Rapid detection kit for large yellow croaker pyramidal worm

Abstract

The invention relates to a rapid detection kit for large yellow croaker pyramidal worm, and belongs to the field of pathogen biological diagnosis in aquaculture. The kit comprises a nucleic acid releasing agent and further comprises a primer and a probe which are used for detecting the large yellow croaker pyramidal worm. The kit comprises a nucleic acid releaser; the formula of the nucleic acid releasing agent comprises tris (hydroxymethyl) aminomethane hydrochloride with the final concentration of 100 mM, sodium chloride with the final concentration of 100 mM, sodium dodecyl sulfate with the final concentration of 1 wt%, sodium alpha-olefin sulfonate with the final concentration of 3 wt%, sodium lauroyl glutamate with the final concentration of 1.2 wt%, betaine with the final concentration of 1 wt% and water as a solvent. The sequences of the primer and the probe for detecting the Pseudosciaena crocea pyramidal are 5 '-TATGGGCAATTCTGAATCCT-3', 5 '-TCTTTTCTGATCCCTGAAT-3' and 5 '-FAM-GTCGGTGGAGTGATTTGTTTG-3'. The kit disclosed by the invention is simple to operate, provides a tool for diagnosis, prevalence monitoring, prevention and control of the Pseudosciaena crocea pyramidal, and has a very high application value.

Description

Technical Field

[0001] The present invention belongs to the field of diagnosis of pathogenic organisms in aquaculture, and particularly relates to a rapid detection kit for Trypanosoma crocea of large yellow croaker. Background Art

[0002] Trypanosoma crocea of large yellow croaker taxonomically belongs to the class Mastigophora, order Kinetoplastida (or order Kinetoplastida), family Trypanosomatidae, and genus Trypanosoma. The size of the parasite is about 2-5μm×15-20μm, slender and curved, resembling a willow leaf or wavy. Its nucleus is elongated and located in the center of the body. There is a long free flagellum at the front end, and an oval kinetoplast at the rear end. The pathogenic effect of Trypanosoma on fish is mainly reflected in the destruction of blood and tissues. They parasitize in the fish blood, consume the nutrients of the host and destroy red blood cells, causing anemia, weight loss and fish body weakness. In addition, Trypanosoma may also invade other tissues and organs of fish, causing more extensive pathological damage. At the peak of the disease, the daily mortality rate can reach 1%, and the cumulative mortality rate approaches 60% after 70 days of infection. Trypanosoma crocea disease of large yellow croaker is a newly emerging disease, and the key issues such as its origin, epidemic law, transmission route and pathogenic mechanism are still unclear, and it is even more impossible to propose effective prevention and control measures. Therefore, establishing a TaqMan probe fluorescence quantitative PCR detection technology for Trypanosoma crocea of large yellow croaker is not only helpful for timely diagnosis, monitoring and prevention of Trypanosoma crocea disease of large yellow croaker, reducing economic losses, but also necessary for strengthening scientific aquaculture management and establishing a seedling quarantine system in the future.

[0003] TaqMan probe real-time fluorescence quantitative PCR is a most commonly used method for detecting bacteria and viruses, and has been applied to the detection of various bacterial and viral fish diseases, including Viral hemorrhagic septicemia virus (VHSV), grass carp reovirus (GCRV), Edwardsiella tarda, Vibrio alginolyticus, Vibrio parahaemolyticus, and Aeromonas hydrophila.

[0004] At present, the detection of Trypanosoma mainly adopts detection methods such as morphology, physiological and biochemical levels, and biotechnology such as polymerase chain reaction (PCR). However, some of these methods are time-consuming and cumbersome to operate, while some have low accuracy and sensitivity. The TaqMan probe real-time fluorescence quantitative PCR technology skillfully utilizes the advantages of high-efficiency DNA amplification of PCR technology, high-specific hybridization of TaqMan probe and DNA template, and the sensitivity and quantitative analysis of spectral technology, overcomes some deficiencies of conventional PCR qualitative detection, greatly improves the accuracy, sensitivity and specificity of detection, and is widely used in microbial detection and pathogen diagnosis. However, there is no report on the application of TaqMan probe real-time fluorescence quantitative PCR in the detection of Trypanosoma crocea. Summary of the Invention

[0005] The purpose of the present invention is to provide a rapid detection kit for Trypanosoma crocea.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions: A rapid detection kit for Trypanosoma crocea, the kit includes a nucleic acid releasing agent; the formula of the nucleic acid releasing agent is: tris(hydroxymethyl)aminomethane hydrochloride with a final concentration of 100 mM, sodium chloride with a final concentration of 100 mM, sodium dodecyl sulfate with a final concentration of 1 wt%, α-olefin sulfonate 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 solvent is water.

[0007] Furthermore, the above kit also includes primers and probes for detecting Trypanosoma crocea; the sequences of the primers and probes for detecting Trypanosoma crocea are as follows: Try-qPCR-2F2: 5’-TATGGGCAATTCTGAATCCT-3’, Try-qPCR-2R2: 5’-TTCTTTCTCGATCCCCTGAAT-3’, Try-qPCR-P2: 5’-FAM-GTCGGTGGAGTGATTTGTTTG-BHQ1-3’.

[0008] Furthermore, the above kit also includes 2×AceQ qPCR Probe Master Mix, 50×ROX Reference Dye 1, positive control samples and negative control samples.

[0009] Furthermore, the PCR amplification reaction system of the above kit is as follows: 0.4 μL of 10 μM Try-qPCR-2F2, 0.4 μL of 10 μM Try-qPCR-2R2, 0.4 μL of 10 μM Try-qPCR-P2, 10 μL of 2×AceQ qPCR Probe MasterMix, 0.4 μL of 50×ROX Reference Dye 1, 2 μL of DNA template, and make up to 50 μL with water.

[0010] Furthermore, the PCR amplification reaction conditions of the above kit are as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 10 s, annealing / extension at 55°C for 30 s, for 40 cycles.

[0011] Furthermore, the above positive control sample is a recombinant vector containing the 18S rRNA gene sequence of Trypanosoma crocea; the above negative control sample is sterilized ultrapure water.

[0012] Furthermore, the above DNA template is obtained by extraction using a nucleic acid releasing agent.

[0013] Furthermore, the result determination basis of the above kit is as follows: If, under the conditions that the Trypanosoma crocea negative control sample has no Ct value and no amplification curve, and the Ct value of the Trypanosoma crocea positive control sample < 35 and has an "S"-shaped amplification curve, when the Ct value of the test sample ≤ 35 and has an "S"-shaped amplification curve, then the test sample is infected with Trypanosoma crocea; If, under the conditions that the Trypanosoma crocea negative control sample has no Ct value and no amplification curve, and the Ct value of the Trypanosoma crocea positive control sample < 35 and has an "S"-shaped amplification curve, when the Ct value of the test sample > 35 or has no amplification curve, then the test sample is not infected with Trypanosoma crocea.

[0014] The remarkable advantages of the present invention are as follows: The present invention provides a specific, simple and rapid detection kit for Trypanosoma crocea. The present invention designs a pair of primers using the gene conserved sequence of Trypanosoma crocea, optimizes the PCR reaction conditions and reaction system, establishes a real-time fluorescence quantitative PCR detection method for detecting Trypanosoma crocea, and on this basis, assembles a simple, rapid, sensitive and specific detection kit for Trypanosoma crocea, with complete reagents and simple operation. This kit can be used for accurate and quantitative tracking and monitoring of aquatic economic animals in mariculture and organisms in the culture water body, so as to effectively prevent and control Trypanosoma crocea disease caused by Trypanosoma crocea in a timely manner, has high practical value, and provides a detection tool for the future epidemic detection and prevention and control of this disease. Description of the Drawings

[0015] Figure 1: Specific amplification of primers Try-qPCR-2F2 and Try-qPCR-2R2. Lane 1: Trypanosoma croceum of Larimichthys crocea; Lane 2: Aspidogaster conchicola; Lane 3: Cryptocaryon irritans; Lane 4: Benedenia sp.; Lane 5: Negative control sample (sterile double-distilled water).

[0016] Figure 2 : Fluorescence quantitative PCR amplification curve of positive control sample using primers Try-qPCR-2F2 and Try-qPCR-2R2 and probe Try-qPCR-P2.

[0017] Figure 3 : Standard curve of fluorescence quantitative PCR amplification of positive control sample using primers Try-qPCR-2F2 and Try-qPCR-2R2 and probe Try-qPCR-P2.

[0018] Figure 4 : Cross-sectional view of the rapid detection kit for Trypanosoma croceum of Larimichthys crocea (i.e., schematic diagram of the composition and position inside the box). 1: Box body; 2: Rapid nucleic acid release agent for aquatic animal samples (DNA type); 3: Fluorescence quantitative PCR reaction solution; 4: Positive control sample; 5: Negative control sample (sterile double-distilled water); 6: Operation manual.

[0019] Figure 5 : Detection results of clinical samples. Specific implementation mode

[0020] To make the content of the present invention easier to understand, the technical solutions of the present invention are further described below in conjunction with specific implementation modes, but the present invention is not limited thereto.

[0021] Example 1: Design of primers and probes Currently, there are few publicly available genomic sequences of Trypanosoma in fish. The present invention selects the 18S rRNA gene as the detection target gene for Trypanosoma croceum of Larimichthys crocea. The 18S rRNA gene sequences of Trypanosoma in fish and Trypanosoma croceum of Larimichthys crocea existing in GenBank are compared and analyzed. Primers and probes are designed using primer premier 6 and Beacon Designer software. The designed primers and probes are synthesized by Fuzhou Qingke Biotechnology Co., Ltd., and the sequences are as follows: Try-qPCR-2F2: 5’-TATGGGCAATTCTGAATCCT-3’, Try-qPCR-2R2: 5’-TTCTTTCTCGATCCCCTGAAT-3’, Try-qPCR-P2: 5’-FAM-GTCGGTGGAGTGATTTGTTTG-BHQ1-3’.

[0022] Using the DNA of positive disease sample drawers of common parasitic pathogens in large yellow croaker such as *Cryptocaryon irritans*, *Aspidogaster conchicola*, and *Benedenia seriolae* and the DNA of positive disease sample drawers of *Trypanosoma croaker* as templates respectively, ordinary PCR was carried out to verify the specificity of the primers. The reaction program of ordinary PCR was: 94°C for 5 min; 94°C for 30 s, 60°C for 35 s, 72°C for 20 s, for 35 cycles; 72°C for 10 min. The amplification reaction system of ordinary PCR was set as: After the PCR reaction, the obtained PCR products were observed by agarose gel nucleic acid electrophoresis. If there was a band at 121 bp in the PCR amplification product, the PCR product was sequenced for confirmation. The results showed that no bands were amplified for the 3 common non-target parasitic pathogens, and a band of 121 bp was amplified for *Trypanosoma croaker* ( Figure 1 , SEQ ID NO.1). The PCR specific amplification product of *Trypanosoma croaker* was analyzed by sequencing and was consistent with the 18S rRNA gene sequence of *Trypanosoma croaker*. It was shown that the primers Try-qPCR-2F2 and Try-qPCR-2R2 had excellent specificity and could be used for the specific detection of *Trypanosoma croaker*.

[0023] Example 2: Preparation of positive control samples and construction of standard curves Using the DNA of positive samples of *Trypanosoma croaker* as a template, the full-length sequence of the 18S rRNA gene of *Trypanosoma croaker* (SEQ ID NO.2) was amplified by PCR. The PCR products were electrophoresed on an agarose gel, and then the target bands of the PCR products were cut and recovered using a GelExtraction Kit (OMEGA, USA) and ligated to the pMD19-T vector (TaKaRa, Dalian, China). It was transformed into Trans-T1 Phage Resistant chemically competent cells (TransGen Biotech, China). Positive clones were screened and verified by sequencing. The constructed recombinant plasmid was extracted, and the quality and concentration of the recombinant plasmid were detected by a spectrophotometer. The copy number of the target gene was calculated according to the following formula, and then it was diluted with sterile ultrapure water to a concentration of 1×10 8 copies / μL as a positive control sample.

[0024] Dilute the positive control sample as a template and perform an amplification reaction on a fluorescence PCR instrument. At the same time, use sterilized ultrapure water as a template-free blank control. The fluorescence quantitative PCR amplification reaction system includes: 0.4 μL of 10 μM Try-qPCR-2F2, 0.4 μL of 10 μM Try-qPCR-2R2, 0.4 μL of 10 μM Try-qPCR-P2, 10 μL of 2×AceQ qPCR Probe Master Mix, 0.4 μL of 50×ROX Reference Dye 1, 2 μL of DNA template, and add water to make up to 50 μL. The fluorescence quantitative PCR amplification reaction conditions include: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 10 s, annealing and extension at 55°C for 30 s, for 40 cycles; measure the fluorescence value at 55°C. After each cycle reaction ends, collect and record the fluorescence signal, and automatically generate a standard curve by computer software. The detection limit is 2.1 copies / μL.

[0025] Example 3: Preparation of a rapid detection kit for Trypanosoma crocea in large yellow croaker 1. Prepare the kit box; 2. Detection reagents: A rapid nucleic acid release agent for aquatic animal samples (DNA type), with the formula: tris(hydroxymethyl)aminomethane hydrochloride with a final concentration of 100 mM, sodium chloride with a final concentration of 100 mM, sodium dodecyl sulfate with a final concentration of 1 wt%, α-olefin sulfonate 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 solvent is water.

[0026] Fluorescence quantitative PCR reaction solution, with the formula: 0.4 μL of 10 μM Try-qPCR-2F2, 0.4 μL of 10 μM Try-qPCR-2R2, 0.4 μL of 10 μM Try-qPCR-P2, 10 μL of 2×AceQ qPCR Probe Master Mix, 0.4 μL of 50×ROX Reference Dye 1; Fluorescence quantitative PCR amplification reaction conditions: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 10 s, annealing and extension at 55°C for 30 s, for 40 cycles; measure the fluorescence value at 55°C.

[0027] Negative control sample: sterilized double-distilled water; Positive control sample: Dilute the recombinant plasmid constructed in Example 2 to a concentration of 1.0×10 8 copies / μL.

[0028] 3 Place the operation manual (6), nucleic acid rapid release agent for aquatic animal samples (DNA type) (2), fluorescence quantitative PCR reaction solution (3), positive control sample (4), and negative control sample (5) into the box body (1) (see Figure 4 ), and the rapid detection kit for Trypanosoma cerebralis of large yellow croaker is obtained.

[0029] Example 4: Application of the rapid detection kit for Trypanosoma cerebralis of large yellow croaker 1 Template DNA preparation ① Take about 50 mg of the tissue sample to be tested (about the size of a mung bean) and put it into a 1.5 mL centrifuge tube.

[0030] ② Add 500 μL of sterile double-distilled water to the centrifuge tube containing the sample tissue and grind it thoroughly to obtain a homogenate of the tissue sample to be tested for standby.

[0031] ③ Take a clean centrifuge tube and add 500 μL of nucleic acid rapid release agent for aquatic animal samples (DNA type). Pipette 20 μL of the prepared homogenate of the tissue sample to be tested and add it thereto, and shake well.

[0032] ④ Lyse at room temperature for 5 min, centrifuge instantaneously for 1 min, take 2 μL of the supernatant as template DNA, and use the kit in Example 3 for amplification and detection.

[0033] 2 Fluorescence quantitative PCR reaction Add the fluorescence quantitative PCR reaction solution and template DNA to the PCR tube, and finally add water to make up to 50 μL. After mixing and instantaneous centrifugation, perform fluorescence quantitative PCR reaction in a PCR amplifier. Positive control samples, negative control samples, and blank controls are set up in the reaction. The PCR reaction parameters are set as follows: pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 10 s, annealing and extension at 55 °C for 30 s, for a total of 40 cycles; measure the fluorescence value at 55 °C. Positive control samples and negative control samples do not require the template DNA preparation process. Directly take 2 μL for the subsequent fluorescence quantitative PCR reaction, and the reaction conditions are the same as those for the previous sample detection.

[0034] 3 Result determination After the PCR reaction is completed, determine whether it is infected with Trypanosoma cerebralis of large yellow croaker by comparing with the fluorescence quantitative PCR amplification curve of the standard product. If under the conditions that the negative control sample of Trypanosoma cerebralis of large yellow croaker has no Ct value and no amplification curve, the Ct value of the positive control sample of Trypanosoma cerebralis of large yellow croaker < 35 and there is an "S"-shaped amplification curve, when the Ct value of the sample to be tested ≤ 35 and there is an "S"-shaped amplification curve, the sample to be tested is infected with Trypanosoma cerebralis of large yellow croaker; if under the conditions that the negative control sample of Trypanosoma cerebralis of large yellow croaker has no Ct value and no amplification curve, the Ct value of the positive control sample of Trypanosoma cerebralis of large yellow croaker < 35 and there is an "S"-shaped amplification curve, when the Ct value of the sample to be tested > 35 or there is no amplification curve, the sample to be tested is not infected with Trypanosoma cerebralis of large yellow croaker.

[0035] Under the condition that the Ct value of the negative control sample of Trypanosoma crocea in large yellow croaker is not detected and there is no amplification curve, and the Ct value of the positive control sample of Trypanosoma crocea in large yellow croaker is less than 35 and there is an "S" type amplification curve, the clinical sample test results are as follows in the table and Figure 5 As shown, the template DNA of the SP and GI groups was prepared using the aquatic animal sample nucleic acid rapid release agent (DNA type) in Example 3, and the template DNA of the SP-1 and GI-1 groups was prepared using the Tiangen marine tissue nucleic acid extraction kit. Under the same reaction system and reaction conditions, the difference in their CT values is not significant, and the test results are basically the same. The samples in the SP, GI, SP-1, and GI-1 groups were all infected with Trypanosoma crocea.

[0036]

Claims

1. A rapid detection kit for Trypanosoma crocea, characterized in that: The kit includes a nucleic acid releasing agent; the formulation of the nucleic acid releasing agent is: Tris (hydroxymethyl) aminomethane hydrochloride with a final concentration of 100 mM, sodium chloride with a final concentration of 100 mM, sodium dodecyl sulfate with a final concentration of 1 wt%, alpha-olefin sulfonate 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 solvent is water.

2. The kit according to claim 1, wherein: The kit further includes primers and probes for detecting *Trypanoplasma crocea*; the primer and probe sequences for detecting *Trypanoplasma crocea* are shown as follows: Try-qPCR-2F2: 5’-TATGGGCAATTCTGAATCCT-3’, Try-qPCR-2R2: 5’-TTCTTTCTCGATCCCCTGAAT-3’, Try-qPCR-P2: 5’-FAM-GTCGGTGGAGTGATTTGTTTG-BHQ1-3’.

3. The kit according to claim 2, wherein: The kit further includes 2× AceQ qPCR Probe Master Mix, 50× ROX Reference Dye 1, a positive control sample and a negative control sample.

4. The kit according to claim 3, wherein: The PCR amplification reaction system of the kit is: 0.4 µL of 10 µM Try-qPCR-2F2, 0.4 µL of 10 µM Try-qPCR-2R2, 0.4 µL of 10 µM Try-qPCR-P2, 10 µL of 2× AceQ qPCR Probe Master Mix, 0.4 µL of 50× ROX Reference Dye 1, 2 µL of DNA template, and add water to make up to 50 µL.

5. The kit according to claim 3, wherein: The PCR amplification reaction conditions of the kit are: pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 10 s, annealing / extension at 55 °C for 30 s, for 40 cycles.

6. The kit according to claim 3, wherein: The positive control sample is a recombinant vector containing the 18S rRNA gene sequence of *Trypanoplasma crocea*; the negative control sample is sterilized ultrapure water.

7. The kit according to claim 4, wherein: The DNA template is obtained by extraction using the nucleic acid releasing agent.

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