SYBR Green I real-time fluorescent quantitative PCR (polymerase chain reaction) detection primer for trypanosoma and kit of SYBR Green I real-time fluorescent quantitative PCR detection primer

Abstract

The invention discloses SYBR Green I real-time fluorescent quantitative PCR (polymerase chain reaction) detection primers and a kit for large yellow croaker pyramidal worm, and belongs to the technical field of biotechnology and diagnostic detection. A pair of specific primers designed by the invention comprises an upstream primer as shown in SEQ ID NO. 1 and a downstream primer as shown in SEQ ID NO. 2. By adopting the kit disclosed by the invention, the rapid quantitative detection of the large yellow croaker pyramidal worm can be realized, the detection specificity is strong, and the sensitivity is high. At present, a research report of a real-time fluorescent quantitative PCR detection method for quantitatively detecting the Pseudosciaena crocea pyramidal based on SYBR Green I does not exist, a basis can be provided for diagnosis of Pseudosciaena crocea pyramidal infection, an important role is played in guarantee of development of Pseudosciaena crocea breeding industry, and the method has a good application prospect.

Description

Technical Field

[0001] The invention relates to the fields of biotechnology, diagnosis and detection technology and identification technology, and in particular to a SYBR Green I real-time fluorescence quantitative PCR detection primer for trypanosomes and a kit thereof. Background Art

[0002] Trypanosomiasis in yellow croaker was first discovered in cage-farmed yellow croaker in Ningde in 2023, and quickly spread to the entire main breeding area of ​​yellow croaker in 2024, becoming one of the most important diseases of yellow croaker. The pathogen is trypanosoma of the genus Trypanosoma. After infecting yellow croaker, it causes ulcers on the surface of the fish, and the mortality rate of infected fish is as high as 40%-70%, causing huge economic losses. Therefore, it is urgent to establish an accurate, rapid and sensitive detection method to monitor its epidemic dynamics and ensure the healthy development of yellow croaker farming industry.

[0003] At present, the traditional detection method for trypanosoma croaker mainly involves making blood smears and conducting optical microscopic examination. The sensitivity and detection rate of this method are low, and it is necessary to manually conduct detailed microscopic observation of the samples, which makes it difficult to achieve early screening of large quantities of samples. To this end, the present invention proposes a SYBR Green I real-time fluorescence quantitative PCR detection primer for trypanosoma croaker and a kit thereof, so as to solve the above-mentioned problems. Summary of the invention

[0004] (1) Technical issues to be solved

[0005] The purpose of the present invention is to provide a SYBR Green I real-time fluorescence quantitative PCR detection primer for trypanosomes;

[0006] Another object of the present invention is to provide a SYBR Green I real-time fluorescence quantitative PCR detection kit for trypanosomes;

[0007] Another object of the present invention is to provide a SYBRGreenⅠ real-time fluorescence quantitative PCR detection method for trypanosomes using the above-mentioned PCR detection primers;

[0008] Another object of the present invention is to provide the use of the above-mentioned PCR detection kit in detecting fish trypanosomiasis.

[0009] The invention can be used for highly sensitive and specific molecular detection of trypanosoma parasites on large yellow croaker, and can also be used for early diagnosis, rapid identification, pathogen molecular epidemiological investigation and early warning of large yellow croaker trypanosoma disease.

[0010] (2) Technical solution

[0011] In order to solve the above technical problems, the first aspect of the present invention provides:

[0012] The invention discloses a SYBR Green I real-time fluorescence quantitative PCR detection primer for trypanosomes. The PCR detection primer comprises a nucleotide sequence of an upstream primer as shown in SEQ ID NO.1 and a nucleotide sequence of a downstream primer as shown in SEQ ID NO.2.

[0013] Different from the prior art, the above technical solution provides PCR specific primers to perform real-time fluorescence quantitative amplification detection of 18S rRNA of trypanosomes by SYBR GreenⅠ method, which is used for the rapid identification of trypanosomiasis in fish, especially trypanosomiasis in large yellow croaker.

[0014] Furthermore, the PCR amplification reaction conditions were: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 5 s, and annealing at 62°C for 30 s, for a total of 40 cycles.

[0015] The second aspect of the present invention provides:

[0016] A SYBR Green I real-time fluorescence quantitative PCR detection kit for trypanosomes, wherein the PCR detection kit comprises the PCR detection primers described in the first aspect of the present application.

[0017] Furthermore, the amplification reaction conditions of the PCR detection kit are: pre-denaturation at 95°C for 30s; denaturation at 95°C for 5s, annealing at 62°C for 30s, for a total of 40 cycles.

[0018] Furthermore, the amplification reaction system of the PCR detection kit is 10 μL of TB Green Premix Ex Taq II (Tli RNaseH Plus), 0.4 μL of upstream primer, 0.4 μL of downstream primer, 2 μL of template, and sterile deionized water is added to a final volume of 20 μL.

[0019] Furthermore, the concentrations of the upstream primer and the downstream primer are both 10 μmol / L.

[0020] Furthermore, the PCR detection kit is used for colony PCR.

[0021] The third aspect of the present invention provides:

[0022] A SYBR Green I real-time fluorescence quantitative PCR detection method for trypanosomes using the PCR detection primers described in the first aspect of the present application, characterized in that it comprises the following steps:

[0023] 1) Extracting genomic DNA from the sample to be tested;

[0024] 2) Real-time fluorescence quantitative PCR reaction;

[0025] The real-time fluorescence quantitative PCR amplification reaction system is: TB Green Premix ExTaq II (TliRNaseH Plus) 10 μL, upstream primer 0.4 μL, downstream primer 0.4 μL, standard plasmid or test template 2 μL, the rest is sterile deionized water, and the total volume of the reaction system is 20 μL; the real-time fluorescence quantitative PCR amplification reaction conditions are: 95°C pre-denaturation for 30 s; 95°C denaturation for 5 s, 62°C annealing for 30 s, a total of 40 cycles;

[0026] 3) Result determination:

[0027] If the nucleic acid concentration of the sample to be tested is higher than the detection limit of 1.0×10 1 When the test result is 0.05 copies / μL, it is judged as positive for Trypanosoma macrocarpa, otherwise it is negative.

[0028] A fourth aspect of the present invention provides:

[0029] The second aspect of the present application provides for the use of the PCR detection kit in detecting fish trypanosomiasis.

[0030] Furthermore, the fish includes large yellow croaker.

[0031] (3) Beneficial effects

[0032] The beneficial effects of the present invention are as follows: the present invention establishes for the first time SYBR GreenⅠ real-time fluorescence quantitative PCR detection primers and a kit for specific detection of trypanosomiasis in large yellow croaker, solving the problem of the lack of SYBR GreenⅠ real-time fluorescence quantitative PCR detection methods for trypanosomiasis in large yellow croaker in the prior art. By adopting the kit of the present invention and using the specific primers, rapid and batch quantitative detection of trypanosomiasis samples in large yellow croaker can be achieved according to the method, with strong detection specificity and high sensitivity. Currently, there are no research reports on the real-time fluorescence quantitative PCR detection method based on SYBR GreenⅠ for quantitative detection of large yellow croaker cones. The present invention provides a basis for the diagnosis of large yellow croaker cone infection, plays an important role in ensuring the development of large yellow croaker breeding industry, has higher reliability, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying drawings are only used to illustrate the principles, implementation methods, applications, features and effects of the specific implementation methods of the present application and other related contents, and cannot be considered as limiting the present application. The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0034] Figure 1 This is the amplification kinetic curve of SYBR GreenⅠ real-time fluorescence quantitative PCR method for Trypanosoma croaker.

[0035] Figure 2This is the melting curve of SYBR GreenⅠ real-time fluorescence quantitative PCR method for Trypanosoma croaker.

[0036] Figure 3 This is the standard curve of SYBR GreenⅠ real-time fluorescence quantitative PCR for Trypanosoma croaker. DETAILED DESCRIPTION

[0037] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0038] The test methods used in the following examples are all conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified; the equipment used are all conventional experimental equipment unless otherwise specified, and the solutions involved are all aqueous solutions unless otherwise specified.

[0039] Trypanosoma jj2024 strain of yellow croaker, swollen cell virus, Vibrio alginolyticus, Vibrio harveyi, Pseudomonas proteus, Nocardia, Photobacterium mermaidii, Cryptocaryon irritans, Shield fibrosis, Kudao worm, and sterile deionized water (ddH2O) were all preserved by the Aquatic Pathogens and Disease Control Laboratory, Institute of Biotechnology, Fujian Academy of Agricultural Sciences. The 10 yellow croaker tissue disease samples were clinical samples collected from marine aquaculture cages in different sea areas of Ningde in 2024.

[0040] Example 1

[0041] 1. Primer design

[0042] The large yellow croaker trypanosoma detection primers described in this embodiment are based on the multiple sequence alignment of the trypanosoma gene sequences (OR934688.1, OR934686.1, OL963935.1, OL963934.1, AJ620549.1) registered in the GenBank database. According to these 18SrRNA V4 variable regions, primers were designed using the primer design software Oligo7.0. The sequences are as follows:

[0043] Upstream primer: 5'-CTGTTGCTGTTAAAGGGTTCGTAG-3' (shown in SEQ ID NO.1)

[0044] Downstream primer: 5'-CGTGTTTCTTGTTTCTGAATGTGT-3' (shown in SEQ ID NO. 2).

[0045] The expected amplified target fragment size of the primers is 127 bp. The above primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0046] 2. Extraction of Trypanosoma genomic DNA from large yellow croaker

[0047] The genomic DNA of the large yellow croaker trypanosoma JJ2024 strain was extracted according to the instructions of the FastPureBlood / Cell / Tissue / Bacteria DNAIsolation Mini Kit (purchased from Nanjing Novozyme Biotechnology Co., Ltd.) and kept at -20°C for later use.

[0048] 3. Construction of positive standard plasmid

[0049] The positive PCR product of 127 bp amplified from the genomic DNA of Trypanosoma crocea JJ2024 strain was connected to the pMD19-T vector using the upstream primer (shown in SEQ ID NO.1) and the downstream primer (shown in SEQ ID NO.2), and then transformed into Escherichia coli DH5α by heat stress. The positive cloned bacteria were screened out, and the positive recombinant plasmid was extracted after culturing overnight in LB broth containing 100 μg / mL ampicillin, and confirmed by sequencing. The concentration of the positive plasmid was determined by NanoDrop microspectrophotometer to be 279.76 ng / μL, which was converted into a copy number of 9.67×10 10 Copy number / μL, stored at -20℃.

[0050] 4. Optimal reaction conditions for SYBR GreenⅠ real-time fluorescence quantitative PCR

[0051] According to the instructions of the TB Green Premix Ex Taq II (Tli RNaseH Plus) kit, 20 μL of real-time fluorescence quantitative PCR reaction system was prepared, and the reaction conditions were optimized at different annealing temperatures (52, 54, 56, 58, 60, 62 and 64°C) and primer concentrations (1.25, 2.5, 5.0, 10 and 20 μmol / L).

[0052] After optimization, the best reaction system (20 μL) was as follows: 10 μL of TB Green Premix Ex Taq II (Tli RNaseHPlus), 0.4 μL of upstream / downstream primers (10 μmol / L each), 2 μL of template, and sterile deionized water was added to a final volume of 20 μL. The best reaction conditions were: 95°C pre-denaturation for 30 s; 95°C for 5 s, 62°C for 30 s, and 40 cycles.

[0053] Example 2 Establishment of SYBR Green I real-time fluorescence quantitative PCR standard curve and its sensitivity

[0054] The positive standard plasmid was serially diluted 10-fold to 1×10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 copies / μL, as the standard template, amplified under the optimized conditions, and obtained the amplification kinetic curve (such as Figure 1 ), dissolution curve (such as Figure 2 ) and standard curve (such as Figure 3 ).from Figure 1 It can be seen that the minimum detection limit of the established real-time fluorescence quantitative PCR method is 10 copies / μL. Figure 2 ) is a single peak at 84.5℃±0.2℃, indicating that there is no nonspecific amplification and primer dimer, and the primer has good specificity. The obtained fluorescence quantitative standard curve ( Figure 3 ) are evenly spaced, the standard curve Slope is -3.335, R 2 The value is 0.999, and the amplification efficiency is 99.474%. This indicates that the standard curve is 1×10 1～ 1×10 8 There is a good linear relationship between the copies / μL range and the Ct value.

[0055] Example 3 Specificity Detection

[0056] The optimized conditions were used to detect the JJ2024 strain of Trypanosoma japonicum and other common pathogens of yellow croaker: swollen cell virus, Vibrio alginolyticus, Vibrio harveyi, Pseudomonas proteus, Nocardia, Photobacterium mermaidii, Cryptocaryon irritans, Shield fibrosis, and Kudau. The results showed that only the JJ2024 strain of Trypanosoma japonicum was positive, while other pathogen samples and negative controls were negative, indicating that the established SYBR GreenⅠ real-time fluorescence quantitative PCR detection method has strong specificity.

[0057] Example 4 Repeatability and Reproducibility Evaluation

[0058] Three replicate tubes were set for the same positive standard, and the SYBR GreenⅠ real-time fluorescence quantitative PCR was used to evaluate its repeatability and calculate the intra-group variation coefficient; the positive standard was stored in a -20℃ refrigerator and retested on the 7th, 14th, and 21st day to evaluate its reproducibility and calculate its inter-group variation coefficient. The calculated intra-group variation coefficient was 0.72% to 2.26%, and the inter-group variation coefficient was 0.93% to 2.57%, indicating that the established method has good repeatability.

[0059] Example 5 Clinical sample detection

[0060] Blood smear microscopy was performed on 10 samples of yellow croaker collected from the seawater cages of yellow croaker aquaculture in the sea area. At the same time, heart tissues were taken from the 10 diseased samples, and total DNA was extracted. The optimized SYBR GreenⅠ real-time fluorescence quantitative PCR method was used for detection, and the genomic DNA of the yellow croaker trypanosome JJ2024 strain in Example 1 was added for detection. The results are shown in Table 1.

[0061] Table 1 Trypanosoma detection

[0062]

[0063]

[0064] The following conclusions were drawn: 7 of the 10 clinical samples of large yellow croaker were positive by microscopic examination using the traditional method, while 9 were detected by SYBR GreenⅠ real-time fluorescence quantitative PCR method, including 2 samples that were negative by microscopic examination. Therefore, the fluorescence quantitative PCR method in this scheme has higher sensitivity.

[0065] Finally, it should be noted that although the above embodiments have been described in the specification and drawings of this application, this does not limit the scope of patent protection of this application. All technical solutions generated by replacing or modifying equivalent structures or equivalent processes based on the essential concept of this application using the contents recorded in the specification and drawings of this application, as well as directly or indirectly implementing the technical solutions of the above embodiments in other related technical fields, are included in the scope of patent protection of this application.

Claims

1. A SYBR Green I real-time fluorescence quantitative PCR detection primer for trypanosomes, characterized in that: The PCR detection primers include the nucleotide sequence of the upstream primer shown in SEQ ID NO.1 and the nucleotide sequence of the downstream primer shown in SEQ ID NO.

2.

2. The SYBR Green I real-time fluorescence quantitative PCR detection primer for trypanosomes according to claim 1, characterized in that: The PCR amplification reaction conditions were as follows: pre-denaturation at 95°C for 30 s, denaturation at 95°C for 5 s, and annealing at 62°C for 30 s, for a total of 40 cycles.

3. A SYBR Green I real-time fluorescence quantitative PCR detection kit for trypanosomes, characterized in that: The PCR detection kit comprises the PCR detection primers described in claim 1.

4. The SYBR Green I real-time fluorescence quantitative PCR detection kit for trypanosomes according to claim 3, characterized in that: The amplification reaction conditions of the PCR detection kit are: pre-denaturation at 95°C for 30s; denaturation at 95°C for 5s, annealing at 62°C for 30s, for a total of 40 cycles.

5. The SYBR Green I real-time fluorescence quantitative PCR detection kit for trypanosomes according to claim 3, characterized in that: The amplification reaction system of the PCR detection kit is 10 μL of TB Green Premix Ex Taq II (Tli RNaseHPlus), 0.4 μL of upstream primer, 0.4 μL of downstream primer, 2 μL of template, and sterile deionized water is added to a final volume of 20 μL.

6. The SYBR Green I real-time fluorescence quantitative PCR detection kit for trypanosomes according to claim 5, characterized in that: The concentrations of the upstream primer and the downstream primer were both 10 μmol / L.

7. The SYBR Green I real-time fluorescence quantitative PCR detection kit for trypanosomes according to claim 3, characterized in that: The PCR detection kit is used for colony PCR.

8. A SYBR Green I real-time fluorescence quantitative PCR detection method for trypanosomes using the PCR detection primers according to claim 1 or 2, characterized in that: The following steps are involved: 1) Extracting genomic DNA from the sample to be tested; 2) Real-time fluorescence quantitative PCR reaction; The real-time fluorescence quantitative PCR amplification reaction system is: TB Green Premix Ex Taq II (Tli RNaseHPlus) 10 μL, upstream primer 0.4 μL, downstream primer 0.4 μL, standard plasmid or test template 2 μL, the rest is sterile deionized water, and the total volume of the reaction system is 20 μL; the real-time fluorescence quantitative PCR amplification reaction conditions are: 95°C pre-denaturation for 30 s; 95°C denaturation for 5 s, 62°C annealing for 30 s, a total of 40 cycles; 3) Result determination: If the nucleic acid concentration of the sample to be tested is higher than the detection limit of 1.0×10 1 When the test result is 0.05 copies / μL, it is judged as positive for Trypanosoma macrocarpa, otherwise it is negative.

9. Use of the PCR detection kit according to any one of claims 3 to 7 in detecting fish trypanosomiasis.

10. The use according to claim 8, characterized in that: The fish include large yellow croaker.

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