Quarantine method for anisakis

Abstract

The invention provides an anisakis quarantine method, which belongs to the technical field of parasite quarantine, and comprises the following steps: S1, sample treatment: placing fish tissues in a stationary liquid for fixation; s2, extracting total DNA: after soaking in a TE buffer solution, extracting genome DNA of the polypide, and storing at-15 to-25 DEG C; s3, designing primers, namely designing and synthesizing an upstream primer Anisakis1 and a downstream primer Anisakis2; s4, carrying out PCR (Polymerase Chain Reaction) amplification, namely carrying out PCR amplification on the polypide genome DNA by using the primer; s5, result judgment: performing result judgment on the target amplified fragment through electrophoresis detection; s6, insect species identification: carrying out real-time PCR detection on the positive sample; and S7, judging a result: judging according to a Ct value and an amplification curve. The anisakis quarantine method provided by the invention not only can determine whether the anisakis belongs to anisakis, but also can determine which anisakis specifically carried.

Description

Technical Field

[0001] This invention belongs to the field of parasite quarantine technology, specifically relating to a quarantine method for Anisakis nematodes. Background Technology

[0002] Anisakis belongs to the family Anisakidae in the order Ascaridia and mainly includes four genera of nematodes: *Anisakis*, *Pseudogeton*, *Pseudocephalocystis*, and *Gymnocytos*. Anisakis commonly parasitizes aquatic products consumed by humans; therefore, quarantine of these products for the Anisakis pathogen is essential to ensure their safety.

[0003] In addition, among the aforementioned pathogenic parasites, *Anisakis simplex* is the core pathogen (accounting for over 90% of cases). Its larvae invade the gastrointestinal mucosa, causing acute abdominal pain, and the secreted Anis 1 antigen can cause anaphylactic shock, requiring emergency endoscopic intervention. Although *Pseudoterranova decipiens* has weaker pathogenicity, its contamination rate in cod and halibut is as high as 30%, making it a mandatory food safety indicator monitored under EU regulations (EC / 2074 / 2005). *Anisakis physeteris* exhibits significant co-parasitism with *Anisakis simplex*, and its cross-antigen Anip 1 can exacerbate allergic reactions; missed detection will lead to treatment failure for refractory allergies.

[0004] Currently, the identification of Anisakis larvae mainly relies on morphological observation. However, due to the large number of Anisakis species and the small size of the larvae that parasitize humans and fish, there are many species with indistinct morphological differences. Therefore, it is difficult to identify the larvae based on traditional anatomical morphological observation. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides a quarantine method for Anisakis nematodes, enabling precise species identification and quantitative analysis of co-infections. The establishment of this detection system provides irreplaceable technical support for precision clinical treatment, food contamination assessment, and allergen control.

[0006] This invention provides a PCR quarantine method for Anisakis simplex, comprising the following steps: S1. Sample processing: The fish tissue is fixed in a fixative solution; S2. Extract total DNA: After soaking in TE buffer, extract the genomic DNA of the insect and store it at -15 to -25°C. S3. Primer design: Design and synthesize upstream primer Anisakis1 and downstream primer Anisakis2; the gene sequence of upstream primer Anisakis1 is shown in SEQ ID NO:1; the gene sequence of downstream primer Anisakis2 is shown in SEQ ID NO:2. S4. PCR amplification: The genomic DNA of the insect was amplified by PCR using upstream primer Anisakis1 and downstream primer Anisakis2. S5. Result determination: The target amplified fragment is detected by electrophoresis to determine the result.

[0007] Furthermore, in step S1, the fixative is a 75% ethanol solution; the fish tissue is the abdominal cavity, stomach, mesentery, liver, gonads, and muscle of the fish; alcohol fixation refers to using ethanol to quickly kill and fix the live worms, preventing them from continuing to move, contract, or autolyze, so that they can be observed, identified, or preserved under a microscope in the future. After fixation, the preservation solution should be replaced in time to ensure that the worm structure is intact and the morphology is natural.

[0008] Furthermore, in step S1, the fixative is a 75% ethanol solution; the fish tissue is the abdominal cavity, stomach, mesentery, liver, gonads, and muscle of a fish.

[0009] Furthermore, in step S2, the method for extracting total DNA is as follows: Ethanol removal treatment: Take the Anisakis sample preserved in 75% ethanol, put it in a 1.5 mL centrifuge tube, add 1 mL TE buffer (pH 8.0), soak overnight at room temperature, remove the TE buffer, and completely discard the residual liquid with a pipette; The preparation method for TE buffer is as follows: S1. Prepare 100×TE stock solution: Take 1 L of commercially available 1 M Tris-HCl (pH 8.0) and 20 mL of commercially available 0.5 M EDTA (pH 8.0), mix well, and obtain 100×TE stock solution.

[0010] S2. Prepare 1×TE working solution: Take 10 mL of the above 100×TE stock solution, add 990 mL of deionized water, mix well, and you will get 1×TE working solution ((10 mM Tris-HCl, 0.1 mM EDTA), i.e. TE buffer).

[0011] Lysis and digestion: Add 500 μL of lysis buffer I, incubate at 55°C with shaking for 3 h, after incubation, transfer to 4°C and let stand for 10 minutes, then cool to room temperature; Nucleic acid extraction: Add 500 μL extraction buffer II, vortex to mix, and then oscillate horizontally for 10 min (200 rpm). Centrifuge at 11,000 ×g for 10 min and collect the supernatant. Repeat the extraction twice and combine the supernatants from the three extractions. Ethanol precipitation: Add 2 times the volume of anhydrous ethanol (0℃), precipitate at -20℃ for 1 h; centrifuge at 5,000 ×g for 10 min, wash the precipitate twice with 70% ethanol, and vacuum dry for 5 min. Dissolution and storage: Dissolve the precipitate in 40 μL TE buffer (pH 8.0) and store at -20°C.

[0012] Furthermore, the lysis buffer I is composed of: 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1% SDS, 1 mM EDTA, and 1 mg / mL proteinase K.

[0013] Furthermore, the extraction buffer II is: 25:24:1 phenol:chloroform:isoamyl alcohol (volume ratio).

[0014] Furthermore, the TE buffer is: 10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA.

[0015] Furthermore, in step S2, the method for extracting total DNA is as follows: extraction is performed using a viral DNA / RNA extraction kit (model: Ex-DNA / RNA Virus 4.0).

[0016] Furthermore, in step S5, the 761 bp target band is determined by detecting it using 1.0% agarose gel electrophoresis.

[0017] On the other hand, the present invention provides a fluorescent PCR method for insect species identification, as follows: S1. Sample processing: The fish tissue is fixed in a fixative solution; S2. Extract total DNA: After soaking in TE buffer, extract the genomic DNA of the insect and store it at -15 to -25°C. S3. Primer design: Design and synthesize upstream primer Anisakis1 and downstream primer Anisakis2; the gene sequence of upstream primer Anisakis1 is shown in SEQ ID NO:1; the gene sequence of downstream primer Anisakis2 is shown in SEQ ID NO:2. S4. PCR amplification: The genomic DNA of the insect was amplified by PCR using upstream primer Anisakis1 and downstream primer Anisakis2. S5. Result Determination: The target amplified fragment is detected by electrophoresis to determine the result. S6. Insect species identification: Real-time PCR detection is performed on positive samples, using the following primers: Piperella salina and / or simple Anisakis detection combination: Upstream primer AF0: SEQ ID NO:3; Downstream primer AR1: SEQ ID NO:4; Probe AT1: SEQ ID NO:5; Detection combination for Anisakis typica: Upstream primer AF0: SEQ ID NO:3; Downstream primer AR2: SEQ ID NO:7; Probe AT2: SEQ ID NO:6; S7. Judgment result: When the Ct value of the tested sample ≤ 38 and a specific amplification curve appears, it is judged as positive; when the tested sample has no Ct value or Ct value ≥ 40, it is judged as negative; when 38 < Ct value < 40 for the tested sample and a specific amplification curve appears, it is judged as suspected positive.

[0018] If the Ct value of the tested sample < 38 and a specific amplification curve appears, it is judged as positive for the nucleic acid of Anisakis pegreffii and / or Anisakis simplex or Anisakis typica; when there is no Ct value or Ct value ≥ 40, it is judged as negative for the nucleic acid of Anisakis; when 38 < Ct value < 40 and a specific amplification curve appears, it is judged as suspected.

[0019] For suspected samples, double the template amount (2 μL DNA template) and conduct 1 re - inspection with 3 replicates; if the Ct values of 2 replicates < 40 and a specific amplification curve appears, it is judged as positive for the nucleic acid, otherwise it is judged as negative for the nucleic acid; Establishment conditions for the fluorescence PCR test: The Ct value of the positive control < 30 and a specific amplification curve appears, the negative control has no Ct value or the Ct value of the negative control > 40 and no specific test amplification curve, and the test result is valid; otherwise, it should be carried out again.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The Anisakis quarantine method provided by this invention can not only determine whether a sample carries Anisakis, but also which specific type of Anisakis it carries. Specifically, by designing and synthesizing upstream primer Anisakis1 and downstream primer Anisakis2 for PCR amplification of the target gene fragment, combined with agarose gel electrophoresis, it is possible to quickly determine whether a sample is infected with Anisakis. At the same time, specific primers and probes are designed for the specific gene sequences of different Anisakis species: *Anisakis pipetterum* and / or *Anisakis simplex* detection combination: upstream primer AF0: SEQ ID NO:3, downstream primer AR1: SEQ ID NO:4, probe AT1: SEQ ID NO:5; typical *Anisakis* detection combination: upstream primer AF0: SEQ ID NO:3, downstream primer AR2: SEQ ID NO:7, probe AT2: SEQ ID NO:6; using probe AT1 (for *Anisakis pipetterum* and / or *Anisakis simplex*) and AT2 (for typical *Anisakis*), the species is directly identified through specific fluorescent signals, avoiding misjudgment based on morphological identification.

[0021] 2. The Anisakis quarantine method provided by this invention has the advantages of being pollution-free, quantitative, and time-saving, enabling highly sensitive, rapid, and automated screening.

[0022] 3. This invention specifies a real-time fluorescent PCR detection method for three species of Anisakis diseases, which is applicable to the diagnosis of Anisakis diseases and can also be used for qualitative detection of Anisakis disease host risk. It can be used as a standard designated method for port inspection and quarantine agencies to quarantine Anisakis diseases.

[0023] 4. Traditional Anisakis quarantine methods rely on morphological observation, but Anisakis larvae are morphologically similar and have cross-antigen reactions, which can easily lead to misjudgment. This invention uses specific primers and fluorescent PCR probes to target conserved gene sequences of the worm species, achieving accurate species differentiation and avoiding interference from cross-reactions. Attached Figure Description

[0024] Figure 1 This is the result of a PCR detection specificity test; Figure 2 This refers to the results of a PCR detection sensitivity test. Figure 3 This is the result of a real-time fluorescent PCR specificity assay; Figure 4 This is a real-time fluorescence PCR sensitivity assay. Detailed Implementation

[0025] The technical solutions of the present invention will be described below with reference to the embodiments of the present invention. The described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0026] Example 1 PCR reaction Table 1 PCR reaction system

[0027] Step 1: Preparation of PCR reaction system Add 10 μl MASTER (2×) (Amplicon), 1 μL 200 μmol / L upstream primer Anisakis1 (SEQ ID NO:1), 1 μL 200 μmol / L downstream primer Anisakis2 (SEQ ID NO:2), 3 μL DNA template, and 5 μL lddH2O water. Mix thoroughly and centrifuge at 4000 r / min for 30 s. Step 2: PCR amplification The first stage is pre-denaturation, with a reaction temperature of 95℃ and a reaction time of 10 min; the second stage is a cyclic reaction, with a denaturation temperature of 95℃ and a reaction time of 10 s, an annealing temperature of 55℃ and a reaction time of 10 s, and an extension temperature of 72℃ and a reaction time of 1 s, for a total of 40 cycles; the third stage is final extension, with a reaction temperature of 40℃ and a reaction time of 30 s; the third stage is storage, maintained at 4℃ until removal. Step 3: Electrophoretic verification of amplification products The amplification reaction products were subjected to agarose gel electrophoresis and observed using a gel imaging system. Positive result: A clear band appears at the 761 bp position; Negative result: No target band found.

[0028] Example 2 Real-time fluorescent PCR reaction This embodiment uses the detection of "typical Anisakis" as an example to demonstrate the usage of the typical Anisakis detection combination (SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:6). If detecting "Anisakis pirae / Anisakis simplex", the corresponding combination should be used instead (upstream primer AF0: SEQ ID NO:3, downstream primer AR1: SEQ ID NO:4, probe AT1: SEQ ID NO:5).

[0029] Table 2 Real-time fluorescence PCR reaction system (taking typical Anisakis nematode detection as an example)

[0030] Step 1: Preparation of PCR reaction system Add 12.5 μL of 2X Taq Master PCR Mix; add 1.5 μL of 10 μmol / L upstream primer AF0 (SEQ ID NO:3); add 1.5 μL of 10 μmol / L downstream primer AR2 (SEQ ID NO:7); add 0.4 μL of 10 μmol / L probe AT2 (SEQ ID NO:6); add 2.0 μL of DNA template (concentration: 0.5 - 100 ng / μL); add 4.6 μL of ddH2O and mix to obtain the reaction solution. Put it into a vortex mixer to mix well, and then perform instantaneous centrifugation; Step 2: Real-time fluorescence PCR amplification program The first stage is pre-denaturation, the reaction temperature is 50°C and the reaction duration is 2 min; the second stage is initial denaturation, the reaction temperature is 95°C and the reaction duration is 10 min; the third stage is amplification cycle, the denaturation temperature is 95°C and the reaction duration is 15 s, the annealing temperature is 62°C and the duration is 1 min (fluorescence signal is collected here), for a total of 40 cycles; the extension reaction temperature is 72°C and the duration is 30 s.

[0031] Step 3: Result determination Conditions for a valid fluorescence PCR test: The Ct value of the positive control < 30 and a specific amplification curve appears, the negative control has no Ct value or the Ct value of the negative control > 40 and no specific test amplification curve, and the test results are valid; otherwise, it should be repeated.

[0032] Step 4: Determination of fluorescence PCR results If the Ct value of the tested sample < 38 and a specific amplification curve appears, it is judged as nucleic acid positive for Anisakis pegreffii and / or Anisakis simplex or Anisakis typica; when there is no Ct value or Ct value ≥ 40, it is judged as nucleic acid negative for Anisakis; when 38 < Ct value < 40 and a specific amplification curve appears, it is judged as suspected. For suspected samples, double the template amount (2 μL of DNA template) and perform 1 recheck, with 3 replicates; if 2 replicates have a Ct value < 40 and a specific amplification curve appears, it is judged as nucleic acid positive, otherwise it is judged as nucleic acid negative.

[0033] Example 3 Specificity test of PCR detection Use the DNA of Hysterothylacium aduncum; Pseudoterranova decipiens as the specific test control, use the previously tested Anisakis DNA as the positive sample, and set ddH2O as the negative control. Perform PCR reaction under the established optimal reaction conditions and reaction system, and analyze the results and take pictures after 1.0% agarose gel electrophoresis, see Figure 1 , from Figure 1 As can be seen, the negative control showed no specific amplification, ruling out reagent contamination or non-specific amplification. Nematode DNA from *Anisakis* and *Pseudogemonia* showed no specific amplification, proving that the primers are highly specific for the *Anisakis* genus. This verifies the effectiveness of the experimental system, demonstrating that the PCR method provided by this invention can accurately distinguish *Anisakis* from other closely related parasites without cross-reaction, meeting the specificity requirements for species identification.

[0034] Example 4: Sensitivity test for PCR detection After determining the concentration of Anisakis DNA using a nucleic acid and protein analyzer, the concentration was quantified to 0.1 ng / μL, and then treated with DEPC water for 10... -1 10 -2 10 -3 10 -4 10 -5 Diluted 10 times to obtain 10 1 10 2 10 3 10 4 10 5 10 6 Perform PCR detection under optimal reaction conditions and system, and observe the results. See Figure 2 The detection limit of PCR is 10 copies / μL, indicating that the conventional PCR method established in this invention has high sensitivity.

[0035] Example 5: Real-time fluorescence PCR detection specificity assay Under the optimized fluorescent PCR reaction system and conditions, using *Nematocystis uteri* and *Nematocystis pseudogemonicus* as specificity controls, fluorescent PCR was performed to test the specificity of the established real-time quantitative PCR method. The experimental results were recorded. (See attached table). Figure 3 The DNA of *C. uterus*, *C. pseudogeli*, and *Anisakis* was detected using the established fluorescent PCR method. The results showed that only *Anisakis* exhibited a strong fluorescent signal, while the other samples were negative, indicating that the qPCR detection method established in this invention has good specificity.

[0036] Example 6 Sensitivity test for real-time fluorescence PCR detection The extracted DNA from *Anisakis p. p.*, *Anisakis typical*, and *Anisakis simplex* was analyzed for concentration using a nucleic acid and protein analyzer, and quantified to a concentration of 0.1 ng / μL. The *Anisakis* DNA concentration was also analyzed using a nucleic acid and protein analyzer, and quantified to a concentration of 0.1 ng / μL. DEPC water was used for 10... -1 10 -2 10 -3 10 -4 Diluted 10 times to obtain 10 1 102 10 3 10 4 10 5 The results are shown Figure 4 The detection limit of fluorescent PCR was 10 copies / μL, indicating that the fluorescent PCR method established in this invention has high detection sensitivity.

Claims

1. A PCR quarantine method for Heterakis sp., characterized by, The method comprises the following steps: S1, sample processing: placing fish tissues in a fixing solution for fixation; S2, extracting total DNA: after soaking in a TE buffer, extracting worm genomic DNA and storing at -15 to -25℃; S3, primer design: designing and synthesizing an upstream primer Anisakis1 and a downstream primer Anisakis2; the gene sequence of the upstream primer Anisakis1 is shown as SEQ ID NO: 1; the gene sequence of the downstream primer Anisakis2 is shown as SEQ ID NO: 2; S4, PCR amplification: using the upstream primer Anisakis1 and the downstream primer Anisakis2 to perform PCR amplification on worm genomic DNA; S5, result determination: performing result determination by electrophoresis detection of a target amplified fragment.

2. A PCR quarantine method for Heterakis sp. according to claim 1, characterized in that, In the step S1, the fixing solution is a 75% ethanol solution; and the fish tissues are fish abdominal cavity, stomach, mesentery, liver, gonad and muscle.

3. The method of PCR quarantine of Heterakis sp. according to claim 1, characterized in that, In the step S2, the method for extracting total DNA is as follows: (1) de-ethanol treatment: soaking the Anisakis simplex sample stored in 75% ethanol in a TE buffer overnight to remove residual liquid; (2) lysis digestion: adding lysis buffer I, oscillating and incubating at 50-60℃ for 2.5-3.5 hours, and centrifuging after cooling; (3) nucleic acid extraction: adding extraction buffer II, oscillating and mixing, and then centrifuging to take supernatant, repeating twice and combining the supernatants; (4) ethanol precipitation: adding 2 times the volume of anhydrous ethanol to precipitate DNA, and then centrifuging, washing and drying; (5) dissolving and storing: dissolving the DNA precipitate in a TE buffer and storing at -20℃.

4. The method of PCR quarantine of Heterakis sp. according to claim 3, characterized in that, The lysis buffer I is a mixture of 50 mM Tris-HCl, 100 mM NaCl, 1% SDS, 1 mM EDTA and 1 mg / mL protease K.

5. The method of PCR quarantine of Heterakis sp. according to claim 3, characterized in that, The extraction buffer II is a mixture of phenol, chloroform and isoamyl alcohol in a volume ratio of 25:24:

1.

6. The method of PCR quarantine of Heterakis sp. according to claim 3, characterized in that, The TE buffer is a mixture of 10 mM Tris-HCl and 0.1 mM EDTA.

7. The method of PCR quarantine of Heterakis sp. according to claim 1, characterized in that, In the step S2, the method for extracting total DNA is as follows: using a viral DNA / RNA extraction kit to extract.

8. The method of PCR quarantine for Heterakis sp. according to claim 1, characterized in that, In the step S5, the result is determined by detecting a 761 bp target band through 1.0% agarose gel electrophoresis.

9. A PCR quarantine method for Heterakis sp. according to any one of claims 1 to 8, characterized in that, The PCR quarantine method further comprises: S6, worm species identification: performing real-time PCR detection on a positive sample, and the PCR detection uses the following primers: Anisakis pegreffii and / or Anisakis simplex detection combination: upstream primer AF0: SEQ ID NO: 3; downstream primer AR1: SEQ ID NO: 4; probe AT1: SEQ ID NO: 5; typical Anisakis detection combination: upstream primer AF0: SEQ ID NO: 3; downstream primer AR2: SEQ ID NO: 7; probe AT2: SEQ ID NO: 6; S7, determination result: when the Ct value of the sample is ≤38 and the specific amplification curve appears, it is determined as positive; when the sample has no Ct value or the Ct value is ≥40, it is determined as negative; when the sample has 38 < Ct value < 40 and the specific amplification curve appears, it is determined as suspected positive.

Images