Preferably, in step (3), the final concentration of polyethylene glycol added to the supernatant in the aqueous solution of polyethylene glycol and NaCl is 50-150 g / L (preferably 100 g / L), and the final concentration of NaCl added to the supernatant is 0.1-1.0 mM (preferably 0.3 mM); preferably, the polyethylene glycol concentration in the aqueous solution of polyethylene glycol and NaCl is 500 g / L, and the NaCl concentration is 87 g / L. The preparation method of the aqueous solution of polyethylene glycol and NaCl is: 500.0 g of polyethylene glycol (preferably PEG8000) and 87.0 g of sodium chloride are stirred and dissolved in ultrapure water, the volume is made up to 1000 mL with ultrapure water, packaged, and sterilized at 121° C. for 15 min.

[0015] Preferably, step (3) is carried out as follows: add an aqueous solution of PEG and NaCl to the supernatant of step (2); shake well for 60 seconds and place on a shaker, oscillate at 60 r / min for 60 minutes at 4°C, centrifuge the solution at 11,000×g for 30 minutes at 4°C, discard the supernatant, and suspend the precipitate with PBS to obtain a suspension.

[0016] Preferably, the fluorescence quantitative PCR detection program in step (4) is as follows: reverse transcription reaction at 42°C for 30 min; pre-denaturation at 95°C for 2 min; denaturation at 95°C for 5 s, annealing and extension at 55°C for 35 s, and 39 cycles.

[0017] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0018] The KNTG elution buffer provided by the present invention is combined with BCAC pre-treatment of fruit and vegetable samples, and can complete enterovirus detection within 6 hours. The average detection limit of enterovirus is reduced by 1-2 log10. It has the advantages of simple operation, short time consumption, rapidity, reliability, high sensitivity, etc. (IV) Description of the drawings

[0019] Figure 1 This is an experimental flow chart of the KNTG-BCAC method for detecting enterovirus in fruits and vegetables in Example 1.

[0020] Figure 2 This is a schematic diagram of the results of evaluating the use of KNTG elution buffer combined with the BCAC method in detecting trace amounts of HuNoV in fruits and vegetables in Example 2. (V) Specific implementation methods

[0021] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0022] The reagents used in the following examples can be purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. and Shenggong Biotechnology (Shanghai) Co., Ltd., and all reagents used were of analytical grade unless otherwise specified.

[0023] The fruit and vegetable samples in the embodiment of the present invention are lettuce (Lactuca sativa L. var. ramosa Hort., Compositae Lactuca), cherry tomatoes (Lycopersicon esculentum var. cerasiforme, Tomato), blueberries (Vaccinium Spp., Vaccinium), strawberries (Fragaria × ananassa Duch., Fragaria) and spinach (Spinacia oleracea L., Spinacia).

[0024] The bacteriophage MS2 and murine norovirus MNV-1 used in the present invention were purchased from the American Tissue Culture Collection, Tulane virus TV was provided by the Agricultural Research Center of the United States Department of Agriculture, and human norovirus HuNoV was provided by the Zhejiang Provincial Center for Disease Control and Prevention. The cellulase activity was 7.5 U / mL; the pectinase activity was 28.5 U / mL.

[0025] The composition of KNTG elution buffer is: 0.05 mM KH2PO4, 1 mM NaCl, 0.1% Triton X-100, 0.05 mM glycine, the solvent is water, the pH is 9.5, packaged, and sterilized at 121°C for 15 minutes.

[0026] The preparation of montmorillonite-coated activated carbon (BCAC) was based on the method of Li et al. (Sheng-Yan Li, Mei Shu, Chan Zhong, Hu Chen, Yan Bi, Peng-Fei Hou, Guo-Ping Wu. Characterization and Kinetic Study of Bentonite-Coated Activated Carbon for Adsorption of DNA Polymerase Inhibitors to Improve the Detection Sensitivity of Salmonella Derived from Vegetables by Rti-LAMP[J]. Food Analytical Methods, 2020, 13(10).), specifically prepared as follows: (1) Take granular activated carbon with a particle size of 1.25-2 mm, wash it with deionized water until the supernatant is clear, and dry it at 60°C for later use; (2) Mix 1g of montmorillonite with 50mL of deionized water, and after mixing, homogenize at 10000rpm and 4°C for 3min to ensure that the montmorillonite is fully mixed, and then centrifuge at 100×g and 4°C for 2min, and take the supernatant as the sealing agent for later use; (3) Preparation of montmorillonite-sealed activated carbon: immerse the granular activated carbon of step (1) with the sealing agent of step (2), seal it at 120r / min in a shaker at 37°C for 3h, then discard the supernatant, and dry it at 60°C to obtain montmorillonite-sealed activated carbon.

[0027] The polyethylene glycol concentration in the aqueous solution of PEG and NaCl is 500 g / L, and the NaCl concentration is 87 g / L. The specific preparation method is: 500.0 g of PEG8000 and 87.0 g of sodium chloride are stirred and dissolved in ultrapure water, and the volume is made up to 1000 mL with ultrapure water, divided into portions, and sterilized at 121° C. for 15 min.

[0028] NaPP elution buffer: volume concentration 1% sodium pyrophosphate (NaPP), 0.05M glycine (Glycine), 3.8mMNa2HPO4, 6.5mM K2HPO4, solvent is water, pH 9.5.

[0029] Example 1: Detection of trace amounts of MNV-1 and TV in fruits and vegetables using KNTG elution buffer combined with BCAC (KNTG-BCAC) method

[0030] 1. KNTG-BCAC method

[0031] Reference Figure 1 The KNTG-BCAC method process shown in the figure detects trace amounts of enterovirus in fruits and vegetables, including the following steps:

[0032] (1) Inoculation of enterovirus in fruit and vegetable samples

[0033] Take fruit and vegetable samples (lettuce, cherry tomatoes, blueberries, strawberries and spinach, the fruit and vegetable samples described below include these five types, and each is treated separately), cut them into small pieces of about 2.5 cm × 2.5 cm × 2.5 cm, clean the surface stains, air-dry them naturally, and irradiate them under a 5 W ultraviolet lamp for 40 seconds to obtain the pretreated fruit and vegetable samples.

[0034] 100 μL of MNV-1 and TV were inoculated into each pretreated fruit and vegetable sample, with the inoculation amount of 3.00×10 5 GC / g and 2.20×10 6 GC / g, and obtain the inoculated fruit and vegetable samples. Place the inoculated fruit and vegetable samples in a bucket and air-dry them overnight at room temperature to obtain the fruit and vegetable samples to be tested.

[0035] (2) Use KNTG elution buffer to elute the enterovirus on the fruit and vegetable samples to be tested:

[0036] Place 25 g of the fruit and vegetable sample to be tested in step (1) in a beaker, add 40 mL of KNTG elution buffer at room temperature. If it is a vegetable sample (lettuce or spinach), add 7.5 U / mL of cellulase to the KNTG elution buffer; if it is a fruit sample (cherry tomatoes, blueberries or strawberries), add 28.5 U / mL of pectinase to the KNTG elution buffer. Place the beaker on a shaker and shake at 120 r / min for 20 min at room temperature. During this period, measure the pH value every 10 min to ensure that the pH is stable at 9.5. Then centrifuge at 11000×g for 30 min at 4°C. Transfer the supernatant to a clean 50 mL centrifuge tube and adjust the pH to 7.0 with 1 mM HCl.

[0037] (3) Enrichment of enterovirus using BCAC combined with PEG / NaCl:

[0038] Add 1.0 g BCAC to the centrifuge tube in step (2), shake at 120 r / min for 15 min, and centrifuge at 100 × g for 2 min. Take 40 mL of the supernatant and add 10 mL of an aqueous solution of PEG8000 and NaCl to make the final solution have a PEG concentration of 100 g / L and a NaCl concentration of 0.3 mM. Shake well for 60 seconds and place on a shaker, shake at 60 r / min for 60 min at 4°C, centrifuge the solution at 11000 × g for 30 min at 4°C, discard the supernatant, and suspend the precipitate with 500 μL PBS to obtain a suspension.

[0039] When the fruit and vegetable samples are vegetables (lettuce or spinach), the suspension in step (3) can be directly transferred to a clean test tube, and the milliliters of the suspension can be measured and recorded, and the template RNA can be extracted using an RNA extraction kit.

[0040] When the fruit and vegetable sample is fruit (cherry tomatoes, blueberries or strawberries), transfer the suspension in step (3) to a chloroform-resistant test tube. Add 500 μL of a chloroform / butanol mixture with a volume ratio of 1:1, vortex and let stand for 5 minutes at room temperature, transfer the liquid portion to a clean centrifuge tube, and then centrifuge at 11,000 × g for 15 minutes at 4°C. Take the supernatant, measure and record the milliliter, and extract the template RNA using an RNA extraction kit.

[0041] (4) RT-qPCR reaction:

[0042] The template RNA extracted in step (3) is amplified with the primers in Table 1 and the PCR conditions described in Table 2.

[0043] Table 1. Primer and probe sequences for enterovirus detection

[0044]

[0045]

[0046] Table 2. 50 μL RT-qPCR reaction system

[0047]

[0048] The RT-qPCR reaction system was placed in a fluorescent quantitative PCR instrument. The first step was to react at 42°C for 30 min, the second step was to react at 95°C for 2 min, the third step was to react at 95°C for 5 s, and the fourth step was to react at 55°C for 35 s. The third and fourth steps were repeated for 39 cycles before the reaction was terminated.

[0049] (5) Result judgment:

[0050] A standard curve of Ct value and target virus concentration was established, and then the concentration of the target virus in the fruit and vegetable samples to be tested was quantitatively detected based on the standard curve.

[0051] The standard curve of MNV-1 is: y = -0.30x + 11.52, R 2 =0.99;

[0052] The standard curve of TV is: y = -0.28x + 11.94, R 2 =0.99;

[0053] The standard curve of HuNoV is y=-0.33x+11.21, R 2 =0.99.

[0054] The recovery rate was calculated according to the formula: R (%) = Q1 / Q2 × 100% (R represents the recovery rate, Q1 represents the amount of virus recovered from the fruit and vegetable matrix, and Q2 represents the original amount of virus).

[0055] 2. NaPP method

[0056] The KNTG elution buffer in step (2) of the KNTG-BCAC method in step 1 was replaced with NaPP elution buffer, and the BCAC in step (3) was removed. The other operations were the same.

[0057] 3. GB 4789.42-2016

[0058] The fruit and vegetable samples to be tested in step (1) of step 1KNTG-BCAC method are tested using the GB 4789.42-2016 method.

[0059] 4. KNTG method

[0060] The BCAC in step (3) of the KNTG-BCAC method in step 1 was omitted, and the other operations were the same.

[0061] 5. GB 4789.42—2016-BCAC method

[0062] In the GB 4789.42-2016 test process, 1.0 g BCAC was added before adding the aqueous solution of PEG8000 and NaCl (1.0 g BCAC was added at step 5.1.1.4 of the standard).

[0063] The experimental results are shown in Tables 3 and 4. The recovery of enterovirus after elution with KNTG elution buffer in step (3) of the KNTG-BCAC method and treatment with BCAC was better than that with KNTG elution buffer or GB alone.

[0064] The recovery effect of KNTG elution buffer combined with BCAC was significantly higher than that of NaPP elution buffer treatment and GB 4789.42-2016. This indicates that KNTG elution buffer combined with BCAC can recover viruses to the greatest extent, among which KNTG elution buffer can fully elute viruses in fruits and vegetables, and BCAC can remove water-soluble inhibitors of RT-qPCR.

[0065] Table 3. Virus recovery rate (%) a

[0066]

[0067] a GC recovery rate / GC original inoculum volume.

[0068] b Mean ± SD calculated from standard curve (n = 3 independent experiments).

[0069] Table 4. Virus recovery rate (%) a

[0070]

[0071] a GC recovery rate / GC original inoculum volume.

[0072] b Mean ± SD calculated from standard curve (n = 3 independent experiments).

[0073] Example 2, KNTG-BCAC method detection sensitivity

[0074] In order to evaluate the detection sensitivity of the KNTG-BCAC method of Example 1, the MNV-1 inoculum was diluted to 1.12×10 3 , 1.12×10 3 , 1.12×10 1GC / g, TV inoculum dilution was 8.88×10 3 ,8.88×10 3 ,8.88×10 1 GC / g, the experiment was carried out according to the steps described in Example 1. The GB 4789.42-2016 method was used as the control group.

[0075] The experimental results show that in the control group GB 4789.42-2016, 10 3 GC / g, but the detection rate is low and can be easily regarded as a false positive result (see Table 5). The experimental group using the KNTG-BCAC method can stably detect 10 3 GC / g of viruses. Most fruit and vegetable samples were within 10 2 It can also be detected in GC / g (see Table 6). Obviously, after elution with KTNG stripping buffer and treatment with BCAC, the detection sensitivity of RT-qPCR increased by 1-2 log, indicating that the KNTG-BCAC method can significantly improve the amplification efficiency of RT-qPCR.

[0076] Table 5. Virus detection rate in GB 4789.42-2016

[0077]

[0078] Table 6. Virus detection rate of KNTG-BCAC method

[0079]

[0080] Example 3: Detection of HuNoV in fruits and vegetables by KNTG-BCAC method

[0081] In order to further evaluate the effect of KNTG-BCAC method for detecting trace amounts of HuNoV virus in actual samples, lettuce and cherry tomatoes irrigated with sewage in abandoned soil were selected, and eluted and amplified by RT-qPCR according to the steps described in Example 1. The GB 4789.42-2016 method was used as the control group.

[0082] The experimental results are shown in Figure 2 As shown in the results, the virus recovery rates of lettuce and cherry tomatoes increased from 9.75PCRU / g to 39.7PCRU / g and from 9.67PCRU / g to 71PCRU / g, respectively, which were 4 times and 7 times that of the control group GB 4789.42-2016. The results showed that the KNTG-BCAC method can improve the sensitivity of RT-qPCR detection of norovirus in vegetables and fruits.

[0083] In summary, the present invention has developed a method for rapid and sensitive detection of norovirus in fruits and vegetables. The optimized method focuses on eluting fruit and vegetable samples with KNTG eluent, and then treating with BCAC to remove PCR inhibitors in the virus-containing eluent, which significantly improves the sensitivity of the detection and can complete the entire detection within 6 hours. The optimized method can be used for eluting and recovering enterovirus in fruit and vegetable samples for future follow-up detection, and has the characteristics of time saving, simple operation, and high sensitivity.

Claims

1. A method for detecting trace amounts of enterovirus in fruits and vegetables, characterized in that: The method comprises the following steps: (1) cutting fresh fruit and vegetable samples into pieces, cleaning the surface stains, and air-drying the pieces to obtain the fruit and vegetable samples to be tested; mixing the fruit and vegetable samples to be tested with KNTG elution buffer, washing and centrifuging, and adjusting the pH of the supernatant to 7.0; if the fruit and vegetable samples to be tested are vegetable samples, adding cellulase to the KNTG elution buffer; if the fruit and vegetable samples to be tested are fruit samples, adding pectinase to the KNTG elution buffer, wherein the amount of cellulase added is 7.5 U / mL based on the volume of the KNTG elution buffer; and the amount of pectinase added is 28.5 U / mL based on the volume of the KNTG elution buffer; the KNTG elution buffer comprises: 0.05 mM KH2PO4, 1 mM NaCl, and a volume concentration of 0.1% Triton X-100, 0.05 mM glycine, the solvent is water, and the pH is 9.5; (2) adding montmorillonite-sealed activated carbon to the supernatant after adjusting the pH value in step (1), shaking and centrifuging, and collecting the supernatant; (3) adding an aqueous solution of polyethylene glycol and NaCl to the supernatant in step (2), shaking and centrifuging, and suspending the precipitate with PBS to obtain a suspension; (4) extracting RNA from the suspension in step (3), and detecting the enterovirus content by RT-qPCR; the enterovirus includes murine norovirus MNV-1, Tulane virus TV or human norovirus HuNoV.

2. The detection method according to claim 1, characterized in that Step (1) The volume of KNTG elution buffer added is 1-6 mL / g based on the mass of the fruit and vegetable sample to be tested.

3. The detection method according to claim 1, characterized in that The post-wash centrifugation method in step (1) is as follows: adding the fruit and vegetable sample to be tested into the KNTG elution buffer, shaking at 120 r / min for 20 min at room temperature, measuring the pH value every 10 min during this period to ensure the pH stability of the KNTG elution buffer; then centrifuging at 11000×g for 30 min at 4°C, and adjusting the pH of the supernatant to 7.0 with 1 mM HCl.

4. The detection method according to claim 1, characterized in that The amount of montmorillonite-sealed activated carbon added in step (2) is 0.001-0.1 g / mL based on the volume of the KNTG elution buffer in step (1); after the montmorillonite-sealed activated carbon is added, the mixture is shaken at 120 r / min for 15 min and centrifuged at 100×g for 2 min.

5. The detection method according to claim 1, characterized in that In step (3), the polyethylene glycol is added to the aqueous solution of polyethylene glycol and NaCl at a final concentration of 50-150 g / L, and the NaCl is added to a final concentration of 0.1-1.0 mM.

6. The detection method according to claim 1, characterized in that Step (3) is carried out as follows: add an aqueous solution of polyethylene glycol and NaCl to the supernatant of step (2); shake well for 60 seconds and place on a shaker, oscillate at 60 r / min for 60 minutes at 4°C, centrifuge the solution at 11,000×g for 30 minutes at 4°C, discard the supernatant, and suspend the precipitate with PBS to obtain a suspension.

7. The detection method according to claim 1, characterized in that Step (4) The detection program of the fluorescence quantitative PCR instrument is as follows: reverse transcription reaction at 42°C for 30 min; pre-denaturation at 95°C for 2 min; denaturation at 95°C for 5 s, annealing and extension at 55°C for 35 s, and 39 cycles.

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