A method for improving the detection rate of norovirus in aquatic products

Abstract

The application provides a method for improving the detection rate of norovirus in aquatic products, which comprises the following steps: first, using a proteinase K solution to enzymolysis the digestive gland of shellfish, then adding a TGBE solution to elute, then adding enzymolysis corn starch to block activated carbon to remove the RT-qPCR inhibitor in the detection sample, taking the supernatant to extract nucleic acid, and using a fluorescent quantitative PCR instrument to detect. The detection method of the application reduces the inhibitor in the shellfish sample which interferes with the fluorescent quantitative PCR by using enzymolysis corn starch. In combination with the proteinase K enzymolysis, the TGBE elution and the RT-qRCR, the norovirus in the shellfish sample is detected, and the detection rate of the shellfish sample is increased by 10-20 times in 4h compared with the national standard method. The virus recovery rate and the detection sensitivity are greatly improved, and the method is suitable for shellfish samples, and has the characteristics of simple operation, high detection sensitivity and short time consumption.

Description

(I) Technical Field

[0001] This invention belongs to the field of microbial detection, specifically relating to a method for improving the detection rate of norovirus in aquatic products. (II) Background Technology

[0002] Norovirus (NoV) is a major foodborne pathogen causing nonbacterial acute gastroenteritis worldwide. It belongs to the Caliciviridae family and is a species of virus in the Norovirus genus. Norovirus is a non-enveloped, single-stranded, positive-sense RNA virus with a genome length of 7.5-7.7 kb. It has three open reading frames (ORFs): ORF1, ORF2, and ORF3. In ORF1, the protease cleaves the encoded polyprotein into six non-structural proteins, including polymerase. ORF2 encodes the major structural protein (VP1), and ORF3 encodes the minor structural protein (VP2). Norovirus can be classified into seven genotypes (GI to GVII) based on the amino acid sequence of its capsid protein. NoV GI, NoV GII, and NoV GIV genotypes are most susceptible to infecting humans. Norovirus is primarily transmitted from person to person, with indirect transmission through contaminated water and food.

[0003] Shellfish, fruits, and vegetables are most susceptible to norovirus contamination, with shellfish being the primary mode of transmission. Shellfish are filter feeders, and their digestive glands can accumulate high concentrations of the virus in water. Therefore, consuming raw shellfish or improper processing greatly increases the risk of norovirus infection. More than 200,000 people die from norovirus each year, especially in semi-enclosed environments such as schools, hospitals, and nursing homes.

[0004] Quantitative real-time PCR (qPCR) is a commonly used method for detecting norovirus. However, nucleic acids contain lipids, polysaccharides, and proteins, which leads to low PCR detection efficiency, and many low-concentration viruses remain undetected. Therefore, improving the detection rate of norovirus in shellfish is of great significance. (III) Summary of the Invention

[0005] The purpose of this invention is to provide a method to improve the detection rate of norovirus in aquatic products. This method combines enzymatic hydrolysis of corn starch to block activated carbon, TGBE buffer to elute norovirus in shellfish samples, reduces the amount of inhibitors in food that interfere with quantitative real-time PCR, and uses real-time quantitative polymerase chain reaction (RT-qPCR) to achieve rapid and specific detection of norovirus in shellfish, which can greatly improve the detection rate of norovirus in aquatic products.

[0006] The technical solution adopted in this invention is as follows:

[0007] This invention provides a method for improving the detection rate of norovirus in aquatic products, the method comprising the following steps:

[0008] (1) Proteinase K enzymatically hydrolyzes the digestive glands in shellfish

[0009] An ultrapure aqueous solution of proteinase K was added to the digestive gland of aquatic products, and the mixture was kept at 30-40℃ and 200-400 rpm for 40-80 min under constant temperature shaking, followed by a water bath at 50-70℃ for 10-20 min to obtain the enzymatically hydrolyzed digestive gland; the aquatic products included shellfish; the ultrapure water was RNase-free ultrapure water.

[0010] (2) Elution with TGBE buffer solution

[0011] Add TGBE elution buffer to the digestive gland after enzymatic hydrolysis in step (1), and shake at 100-300 rpm for 20-40 min at room temperature; then centrifuge at 4℃ and centrifugation force of 5000-15000×g for 20-40 min, and collect the supernatant.

[0012] (3) Enzymatic hydrolysis of corn starch and blocking activated carbon (SCAC) to remove RT-PCR interference in the test samples.

[0013] Add the enzymatically hydrolyzed corn starch-blocked activated carbon (SCAC) to the supernatant collected in step (2), shake at 26℃ and 100-300 rpm for 20-40 min, extract RNA, and detect norovirus using real-time quantitative polymerase chain reaction (RT-qPCR); the enzymatically hydrolyzed corn starch-blocked activated carbon is obtained by mixing corn starch enzymatically hydrolyzed with water, centrifuging, taking the supernatant and shaking it with activated carbon for 4-5 h, drying, and obtaining the enzymatically hydrolyzed corn starch-blocked activated carbon.

[0014] Furthermore, in step (1), the concentration of proteinase K in the proteinase K ultrapure aqueous solution is 3-4 U / mL, preferably 3.4 U / mL. The volume of the proteinase K ultrapure aqueous solution used is 0.5-5 mL / g based on the mass of the digested gland, preferably 1 mL / g.

[0015] Furthermore, in step (1), it is preferable to use a constant temperature oscillation at 37°C and 320 rpm for 60 min, followed by a water bath at 60°C for 15 min.

[0016] Furthermore, in step (2), the volume of TGBE elution buffer used is 10-30 mL / g based on the mass of the digested gland in step (1), preferably 15 mL / g.

[0017] Furthermore, in step (2), the mixture is shaken at 150 rpm for 30 min at room temperature; then it is centrifuged at 4℃ and centrifugal force of 10000×g for 30 min.

[0018] Further, in step (2), the TGBE elution buffer consists of: 0.1 mM Tris-HCl, 0.05 mM glycine, 30 g / L beef extract, diluted to 1 L with deionized water, pH 9.5, dispensed, and autoclaved at 121°C for 15 min.

[0019] Further, in step (3), shake at 26°C and 150 rpm for 30 minutes.

[0020] Furthermore, in step (3), the mass ratio of the enzymatically hydrolyzed corn starch-sealed activated carbon to the digestive gland in step (1) is 0.5-1.5:1, preferably 0.75:1.

[0021] Furthermore, the enzymatically hydrolyzed corn starch-blocked activated carbon in step (3) was prepared as follows:

[0022] ① Pretreatment of activated carbon: Activated carbon is washed with deionized water until the supernatant is clear, and then dried (preferably at 60℃ for 15 hours) to obtain pretreated activated carbon;

[0023] ② Preparation of enzymatically hydrolyzed corn starch: Corn starch is added to Na2HPO4-citric acid buffer at pH 6, and after ultrasonic mixing, α-amylase is added and the mixture is incubated in a water bath at 45-65℃ for 10-20 hours. After cooling to room temperature, the pH is adjusted to 6-7 with NaOH, and the mixture is centrifuged (10000 rpm, 10 min, preferably the precipitate is washed with distilled water and centrifuged 3 times). The mixture is then dried (preferably at 60℃ for 15 hours) to obtain enzymatically hydrolyzed corn starch.

[0024] ③ Enzymatic hydrolysis of starch-blocked activated carbon: After mixing the enzymatically hydrolyzed corn starch from step ② with deionized water, homogenize at high speed, centrifuge (700 rpm for 2 min), take the supernatant (as a blocking agent) and add it to the activated carbon pretreated in step ①. Block at 32-38℃ and 110-160 rpm for 3-6 h, filter, and dry the filter cake (preferably at 37℃ for 4 h) to obtain enzymatically hydrolyzed corn starch-blocked activated carbon.

[0025] Furthermore, in step ①, the activated carbon is coconut shell activated carbon with a particle size of 1.25-2 mm.

[0026] Furthermore, in step ②, ultrasonic mixing is performed at 300-500W for 20-40 minutes, preferably at 400W for 30 minutes. Then, the mixture is in a 55℃ water bath for 15 hours.

[0027] Furthermore, in step ②, the volume of Na2HPO4-citric acid buffer is 1-5 mL / g based on the mass of corn starch, preferably 4 mL / g; the amount of α-amylase is 20-40 μg / 30g based on the mass of corn starch, preferably 31.5 μg / 30g.

[0028] Furthermore, in step ③, the amount of deionized water used is 40-60 mL / g based on the mass of the enzymatically hydrolyzed corn starch, preferably 50 mL / g; the mass ratio of pretreated activated carbon to enzymatically hydrolyzed corn starch is 3-5:1, preferably 4:1. It is preferred to seal the mixture at 37℃ and 150 rpm in a shaker for 4-5 hours.

[0029] Furthermore, in step (3), RNA was extracted using a kit; the RT-qPCR program was as follows: reverse transcription at 42℃ for 30 min; pre-denaturation at 95℃ for 2 min; denaturation at 95℃ for 5 s; annealing and extension at 55℃ for 35 s; repeated 39 times. The total volume of the RT-qPCR reaction system was 50 μL.

[0030] Furthermore, the viruses in the aquatic products include murine norovirus-1 (MNV-1) and Tulane virus (TV).

[0031] Compared with existing technologies, the beneficial effects of this invention are mainly reflected in the following aspects: This invention first uses proteinase K solution to enzymatically hydrolyze the digestive glands of shellfish, then adds TGBE solution for elution, and then adds enzymatically hydrolyzed corn starch-blocked activated carbon to remove RT-qPCR inhibitors from the test samples. The supernatant is then used for nucleic acid extraction, and detection is performed using a real-time PCR instrument. This invention uses enzymatically hydrolyzed corn starch-blocked activated carbon to remove inhibitors that interfere with PCR polymerase activity in shellfish samples. By pretreating shellfish samples with enzymatically hydrolyzed corn starch-blocked activated carbon combined with proteinase K solution and TGBE elution buffer, the detection of shellfish samples can be completed rapidly within 4 hours, which is 10-20 times faster than the national standard method. This invention significantly improves virus recovery rate and detection sensitivity, is suitable for shellfish samples, and features simple operation, short processing time, rapid reliability, and high sensitivity. (iv) Description of the attached drawings

[0032] Figure 1 A flowchart for enzymatic hydrolysis of corn starch-blocked activated carbon.

[0033] Figure 2 Electron micrographs of AC(a) and SCAC(b) in Example 1.

[0034] Figure 3 Virus recovery rates of blocked activated carbon products prepared from different starch enzymatic hydrolysis methods (V) Detailed Implementation

[0035] The following examples are used to explain the technical solutions of the present invention in a non-limiting manner.

[0036] The reagents used in the following examples were purchased from Tiangen Biotech (Beijing) Co., Ltd. and Sangon Biotech (Shanghai) Co., Ltd. Unless otherwise specified, all reagents used were of analytical grade.

[0037] The α-amylase used in this invention has an activity of 2100 U / g. The room temperature is 25-30°C. The proteinase K has an activity of 30 U / mg.

[0038] Example 1: Enzymatic hydrolysis of corn starch blocked activated carbon (SCAC)

[0039] 1. Enzymatic hydrolysis of corn starch with sealed activated carbon

[0040] (1) Enzymatic hydrolysis of corn starch

[0041] Weigh 30g of corn starch into a beaker, add 120mL of pH 6 Na₂HPO₄-citric acid buffer, mix well, place the beaker in an ultrasonic instrument, sonicate at 400W for 30min, then add 4.5mL (31.5μg) of α-amylase, place the beaker in a 55℃ water bath for 15h, remove and allow to cool to room temperature, add NaOH to adjust the pH to 6-7, centrifuge at 10000rpm for 10min, discard the supernatant, add 200mL of deionized water, mix well, and centrifuge again at 10000rpm for 10min. Repeat three times. Dry the precipitate at 60℃ for 15h to obtain 18g of enzymatically hydrolyzed corn starch.

[0042] (2) Preparation of activated carbon

[0043] Coconut shell activated carbon with a diameter of 1.25-2 mm was sieved, washed with deionized water until clear and transparent, and then dried in an oven at 60℃ to obtain pretreated activated carbon. See electron micrograph for details. Figure 2 a.

[0044] (3) Preparation of SCAC

[0045] Weigh 4g of the enzymatically hydrolyzed corn starch prepared in step (1), add 200mL of water, and stir at 10000rpm for 2min to ensure the corn starch is thoroughly mixed. Pour into a 250-mL centrifuge tube, centrifuge at 700rpm for 2min, collect the supernatant and add it to 16g of the pretreated activated carbon prepared in step (2). Shake at 37℃ and 150rpm for 4-5h. Filter, place the filter cake in an oven, and dry at 60℃ for 15h to obtain 18g of enzymatically hydrolyzed corn starch-blocked activated carbon. See electron micrograph for details. Figure 2 b.

[0046] 2. Enzymatic hydrolysis of other starches and sealed activated carbon

[0047] Referring to step 1, corn starch was replaced with potato starch, pea starch, and gluten starch, with all other operations remaining the same. Three types of starch-blocked activated carbon were obtained respectively. Using the method in Example 2 to detect norovirus MNV-1 in clams, corn starch-blocked activated carbon showed the highest virus recovery efficiency, reaching 71.39 ± 5.73%. Figure 3 ).

[0048] Example 2: Detection of Norovirus

[0049] 1. Duran Virus TV

[0050] (1) Inoculation of shellfish samples with virus

[0051] Wearing protective gloves, use a sterile knife to open the oyster, remove the digestive gland and place it on a clean culture dish. Collect 2.0g of digestive gland, homogenize it with a sterile blade, and transfer it to a 10-mL centrifuge tube. Add 10μL of Dulan virus (TV) to achieve an inoculum size of 3.5×10⁻⁶. 5 GC / g, refrigerate overnight at 4°C after inoculation.

[0052] (2) Proteinase K solution enzymatically digests the digestive glands

[0053] Step (1): Add 2 mL of 3.4 U / mL proteinase K-free RNase-free ultrapure water to the refrigerated centrifuge tube and shake at 37°C and 320 rpm for 60 min on a constant temperature shaker. After removing the tube, place it in a water bath at 60°C for 15 min to obtain the enzymatically hydrolyzed digestive gland.

[0054] (3) Elution with TGBE elution buffer

[0055] Transfer the digested glands from the centrifuge tube in step (2) to a 50-mL sterile centrifuge tube, add 30mL of TGBE elution buffer, place the centrifuge tube on a shaker, and shake at 150rpm for 30min at room temperature. Then centrifuge for 30min at a centrifugal force of 10000×g at 4℃, and collect the supernatant. The TGBE elution buffer consists of 0.1mM Tris-HCl, 0.05mM glycine, 30g / L beef extract, and deionized water to a final volume of 1L, with a pH of 9.5. Aliquot the solution and autoclave at 121℃ for 15min.

[0056] (4) Add SCAC processing

[0057] Add 1.5g of the enzymatically hydrolyzed corn starch blocking activated carbon prepared in Example 1 to the supernatant collected in step (3), and shake at 26°C and 150 rpm for 30 min. Then extract RNA using the kit. Use the control without adding the enzymatically hydrolyzed corn starch blocking activated carbon.

[0058] (5) RT-qPCR reaction

[0059] Using the RNA extracted in step (4) as a template, according to the primers and probes in Table 1 and Table 2, prepare a 50 μL RT-qPCR reaction system. Place the prepared PCR solution into a real-time PCR instrument. The detection program of the real-time PCR instrument is as follows: reverse transcription reaction at 42℃ for 30 min; pre-denaturation at 95℃ for 2 min, denaturation at 95℃ for 5 s, annealing at 55℃ for 35 s, and repeat 39 times.

[0060] Table 1. Virus detection primer and probe sequences.

[0061]

[0062]

[0063] Table 2. RT-qPCR reaction system

[0064]

[0065] 2. Norovirus MNV-1

[0066] Under the same conditions as in step 1, replace Dulan virus TV with murine norovirus MNV-1, with an inoculation dose of 7.5 × 10⁻⁶. 6 GC / g, other operations are the same.

[0067] 3. Other shellfish

[0068] Under the same conditions as in step 1, replace oysters with razor clams or clams, and follow the same procedure.

[0069] 4. Result Judgment

[0070] Establish a standard curve of virus concentration versus ct value, calculate the virus concentration in the sample based on the standard curve, and then calculate the virus recovery rate according to formula (1).

[0071] The standard curve for MNV-1 is y = -3.437x + 43.642, R0 2 =0.99.

[0072] The standard curve for TV is: y = -3.297x + 43.998, R0 2 =0.99;

[0073] R(%)=Q1 / Q2×100% Formula (1)

[0074] R represents the recovery rate, Q1 represents the virus recovered from shellfish, and Q2 represents the original amount of virus.

[0075] The experimental results are shown in Table 3. After treatment with SCAC, the recovery rate was significantly higher than that of GB 4789.42—2016, which indicates that SCAC can remove PCR inhibitors.

[0076] As shown in Table 3, norovirus alternatives (MNV-1, TV) were selected to evaluate the detection efficacy of the SCAC method. The assays were performed using 3.5 × 10⁻⁶ cells. 5 MNV-1(GC / g), 7.5×10 5 When TV(GC / g) is inoculated onto oysters, razor clams, and clams, the recovery rate can reach 50%-70%, which is 8-24 times that of the control group GB 4789.42-2016. The results show that the SCAC method can significantly improve the recovery rate of norovirus in shellfish and enhance its sensitivity.

[0077] Table 3. Results of MNV-1 and TV detected by the SCAC method

[0078]

[0079]

[0080] a GC recovery rate / GC initial inoculation amount.

[0081] b Mean ± standard deviation calculated from the standard curve (n = 3 independent experiments).

[0082] Example 3: Detection capability and sensitivity of the SCAC method

[0083] The norovirus MNV-1 inoculation dose in Example 2 was changed to 0.6 × 10⁻⁶. 3 0.6×10 2 0.6×10 1 GC / g, the Dulan virus TV inoculation dose was changed to 1.1×10⁻⁶. 3 1.1×10 2 1.1×10 1 GC / g, other operations are the same as in Example 2, and the results are shown in Table 4.

[0084] Table 4. Schematic diagram of the detection sensitivity results of the SCAC method

[0085]

[0086] Experimental results showed that, in the control group GB 4789.42—2016, a maximum of 10 could be detected in shellfish samples. 3 GC / g, but the detection rate is low and unstable. The experimental group using the SCAC method can stably detect 10 g in shellfish samples. 3 Virus GC / g. Most shellfish samples were at 10.1 It can also be detected in GC / g. Clearly, SCAC treatment increased the detection sensitivity of RT-qPCR by 1-3 log, indicating that the SCAC method can significantly improve the inhibitory activity of RT-qPCR and increase amplification efficiency.

[0087] In summary, this invention utilizes enzymatically hydrolyzed corn starch-blocked activated carbon for the detection of norovirus in shellfish. The enzymatic hydrolysis of corn starch-blocked activated carbon eliminates PCR inhibitors, allowing for highly efficient detection of viruses in shellfish. It is characterized by its ease of operation and high sensitivity.

Claims

1. A method for improving the detection rate of norovirus in aquatic products, characterized in that, The method includes the following steps: (1) Proteinase K digestion An ultrapure aqueous solution of proteinase K was added to the digestive gland of aquatic products, and the mixture was kept at a constant temperature of 30-40 °C and 200-400 rpm for 40-80 min, followed by a water bath at 50-70 °C for 10-20 min to obtain the enzymatically hydrolyzed digestive gland; the aquatic products included shellfish; the ultrapure water was RNase-free ultrapure water. (2) Elution with TGBE buffer solution Add TGBE elution buffer to the digestive gland after enzymatic hydrolysis in step (1), and shake at 100-300 rpm for 20-40 min at room temperature; then centrifuge at 4℃ and centrifugation force of 5000-15000×g for 20-40 min, and collect the supernatant; (3) Enzymatic hydrolysis of corn starch and blocking with activated carbon to remove interference from RT-PCR in the test samples Add the enzymatically hydrolyzed corn starch-blocked activated carbon to the supernatant collected in step (2), shake at 26℃ and 100-300 rpm for 20-40 min, extract RNA, and detect norovirus using RT-qPCR; The enzymatically hydrolyzed corn starch-blocked activated carbon is prepared according to the following method: ① Pretreatment of activated carbon: Activated carbon is washed with deionized water until the supernatant is clear, and then dried to obtain pretreated activated carbon; ② Preparation of enzymatically hydrolyzed corn starch: Corn starch was added to Na2HPO4-citric acid buffer at pH 6, and after ultrasonic mixing, α-amylase was added and the mixture was incubated in a water bath at 45-65℃ for 10-20 hours. After cooling to room temperature, the pH was adjusted to 6-7 with NaOH, centrifuged, and dried to obtain enzymatically hydrolyzed corn starch. ③ Enzymatic hydrolysis of starch-blocked activated carbon: After mixing the enzymatically hydrolyzed corn starch from step ② with deionized water, homogenize at high speed, centrifuge, take the supernatant and add it to the activated carbon pretreated in step ①, block at 32-38℃ and 110-160rpm for 3-6 hours, filter, dry the filter cake to obtain enzymatically hydrolyzed corn starch-blocked activated carbon.

2. The method for improving the detection rate of norovirus in aquatic products as described in claim 1, characterized in that, In step (1), the proteinase K concentration in the proteinase K ultrapure aqueous solution is 3-4 U / mL, and the volume of the proteinase K ultrapure aqueous solution used is 0.5-5 mL / g based on the mass of the digested gland.

3. The method for improving the detection rate of norovirus in aquatic products as described in claim 1, characterized in that, In step (2), the volume of TGBE elution buffer used is 10-30 mL / g based on the mass of the digested gland in step (1).

4. The method for improving the detection rate of norovirus in aquatic products as described in claim 1, characterized in that, The TGBE elution buffer in step (2) consists of: 0.1 mM Tris-HCl, 0.05 mM glycine, 30 g / L beef extract, and deionized water to a final volume of 1 L, with a pH of 9.

5.

5. The method for improving the detection rate of norovirus in aquatic products as described in claim 1, characterized in that, In step (3), the mass ratio of the enzymatically hydrolyzed corn starch-sealed activated carbon to the digestive gland in step (1) is 0.5-1.5:

1.

6. The method for improving the detection rate of norovirus in aquatic products as described in claim 1, characterized in that, In step ①, the activated carbon is coconut shell activated carbon with a particle size of 1.25-2mm.

7. The method for improving the detection rate of norovirus in aquatic products as described in claim 1, characterized in that, In step ②, ultrasonic mixing is performed at 300-500W for 20-40 minutes.

8. The method for improving the detection rate of norovirus in aquatic products as described in claim 1, characterized in that, In step ②, the volume of Na2HPO4-citric acid buffer is 1-5 mL / g based on the mass of corn starch; the amount of α-amylase is 20-40 μg / 30g based on the mass of corn starch.

9. The method for improving the detection rate of norovirus in aquatic products as described in claim 1, characterized in that, In step ③, the amount of deionized water used is 40-60 mL / g based on the mass of enzymatically hydrolyzed corn starch; the mass ratio of pretreated activated carbon to enzymatically hydrolyzed corn starch is 3-5:1.

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