Pseudosciaena crocea pyramidal RPA detection kit, RPA-LFD detection system and detection method

Abstract

The invention provides an RPA (recombinase polymerase amplification) detection kit, an RPA-LFD (lateral flow dipstick) detection system and an RPA-LFD detection method. The sequences of an upstream primer and a downstream primer used in the detection are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2; the sequence of the probe is as shown in SEQ ID NO.3, and tetrahydrofuran is inserted into the probe. The invention further provides a trypanosome RPA (recombinase polymerase amplification) detection kit and a trypanosome RPA-LFD (lateral flow dipstick) detection system containing the detection kit, and also provides a corresponding detection method. The whole detection process can be completed within 23 minutes, the operation is simple and convenient, and the result is visible. Compared with an existing detection method, the method has the advantages of being rapid, sensitive, high in specificity, suitable for on-site operation and the like, and is suitable for early diagnosis and epidemiological investigation of the trypanosomiasis in the large yellow croaker culture process.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and aquatic disease detection technology, and relates to the rapid detection of trypanosoma sp. in large yellow croaker. In particular, it relates to a primer and probe set, kit and detection method for rapid detection of trypanosoma sp. in large yellow croaker based on RPA-LFD (recombinase polymerase amplification-lateral chromatography strip) technology. It is suitable for rapid on-site diagnosis and epidemiological monitoring of trypanosoma diseases in aquaculture. Background Technology

[0002] Large yellow croaker Larimichthys crocea Trypanosoma japonicum is one of the important marine aquaculture fish species in my country. In recent years, with the expansion of aquaculture scale, disease problems have become increasingly serious. Among them, trypanosoma japonicum (…) Trypanosoma sp. Parasitic diseases caused by infection have become a significant threat to the healthy aquaculture of large yellow croaker. These diseases can lead to anemia, extensive ulceration of the body surface, and even death, causing substantial economic losses to the aquaculture industry.

[0003] Currently, the detection of trypanosomiasis in large yellow croaker mainly relies on tissue smear microscopy and molecular biological methods (such as PCR or qPCR). However, traditional microscopic methods have low sensitivity, especially when the number of trypanosomiasis parasites is small or in the early stages of infection, making them prone to missed detection. Furthermore, they are highly dependent on the operator's experience and skill level, making it easy for beginners to misdiagnose or miss cases. Although molecular diagnostic techniques based on nucleic acid detection, such as PCR and qPCR, can overcome the shortcomings of traditional diagnostic methods and achieve better specificity and sensitivity, these methods require sophisticated equipment and skilled personnel, are cumbersome to operate, and are time-consuming, making them unsuitable for rapid on-site diagnosis. Therefore, there is an urgent need to develop a convenient, rapid, sensitive, and highly specific molecular detection method to meet the rapid detection needs of trypanosomiasis patients in aquaculture.

[0004] Isothermal amplification, unrestricted by instruments and laboratory conditions, is gaining traction in the field of rapid diagnostics. Recombinase polymerase amplification (RPA) technology, a recently emerging isothermal amplification technique, eliminates the need for thermal cycling devices compared to traditional PCR, enabling rapid DNA amplification under isothermal conditions. Compared to other techniques, RPA offers better specificity, sensitivity, and ease of operation. It utilizes a modified DNA enzyme that binds to primers to form a protein-DNA complex, searching for homologous sequences in double-stranded DNA and initiating DNA synthesis. This leads to exponential amplification of the target gene on the template, yielding a large quantity of amplified products in approximately 20 minutes at 37-40°C, making it suitable for rapid detection of pathogens in aquaculture.

[0005] RPA amplification products can be detected using gel electrophoresis, fluorescence detectors, and latRPA flow strips (LFD). However, outside the laboratory, gel electrophoresis and fluorescence detection have limited sensitivity and operability. In contrast, LFD is suitable for simple and intuitive detection, and its result interpretation does not rely on complex equipment or the technical skills of the testing personnel, making it more suitable for rapid on-site detection. Therefore, establishing a rapid detection method for trypanosomes in large yellow croaker using RPA-LFD technology provides a feasible technical means for the detection of parasitic infections in primary laboratories and on-site, and has good application prospects. Summary of the Invention

[0006] This invention aims to establish a rapid detection technology for trypanosomes in large yellow croaker using RPA-LFD technology, enabling rapid detection of parasite infections in primary laboratories and on-site.

[0007] To achieve the above objectives, the specific technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a probe-primer composition for detecting trypanosome RPA in large yellow croaker, having the following technical features: the sequences of its upstream and downstream primers are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively; the probe sequence is shown in SEQ ID NO.3, wherein tetrahydrofuran is inserted, preferably between the 30th and 31st bases; furthermore, a FITC tag is provided at the 5' end of the probe, and a C3 blocking site is provided at the 3' end.

[0009] The specific sequences of the probe primer composition are as follows:

[0010] Upstream primer, 5′-GATAGACGAGAACGACAAGAGCACTATCAA-3′ (SEQ ID NO.1);

[0011] Downstream primer, 5′-biotin-GCATACCACCAGGCATACCGCCGGGCATAC-3′ (SEQ ID NO.2);

[0012] Probe, [5′-6-FAM]-ACGACCACCACCAGAAGGAGCTCGAGGGCAT[THF]TGCACGCCCATCATGA-[3′-C3-spacer] (SEQ ID NO. 3).

[0013] In a second aspect, the present invention provides a large yellow croaker trypanosome RPA detection kit, comprising the probe primer composition, buffer, activator and double-distilled water described above.

[0014] The upstream and downstream primers, as well as the probe, are all in powder form and are dissolved in double-distilled water at a concentration of 10 μmol / L. The buffer and activator are products from existing RPA detection kits.

[0015] In a third aspect, this invention provides a large yellow croaker trypanosome RPA-LFD detection system, comprising a large yellow croaker trypanosome RPA detection kit and an LFD flow measurement strip. The large yellow croaker trypanosome RPA detection kit is as described above, and the LFD flow measurement strip has a gold nanoparticle-labeled anti-FITC antibody on its conjugate pad, and streptavidin coated on the detection line.

[0016] LFD visualization relies on the 5' fluorescein isothiocyanate (FITC) tag on the probe and the C3 retardation site at the 3' end, as well as the affinity (e.g., biotin) label on the reverse primer. In the middle of the probe, a base is substituted with tetrahydrofuran (THF). Binding of the probe to the amplification strand begins with the cleavage of endonuclease IV (Nfo), which releases the 3' end of the probe for extension, amplifying the double-labeled amplification product along with the reverse primer. Anti-FITC antibodies labeled with gold nanoparticles (AuNP-labeled) on the LFD conjugated pad bind to the amplification product, which is then captured and aggregated by a streptavidin-coated detection line, resulting in a red positive signal.

[0017] In a fourth aspect, the present invention provides a method for rapid detection of trypanosomes in large yellow croaker using the above-mentioned RPA-LFD detection system, characterized by comprising the following steps:

[0018] (1) RPA amplification

[0019] Mix 29.4 μL buffer, 2 μL upstream primer, 2 μL downstream primer, 0.6 μL probe, and 5 μL trypanosome nucleic acid.

[0020] The template, 8.5 μL of double-distilled water, and 2.5 μL of activator were added to the PCR tube. The tube was inverted 8-10 times and then briefly centrifuged. The reaction was carried out at 42°C for 20 min. The concentrations of the upstream primer, downstream primer, and probe were all 10 μmol / L.

[0021] (2) LFD flow meter strip detection

[0022] Calculate the sample copy number when it is within a predetermined range (≥1.1×10⁻⁶). 1 When the number of copies / μL is within a certain range, test strips are used for detection.

[0023] The beneficial protections and effects of this invention are as follows:

[0024] In terms of detection efficiency, this invention first performs RPA amplification and then LFD flow strip detection. The RPA amplification detection time is 20 minutes, the LFD flow strip detection time is about 3 minutes, and the entire process takes about 23 minutes. This allows for on-site sampling and detection at any time, with results available immediately, which is helpful for the early diagnosis and epidemiological investigation of trypanosomiasis in large yellow croaker.

[0025] In terms of detection conditions, RPA amplification no longer requires a thermal cycling device. A large amount of amplification products can be obtained by reacting at a constant temperature of 35-45℃ for 20 minutes. Detection can be achieved by using a water bath or other devices that can provide this constant temperature condition. This reduces detection costs and avoids reliance on expensive instruments, which helps to promote the application of RPA in grassroots laboratories and on-site testing, and improves the early diagnosis rate of trypanosomiasis in large yellow croaker.

[0026] In terms of specificity, the detection system of this invention can only detect trypanosomes in large yellow croaker. It does not detect other common pathogens in large yellow croaker, such as Cryptocaryon irritans, Vibrio harveyi, Pseudomonas aeruginosa, iridovirus, and Myocystis suis in swimming crab.

[0027] In terms of sensitivity, the detection limit can be as low as 1.1 × 10⁻⁶. 1 It has excellent sensitivity, with a viral nucleic acid content of copies / μL. Attached Figure Description

[0028] Figure 1 RPA results at different reaction temperatures.

[0029] Figure 2 The results show the optimization of RPA response time.

[0030] Figure 3 The results of RPA-LFD specificity verification are as follows: 1: Positive control using trypanosome DNA from large yellow croaker as a template; 2-6: Detection results of Cryptocaryon stimulans, Vibrio harveyi, Pseudomonas aeruginosa, large yellow croaker iridovirus, and Myocystis suis from swimming crab; 7: Negative control using gill DNA from healthy large yellow croaker as a template; 8: Blank control using ddH2O as a template.

[0031] Figure 4 The results are for RPA-LFD sensitivity verification. Detailed Implementation

[0032] The implementation of the present invention will be described in detail below with reference to the embodiments of the present invention. The following embodiments are implemented under the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0034] 1. Experimental Materials

[0035] Blood samples were taken from naturally infected large yellow croakers from disease-affected areas in Fujian Province. Microscopic examination confirmed trypanosomiasis infection. Total DNA was extracted using the Tiangen Marine Animal Genomic DNA Extraction Kit, and the concentration of trypanosomiasis DNA in the extracted total DNA was determined by qPCR to be 4.3 × 10⁻⁶. 5 copies / μL.

[0036] 2. Primer and probe design

[0037] Based on the trypanosome gene sequence of large yellow croaker, RPA primers and probes were designed to select specific sites. The designed primers amplified the target band of 236 bp.

[0038] Forward primer: 5'-GATAGACGAGAACGACAAGAGCACTATCAA-3' (SEQ ID NO.1);

[0039] Reverse primer: [5'-Biotin]-GCATACCACCAGGCATACCGCCGGGCATAC-3' (SEQ ID NO.2);

[0040] The sequence of the probe used is: [5'-6-FAM]-ACGACCACCACCAGAAGGAGCTCGAGGGCAT[THF]TGCACGCCCATCATGA-[3'-C3-spacer] (SEQ ID NO.3).

[0041] 3. RPA reaction temperature optimization

[0042] Preparation of 50 μL RPA amplification reaction system: Add 29.4 μL buffer, 2 μL upstream primer, 2 μL downstream primer, 5 μL Trypanosoma nucleic acid template, 9.1 μL ddH2O, and 2.5 μL activator to a PCR tube, invert and mix 8-10 times, then centrifuge briefly. Set up a blank control using ddH2O as the template.

[0043] The prepared mixture was incubated at different temperatures (35℃, 38℃, 40℃, 42℃, 45℃, 48℃, 50℃) for 15 min each, and then the reaction was terminated at 60℃ for 5 min. After the reaction, Tris-saturated phenol / chloroform / isoamyl alcohol (25:24:1) DNA extraction buffer was added at a 1:1 ratio to extract the DNA fragments, which were then verified by 2% agarose gel electrophoresis. The electrophoresis results are shown below. Figure 1 As shown, the optimal reaction temperature is 42℃.

[0044] 4. RPA Response Time Optimization

[0045] Following the RPA temperature optimization method described above, a 50 μL reaction system was prepared. Amplification was performed at the optimal temperature of 42℃ for different time periods (10 min, 15 min, 18 min, 20 min, 22 min, 25 min, 28 min, 30 min, 32 min, and 35 min), with the reaction terminated at 60℃ for 5 min. A blank control group was also included. The amplified products were validated by DNA extraction and 2% agarose gel electrophoresis. The results are as follows: Figure 2 As shown, the results indicate that better amplification results can be achieved after 15 minutes. To improve the sensitivity of the detection method and shorten the rapid detection time as much as possible, the optimal reaction time was determined to be 20 minutes.

[0046] 5. RPA-LFD Specificity Test

[0047] To evaluate the specificity of the RPA-LFD method, five common pathogens of large yellow croaker and other pathogens present in the aquaculture environment were selected: Cryptocaryon irritans, Vibrio harveyi, Pseudomonas aeruginosa, iridovirus, and Myocystis suis from swimming crab. Their DNA was used for cross-reaction verification. At the same time, a negative control with DNA template from the gills of healthy large yellow croaker, a positive control with DNA template from trypanosomes of large yellow croaker, and a blank control with ddH2O template were set up.

[0048] RPA isothermal amplification: The 50 μL reaction system included 29.4 μL buffer, 2 μL upstream primer, 2 μL downstream primer, 0.6 μL probe, 5 μL Trypanosoma nucleic acid template, 8.5 μL ddH2O, and 2.5 μL activator. The optimal reaction temperature and time were selected and optimized, i.e., 42℃ for 20 min.

[0049] LFD visualization detection: The reaction product is diluted 10 times with ddH2O and mixed evenly. It is then dropped into the sample well of the test strip. The color development of the control line and the detection line is observed within 3 minutes, and the results are interpreted.

[0050] The results are as follows Figure 3The results showed that no detection lines were observed in the five pathogens, as well as in the negative and blank controls, indicating that the method has good specificity.

[0051] 6. Sensitivity Test

[0052] With a concentration of 4.3 × 10 5 Using Trypanosoma DNA copies / μL as a template, five gradients (10) were performed. -1 10 -2 10 -3 10 -4 and 10 -5 The concentrations of trypanosome DNA in each dilution were calculated using qPCR and found to be 5.6 × 10⁻⁶. 4 6.5×10 3 6.3×10 2 7.1×10 1 1.1×10 1 Copies / μL were then analyzed using RPA-LFD to determine the limit of detection.

[0053] The results are as follows Figure 4 As shown, the detection sensitivity of RPA-LFD can reach 1.1 × 10⁻⁶. 1 copies / μL of pathogenic nucleic acid.

[0054] In summary, this invention exhibits high specificity and a low detection limit for the detection of trypanosomes in large yellow croaker, with the detection limit as low as 1.1 × 10⁻⁶. 1 The amplification reaction requires minimal hardware, using only a small temperature control device, and the entire process takes approximately 23 minutes. The results can be read from the test strip, making it ideal for on-site testing.

[0055] The undescribed parts of this invention are the same as or implemented using existing technology. The applicant declares that this invention is illustrated through the above embodiments, but the invention is not limited to the above detailed methods, i.e., it does not mean that the invention must rely on the above detailed methods to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the product of this invention, additions of auxiliary components, and selection of specific methods all fall within the protection and disclosure scope of this invention.

Claims

1. A probe and primer composition for detecting trypanosomes RPA in large yellow croaker, characterized in that, The sequences of the upstream and downstream primers are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively; the probe sequence is shown in SEQ ID NO.3, in which tetrahydrofuran is inserted.

2. The probe and primer composition for detecting trypanosome RPA in large yellow croaker according to claim 1, characterized in that, Tetrahydrofuran was inserted between the 31st and 32nd bases in the probe sequence.

3. The probe and primer composition for detecting trypanosome RPA in large yellow croaker according to claim 1, characterized in that, The probe has a FAM tag at its 5' end and a C3 blocking site at its 3' end.

4. A test kit for detecting trypanosomes RPA in large yellow croaker, characterized in that, The composition comprises the probe primer composition according to any one of claims 1 to 3.

5. The large yellow croaker trypanosome RPA detection kit according to claim 4, characterized in that, It also includes buffer solutions, activators, and double-distilled water.

6. The large yellow croaker trypanosome RPA detection kit according to claim 5, characterized in that: in, The concentrations of both the upstream and downstream primers were 10 μmol / L, and the probe concentration was 10 μmol / L.

7. A large yellow croaker trypanosome RPA-LFD detection system, characterized in that, This includes a large yellow croaker trypanosome RPA detection kit and LFD flow measurement strips. The large yellow croaker trypanosome RPA detection kit, as described in any one of claims 4 to 6, has gold nanoparticle-labeled anti-FITC antibody on the conjugate pad of the LFD flow measurement strip, and streptavidin is coated on the detection line.

8. A method for rapid detection of trypanosomes in large yellow croaker using the RPA-LFD detection system for trypanosomes in large yellow croaker as described in claim 7, characterized in that, Includes the following steps: (1) RPA amplification Mix 29.4 μL buffer, 2 μL upstream primer, 2 μL downstream primer, 0.6 μL probe, and 5 μL trypanosome nucleic acid. Add template, 8.5 μL of double-distilled water and 2.5 μL of activator to the PCR tube, invert and mix 8-10 times, centrifuge briefly, and react at 42℃ for 20 min. (2) LFD flow meter strip detection Calculate the sample copy number, and when it is within the predetermined range, perform test strip testing.

9. The method for rapid detection of trypanosomes in large yellow croaker using the RPA-LFD detection system according to claim 8, characterized in that: in, The predetermined range for the sample copy number is ≥1.1×10⁻⁶. 1 copies / μL.

Images