Rapid detection kit for large yellow croaker pyramidal worm based on MIRA technology

Abstract

The invention relates to a large yellow croaker pyramidal worm rapid detection kit based on an MIRA technology, and belongs to the field of aquaculture pathogen biological diagnosis. The rapid detection kit for the large yellow croaker pyramidal worm comprises a nucleic acid releasing agent, a primer probe group for MIRA amplification and a nucleic acid detection test strip for detecting an MIRA amplification product in a matched manner, the nucleic acid releasing agent is used for releasing nucleic acid in a sample to be detected; the primer probe group comprises an upstream primer, a downstream primer and a probe; the nucleic acid test strip is a colloidal gold lateral flow chromatography test strip. The rapid detection kit for the Pseudosciaena crocea pyramidal can rapidly, efficiently and sensitively detect the Pseudosciaena crocea pyramidal, and provides an effective technical means for differential diagnosis of the Pseudosciaena crocea pyramidal.

Description

Technical Field

[0001] The invention belongs to the field of aquaculture pathogen diagnosis, and particularly relates to a large yellow croaker trypanosoma rapid detection kit based on MIRA technology. Background Art

[0002] From September to October 2023, trypanosomiasis was first discovered in farmed large yellow croaker. The infected individuals showed typical symptoms such as loss of appetite, pale gill filaments, abnormal proliferation of body mucus, enlarged and necrotic spleen, compensatory hyperplasia of the kidneys and body surface ulcers. It can infect large yellow croaker at different stages, including fry (less than 100g), medium fish (between 100g and 400g) and adult fish (greater than 400g); during the peak of the epidemic, the daily mortality rate can reach 1%, and the cumulative mortality rate can be close to 30%-40% of the farmed fish population, causing significant economic losses. There is no effective treatment yet, and it mainly relies on the comprehensive prevention and control strategy of "prevention first". Therefore, accurate diagnosis is of paramount importance.

[0003] Trypanosomes are a group of flagellated protozoa that parasitize the blood of fish. They belong to the class Mastochorata, order Kinetoplastida, family Trypanosomatida, and genus Trypanosoma. Both marine and freshwater fish can be parasitized by trypanosomes. Currently, the etiology and biology of trypanosomiasis in large yellow croaker remain largely unexplained, and its prevalence, transmission pathways, and pathogenic mechanisms remain largely unknown. Effective prevention and control measures are also lacking. To mitigate the threat of the disease, aquaculture operators are generally adopting a shortened breeding cycle and phased farming practices to reduce risks. Due to climatic constraints, large yellow croaker farmed in Zhejiang Province must be transported across regions to Fujian and other places for overwintering. However, the frequent cross-regional migration of large yellow croaker has objectively accelerated the spread of trypanosomes. To curb the spread of the disease, governments at all levels are actively promoting the establishment of quarantine systems for the cross-regional transportation of large yellow croaker and for seedling and seedling production. However, this currently faces a challenge: standardized diagnostic criteria and practical detection methods for fish trypanosomiasis are lacking at the national, local, and industry levels. Therefore, the development of accurate and rapid early diagnosis technology has become the core strategy for the prevention and control of yellow croaker trypanosomiasis.

[0004] Currently, the detection of trypanosomes in large yellow croaker mainly relies on microscopic examination and PCR technology, but the above two methods have obvious limitations. Microscopic examination is prone to missed detection and wrong detection, and PCR detection is complicated, time-consuming (about 3 to 4 hours), and relies on professional equipment and technicians. These limitations make it difficult to meet the needs of rapid on-site screening in grassroots farms. In contrast, colloidal gold immunochromatography technology has become an ideal alternative due to its advantages such as simple operation (no instruments required), rapid detection (results within 20 minutes), low cost and suitability for on-site application. The establishment of trypanosome detection technology based on colloidal gold test strips can not only improve the efficiency of disease monitoring and reduce aquaculture risks, but also provide key technical support for the standardization and intelligence of the healthy aquaculture system of large yellow croaker. Summary of the Invention

[0005] The purpose of the present invention is to provide a large yellow croaker trypanosoma detection kit based on MIRA technology.

[0006] The technical solution adopted in the present invention is as follows: A large yellow croaker trypanosoma rapid detection kit based on MIRA technology, the large yellow croaker trypanosoma detection kit comprising a nucleic acid releaser, a primer probe set for MIRA amplification, and a nucleic acid detection test strip for detecting MIRA amplification products; the nucleic acid releaser is used to release nucleic acid in a sample to be detected; the sequence of the primer probe set is: Upstream primer: 5'-TGTTCTTTCTCGAAGTCCCCTTCCCCTGAA-3', Downstream primer: 5'-biotin-ATCCCGCAGAGAAGGATACAAACGAATACC-3', Probe: 5'-FAM-AGATCCAAGCTGCCCAGGATGATTCAGAATTGCACATAGGATAGCA-C3spacer-3'; the nucleic acid detection test strip is a colloidal gold lateral flow chromatography test strip.

[0007] Furthermore, the formula of the above-mentioned nucleic acid releaser is: tris(hydroxymethyl)aminomethane hydrochloride with a final concentration of 100mM, sodium chloride with a final concentration of 100mM, sodium dodecylsulfonate with a final concentration of 1wt%, sodium α-olefinsulfonate with a final concentration of 3wt%, sodium lauroyl glutamate with a final concentration of 1.2wt%, betaine with a final concentration of 1wt%, and the rest is water.

[0008] Furthermore, the colloidal gold lateral flow chromatography test strip comprises a sample pad, a conjugation pad, a nitrocellulose membrane, an absorption pad and a base plate, wherein the sample pad, conjugation pad, nitrocellulose membrane and absorption pad are sequentially pasted on the base plate; the conjugation pad contains colloidal gold-labeled streptavidin; and the nitrocellulose membrane is provided with an anti-carboxyfluorescein FAM antibody detection line and a biotinylated antibody quality control line.

[0009] The use of the above-mentioned large yellow croaker trypanosoma rapid detection kit in detecting large yellow croaker trypanosoma is not for the purpose of disease diagnosis and / or treatment.

[0010] Furthermore, the above application includes the following steps: (1) releasing nucleic acid in the sample to be detected by a nucleic acid releasing agent; (2) using the product obtained in step (1) as a template, using upstream primers, downstream primers and probes, and performing a MIRA amplification reaction in a MIRA dry powder reaction tube; (3) Using a nucleic acid detection test strip to detect the MIRA amplification product obtained in step (2).

[0011] Furthermore, the method for using the nucleic acid releaser is as follows: take a sample to be tested, add the nucleic acid releaser, grind it thoroughly, shake and mix it, let it stand at room temperature, centrifuge and collect the supernatant to obtain a product.

[0012] Furthermore, the system of the MIRA amplification reaction is as follows: template 10 μL, E Buffer 29.4 μL, B Buffer 2.5 μL, upstream primer 2 μL, downstream primer 2 μL, probe 0.6 μL, and water is added to 50 μL.

[0013] Furthermore, the conditions of the MIRA amplification reaction are: 38-42° C. for 15-20 min.

[0014] Furthermore, the method for using the above-mentioned nucleic acid detection test strip is as follows: drop the MIRA amplification product onto the sample pad, observe the results after 5 minutes, and make a judgment; if there is no band on the biotinylated antibody quality control line, the test strip is invalid; if a band appears on the biotinylated antibody quality control line and no band appears on the anti-carboxyfluorescein FAM antibody detection line, the sample to be tested is negative; if a band appears on the biotinylated antibody quality control line and a band appears on the anti-carboxyfluorescein FAM antibody detection line, the sample to be tested is positive.

[0015] The significant advantages of the present invention are: The application provides a large yellow croaker pyramida rapid detection kit based on MIRA technology, which comprises a nucleic acid releasing agent, a primer probe set for MIRA amplification, and a nucleic acid detection test strip matched with the MIRA amplification product; the nucleic acid releasing agent is used for releasing nucleic acid in a sample to be detected; the primer probe set comprises an upstream primer, a downstream primer and a probe; and the nucleic acid detection test strip is a colloidal gold lateral flow chromatographic test strip; the colloidal gold lateral flow chromatographic test strip comprises a sample pad, a conjugate pad, a nitrocellulose membrane, an absorbent pad and a base plate, and the sample pad, the conjugate pad, the nitrocellulose membrane and the absorbent pad are sequentially pasted on the base plate; the conjugate pad contains colloidal gold-labeled streptavidin; and the nitrocellulose membrane is provided with an anti-carboxyfluorescein FAM antibody detection line and a biotinylated antibody quality control line. The kit has extremely high detection sensitivity, can achieve 0.01 fg / ul, has fast detection speed (results can be obtained within 20 minutes), can accurately detect during the latent period and low pathogen amount infection period of pyramida, can lay a solid foundation for early prevention and control, has important significance for the prevention and control of pyramida disease of large yellow croaker, does not need professional equipment, is suitable for on-site screening in a breeding farm, provides key technical support for real-time monitoring and green prevention and control of pyramida disease of large yellow croaker, and greatly reduces the detection threshold of pyramida disease. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 : result interpretation schematic diagram.

[0017] Figure 2 : primer probe set screening result.

[0018] Figure 3 : sensitivity experiment result.

[0019] Figure 4 : specificity experiment result. DETAILED DESCRIPTION

[0020] In order to make the content of the application more convenient to understand, the technical solutions of the application are further described below in combination with specific embodiments, but the application is not limited to this.

[0021] Embodiment 1: Composition of the large yellow croaker pyramida detection kit A large yellow croaker pyramida detection kit based on MIRA technology, which comprises: a box body, the box body is a cuboid; a nucleic acid releasing agent, a MIRA freeze-dried enzyme powder reaction tube, an EBuffer, a BBuffer, a primer probe set for MIRA amplification, a nucleic acid detection test strip matched with the MIRA amplification product, a negative control sample, a positive control sample and a kit instruction manual arranged in the box body.

[0022] The nucleic acid releaser is used to release nucleic acids in a sample to be detected. The formula is: tris(hydroxymethyl)aminomethane hydrochloride with a final concentration of 100 mM, sodium chloride with a final concentration of 100 mM, sodium dodecylsulfonate with a final concentration of 1 wt%, sodium α-olefinsulfonate with a final concentration of 3 wt%, sodium lauroyl glutamate with a final concentration of 1.2 wt%, betaine with a final concentration of 1 wt%, and the remainder is ultrapure water.

[0023] The MIRA freeze-dried enzyme powder reaction tube was purchased from Anpu Future (Changzhou) Biotechnology Co., Ltd. with the product number A0203D103; E Buffe was purchased from Anpu Future (Changzhou) Biotechnology Co., Ltd. with the product number A0203D104; BBuffer was purchased from Anpu Future (Changzhou) Biotechnology Co., Ltd. with the product number A0203D102.

[0024] The primer-probe set consists of an upstream primer, a downstream primer and a probe.

[0025] The nucleic acid detection test strip is a colloidal gold lateral flow chromatography test strip with product number WLFS8204 purchased from Anpu Future (Changzhou) Biotechnology Co., Ltd.; the colloidal gold lateral flow chromatography test strip includes a sample pad, a conjugation pad, a nitrocellulose membrane, an absorption pad and a base plate, and the sample pad, conjugation pad, nitrocellulose membrane and absorption pad are sequentially pasted on the base plate; the conjugation pad contains colloidal gold-labeled streptavidin; the nitrocellulose membrane is provided with an anti-carboxyfluorescein FAM antibody detection line and a biotinylated antibody quality control line.

[0026] The negative control sample is sterile double-distilled water, and the positive control sample is the recombinant vector pMD19-T-18SrRNA; the recombinant vector pMD19-T-18SrRNA is obtained by inserting the large yellow croaker trypanosoma 18S rRNA gene sequence shown in SEQ ID NO.1 into the pMD19-T vector.

[0027] The method of using the kit is as follows: (1) Release nucleic acid from the sample to be tested: 10-50 mg of the sample to be tested was placed in a centrifuge tube. 500 μL of nucleic acid release agent was added to the centrifuge tube to completely immerse the sample. The sample was then ground 10-15 times with a grinding rod, shaken and mixed, and allowed to stand at room temperature for 5 min. The sample was then centrifuged at 8000 rpm for 1 min, and the supernatant was collected. (2) MIRA amplification reaction: Using the supernatant collected in step (1) as a template, upstream primers, downstream primers and probes, MIRA amplification reaction was carried out in a MIRA freeze-dried enzyme powder reaction tube; the MIRA amplification reaction system was: template 10 μL, EBuffer 29.4 μL, B Buffer 2.5 μL, upstream primer 2 μL, downstream primer 2 μL, probe 0.6 μL, and sterilized double-distilled water was used to make up to 50 μL; the MIRA amplification reaction conditions were: 39°C for 20 min.

[0028] (3) MIRA amplification product detection: After diluting the MIRA amplification product 10 to 20 times, take 50 μL and drop it onto the sample pad of the nucleic acid detection test strip. Observe the results after 5 minutes and make a judgment. If there is no band on the biotinylated antibody quality control line, the test strip is invalid. If there is a band on the biotinylated antibody quality control line and no band on the anti-carboxyfluorescein FAM antibody detection line, the sample to be tested is negative. If there is a band on the biotinylated antibody quality control line and a band on the anti-carboxyfluorescein FAM antibody detection line, the sample to be tested is positive. Figure 1 ).

[0029] Example 2: Screening of primer probe sets The 18S rRNA gene was selected as the target gene for detecting trypanosomes in large yellow croaker. 18S rRNA gene sequences of fish trypanosomes and large yellow croaker trypanosomes were compared and analyzed in GenBank. Primers and probes were designed using Primer Premier 6 and BeaconDesigner software. The 18S rRNA gene of large yellow croaker trypanosomes was divided into three regions (n1, n2, and n3), and upstream primers, downstream primers, and probes were designed for each of these three regions; one upstream primer, three downstream primers, and one probe were designed for each region.

[0030] N1 area: Upstream primer Try-n1F1: 5'-TGAAACTTAAAGAAATTGACGGAATGGCAC-3', Downstream primer Try-n1R1: 5'-biotin-ACTCAATCTGTCAATCCTCACCCTGTCCGG-3', downstream primer Try-n1R2: 5'-biotin-TCAGGGGAAGGGGACTTCGAGAAAGAACACTCAATCTG-3', Downstream primer Try-n1R3: 5'-biotin-ACCAAAAGCGGCCATGCACCACCATTCAGG-3', probe Try-nP1: 5'-FAM-CACAAGACGTGGAGCGTGCGGTTTAATTTGACTCAACACGGGGAAC-C3spacer-3'; n2 region: Upstream primer Try-n2F1: 5'-TGTTCTTTCTCGAAGTCCCCTTCCCCTGAA-3', Downstream primer Try-n2R1: 5'-biotin-ATCCCGCAGAGAAGGATACAAACGAATACC-3', downstream primer Try-n2R2: 5'-biotin-AAATCTCACCTTGTGCAAAGTCAAGGAATC-3', downstream primer Try-n2R3: 5'-biotin-CATTGAGGAGCATCACAGACCTGCTGTTGC-3', probe Try-nP2: 5'-FAM-AGATCCAAGCTGCCCAGGATGATTCAGAATTGCACATAGGATAGCA-C3spacer-3'; n3 region: Upstream primer Try-n3F1: 5'-CTGCTTCCAGGAATGAAGGAGGGTAGTTCG-3', Downstream primer Try-n3R1: 5'-biotin-ATCCTTGAAGAATGCCTTCGCTGTAGTTCG-3', downstream primer Try-n3R2: 5'-biotin-CACTTTGGTTCTTGATTGAGGAAGGTATCC-3', downstream primer Try-n3R3: 5'-biotin-ACTACAATGGTCTCTAATCATCTTCGATCC-3', probe Try-nP3: 5'-FAM-AGAACGTACTGGTGCGTCAGAGGTGAAATTCTTAGACCGCACCAAG-C3spacer-3'; Using sterile double-distilled water as a template, MIRA amplification reaction was carried out in a MIRA freeze-dried enzyme powder reaction tube according to the method of Example 1, and the obtained MIRA amplification product was detected using a nucleic acid detection test strip. The results showed that the primer probe set Try-n2F1 / Try-n2R1 / Try-nP2 and the primer probe set Try-n2F1 / Try-n2R3 / Try-nP2 were able to develop color normally, with a band appearing on the biotinylated antibody quality control line and no band appearing on the anti-carboxyfluorescein FAM antibody detection line; other primer probe sets such as Try-n1F1 / Try-n1R1 / Try-nP1 developed color abnormally, with a band appearing on the biotinylated antibody quality control line and a band appearing on the anti-carboxyfluorescein FAM antibody detection line ( Figure 2 Subsequent experiments were performed using primer probe sets Try-n2F1 / Try-n2R1 / Try-nP2 and primer probe set Try-n2F1 / Try-n2R3 / Try-nP2.

[0031] Primer probe set Color development results Try-n1F1 / Try-n1R1 / Try-nP1 abnormal Try-n1F1 / Try-n1R2 / Try-nP1 abnormal Try-n1F1 / Try-n1R3 / Try-nP1 abnormal Try-n2F1 / Try-n2R1 / Try-nP2 normal Try-n2F1 / Try-n2R2 / Try-nP2 abnormal Try-n2F1 / Try-n2R3 / Try-nP2 normal Try-n3F1 / Try-n3R1 / Try-nP3 abnormal Try-n3F1 / Try-n3R2 / Try-nP3 abnormal Try-n3F1 / Try-n3R3 / Try-nP3 abnormal Example 3: Sensitivity experiment The recombinant vector pMD19-T-18S rRNA was diluted to 0.01-1 fg / μL with sterile ultrapure water as a template. The MIRA amplification reaction was carried out in a MIRA freeze-dried enzyme powder reaction tube according to the method of Example 1, and the MIRA amplification product was detected using a nucleic acid detection test strip; the negative control sample (NC) was sterile double-distilled water. The results showed that the primer probe group Try-n2F1 / Try-n2R1 / Try-nP2 and the primer probe group Try-n2F1 / Try-n2R3 / Try-nP2 were able to develop color normally, the biotinylated antibody quality control line showed a band, and the anti-carboxyfluorescein FAM antibody detection line had a faint band when the recombinant vector pMD19-T-18S rRNA concentration was 0.01 fg / μL, indicating that the detection system can detect the pathogen when the template concentration is 0.01 fg / μL ( Figure 3 ). The primer probe set Try-n2F1 / Try-n2R1 / Try-nP2 was used for subsequent experiments.

[0032] Example 4: Specificity Experiment For clinical samples containing Shield Ciliate, Cryptocaryon irritans, Benedenia or Trypanosoma spp., nucleic acid release agents were used to release nucleic acids in the samples according to the method of Example 1. MIRA amplification reaction was carried out in MIRA freeze-dried enzyme powder reaction tubes, and the obtained MIRA amplification products were detected using nucleic acid detection test strips; the negative control sample (NC) was sterile double-distilled water. The results showed that the primer probe group Try-n2F1 / Try-n2R1 / Try-nP2 only showed a positive reaction to Trypanosoma spp., with bands appearing on the biotinylated antibody quality control line and bands appearing on the anti-carboxyfluorescein FAM antibody detection line ( Figure 4 ).

Claims

1. A rapid detection kit for trypanosoma spp. based on MIRA technology, characterized in that: The large yellow croaker trypanosoma detection kit includes a nucleic acid releaser, a primer probe set for MIRA amplification, and a nucleic acid detection test strip for detecting MIRA amplification products; the nucleic acid releaser is used to release nucleic acid in the sample to be detected; the sequence of the primer probe set is: Upstream primer: 5'-TGTTCTTTCTCGAAGTCCCCTTCCCCTGAA-3', Downstream primer: 5'-biotin-ATCCCGCAGAGAAGGATACAAACGAATACC-3', Probe: 5'-FAM-AGATCCAAGCTGCCCAGGATGATTCAGAATTGCACATAGGATAGCA-C3spacer-3'; the nucleic acid detection test strip is a colloidal gold lateral flow chromatography test strip.

2. The large yellow croaker trypanosoma rapid detection kit according to claim 1, wherein: The formula of the nucleic acid releaser is: tris(hydroxymethyl)aminomethane hydrochloride with a final concentration of 100mM, sodium chloride with a final concentration of 100mM, sodium dodecyl sulfonate with a final concentration of 1wt%, sodium α-olefin sulfonate with a final concentration of 3wt%, sodium lauroyl glutamate with a final concentration of 1.2wt%, betaine with a final concentration of 1wt%, and the rest is water.

3. The large yellow croaker trypanosoma rapid detection kit according to claim 1, wherein: The colloidal gold lateral flow chromatography test strip comprises a sample pad, a conjugation pad, a nitrocellulose membrane, an absorption pad and a base plate, wherein the sample pad, conjugation pad, nitrocellulose membrane and absorption pad are sequentially adhered to the base plate; the conjugation pad contains colloidal gold-labeled streptavidin; and the nitrocellulose membrane is provided with an anti-carboxyfluorescein FAM antibody detection line and a biotinylated antibody quality control line.

4. The use of the large yellow croaker trypanosoma rapid detection kit according to any one of claims 1 to 3 in detecting large yellow croaker trypanosoma, characterized in that: The application is not intended for disease diagnosis and / or treatment.

5. The use according to claim 4, characterized in that: The following steps are involved: (1) Release the nucleic acid in the sample to be tested through a nucleic acid release agent; (2) Using the product obtained in step (1) as a template, an upstream primer, a downstream primer, and a probe, a MIRA amplification reaction is performed in a MIRA dry powder reaction tube; (3) Using a nucleic acid test strip to detect the MIRA amplification product obtained in step (2).

6. The use according to claim 5, characterized in that: The method for using the nucleic acid releaser is as follows: taking a sample to be tested, adding the nucleic acid releaser, grinding the sample thoroughly, shaking and mixing, standing at room temperature, and centrifuging to collect the supernatant to obtain a product.

7. The use according to claim 5, characterized in that: The MIRA amplification reaction system is as follows: template 10µL, EBuffer 29.4µL, B Buffer 2.5µL, upstream primer 2µL, downstream primer 2µL, probe 0.6µL, and water is added to 50µL.

8. The use according to claim 5, characterized in that: The conditions of the MIRA amplification reaction are: 38-42° C. for 15-20 min.

9. The use according to claim 5, characterized in that: The method of using the nucleic acid detection test strip is as follows: drop the MIRA amplification product onto the sample pad, observe the results after 5 minutes, and make a judgment; if there is no band on the biotinylated antibody quality control line, the test strip is invalid; if a band appears on the biotinylated antibody quality control line and no band appears on the anti-carboxyfluorescein FAM antibody detection line, the sample to be tested is negative; If a band appears on the biotinylated antibody control line and a band appears on the anti-carboxyfluorescein FAM antibody test line, the sample to be tested is positive.

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