A dual PCR primer, kit and detection method for detecting pathogenic bacteria of fish gill nodule disease

Abstract

The present invention discloses a dual PCR primer, a kit and a detection method for detecting pathogenic bacteria of gill nodule disease in fish, wherein the primers include a primer pair 1 and a primer pair 2, and the sequences thereof are shown in SEQ ID NO.1‑4. The detection method includes: extracting genomic DNA of a sample to be tested, using the genomic DNA as a template, performing PCR amplification using the dual PCR primers, and performing electrophoresis detection on the amplified product, and if electrophoresis bands appear at 263 bp and 593 bp, it indicates that the sample to be tested is infected with pathogenic bacteria of gill nodule disease. The dual PCR detection method provided by the present invention detects DNA of gill tissue of fish samples infected with Nocardia, and can quickly and accurately detect the pathogen of Nocardia amberjack, with high sensitivity, good specificity and good repeatability.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial detection, and in particular to a dual PCR primer, a kit and a detection method for detecting pathogenic bacteria of fish gill nodule disease. Background Art

[0002] Fish gill sarcoidosis is a disease characterized by the appearance of macroscopically visible, multifocal white nodules of varying sizes on the gills. In most cases, it can spread to multiple organs throughout the body. It is essentially a granulomatous inflammatory disease. Because the gills are most easily observed clinically, it is referred to as "fish gill sarcoidosis" or "fish sarcoidosis." It is a common disease in aquaculture. In recent years, sarcoidosis has become common in marine and freshwater fish, most notably snakehead, California sea bass, large yellow croaker, and ovate pomfret. The main manifestations of sarcoidosis include sluggish movement in the early stages of the disease, a significant decrease in vitality, and a reduced or absent feeding ability. While the affected fish may show no obvious symptoms on the surface, clinical dissection reveals numerous white nodules on the gills and internal organs. The disease is characterized by a wide range of prevalence, high infectiousness, a high mortality rate, and a long incubation period. The bacteria that cause fish nodular disease mainly include Nocardia seriola, Photobacterium mermanii and Vibrio harveyi. However, through epidemiological surveys, we found that in recent years, due to global warming, nodular disease caused by Nocardia seriola has become particularly common and serious, and has become the main epidemic trend of chronic diseases in fish farming.

[0003] Nocardia seriolae is a Gram-positive, facultative intracellular mycobacterium that can grow in water temperatures ranging from 15°C to 32°C, with an optimum temperature of 25°C to 28°C. In harsh environments, such as when fish are undernourished, have poor physical fitness, or have a weakened immune system, Nocardia seriolae primarily infects fish through wounds on the skin, gills, or digestive tract. As the bacteria continue to invade and colonize tissues, nodular lesions multiply, severely impairing the normal physiological function of organs such as the gills, leading to fish death. Since Nocardia seriola is a facultative intracellular parasite, under natural infection conditions, its growth and reproduction rate in fish is very slow, and the infection lasts for a long time. The sick fish have no obvious symptoms in the early stage. As the infection time prolongs, typical symptoms are multifocal white nodules in tissues such as gills, liver, spleen, kidney, and head kidney. The gills are the invasion channel for pathogens, so nodular lesions occur earlier and are easier to be discovered. Once a large number of nodules are formed, treatment is very difficult, which can lead to serious economic losses.

[0004] In summary, gill nodule disease in fish, caused by Nocardia septentrifica, progresses slowly, and by the time obvious symptoms appear, treatment is difficult. This severely impacts the economic value of adult fish and causes significant losses to the aquaculture industry. Therefore, establishing a rapid and accurate method to detect the pathogen responsible for gill nodule disease in fish is crucial for treating the disease.

[0005] Traditional bacterial isolation and culture combined with morphological identification is the most direct method, but the dominant pathogens must be isolated and purified through culture medium, and then the results can be obtained through bacterial 16S rRNA sequencing and identification. This is not only labor-intensive, but also the growth of Nocardia septempunctata is slow. The entire detection process takes at least 7-10 days, which is not suitable for the detection of the bacteria and the treatment of fish diseases.

[0006] Current methods for detecting Nocardia seriola include conventional PCR, real-time quantitative PCR, and loop-mediated isothermal amplification (LAMP). Conventional PCR is prone to cross-contamination and false positives. For latent or mixed infections of seriola with low levels of Nocardia seriola, the detection rate is lower than the actual infection rate. Real-time quantitative PCR is cumbersome, time-consuming, and costly. While LAMP has many advantages, it is limited in its ability to produce either amplified or non-amplified results. Furthermore, when amplified at the same temperature, primers may complement each other, amplifying nonspecific bands and causing false positives. A latex agglutination antibody test kit has been developed to detect serum antibodies produced after Nocardia seriola infection. Although this kit is highly sensitive, it is currently unknown when antibodies are produced in California bass infected with Nocardia seriola. Furthermore, the experimental procedure is complex and costly, hindering widespread use.

[0007] Based on this, there is an urgent need to develop a dual PCR detection method that can be used to quickly, accurately and cost-effectively detect the pathogens of fish gill nodule disease. Summary of the Invention

[0008] In order to address the above-mentioned deficiencies in the prior art, the present invention aims to provide a dual PCR primer, a kit and a detection method for detecting the pathogen of fish gill nodule disease. The present invention designs two pairs of specific primers for Nocardia aurea and applies dual PCR technology to establish a method for rapid and accurate detection of Nocardia aurea. This method has strong specificity, good repeatability and high sensitivity, providing strong technical support for the clinical differential diagnosis of Nocardia aurea.

[0009] The technical solution of the present invention to solve the above technical problems is as follows: The first object of the present invention is to provide a dual PCR primer for detecting pathogens of fish gill nodule disease, comprising:

[0010] Primer pair 1:

[0011] Secretory A1 F: 5'-TGCATTACATGGCACCGAGT-3' (SEQ ID NO. 1);

[0012] Secretory A1 R: 5'-GCTACTGGATTCGGAGACCG-3' (SEQ ID NO. 2);

[0013] Primer pair 2:

[0014] 16S rRNA F: 5'-TGAGTAGTGGCGAGCGAAAG-3' (SEQ ID NO. 3);

[0015] 16S rRNA R: 5'-GGGTGGTCCCGACAGATT-3' (SEQ ID NO. 4).

[0016] Furthermore, the pathogen of fish gill nodule disease is Nocardia septempunctata.

[0017] The present invention uses the Secretory A1 and 16S rRNA gene sequences of Nocardia aurea, the pathogen of fish gill tuberculosis, as templates to design specific primers. After screening, two pairs of primers with high efficiency and strong specificity, namely the primer pair 1 and primer pair 2, were determined.

[0018] The second object of the present invention is to use the above-mentioned dual PCR primers in the preparation of a reagent or a kit for detecting pathogens of fish gill nodule disease.

[0019] The third object of the present invention is to provide a reagent / kit for detecting pathogens of fish gill nodule disease, wherein the reagent / kit comprises the above-mentioned dual PCR primers.

[0020] Furthermore, the above reagents / kits also include 2×PCR Hero TM Mix (dye) and DNase / RNase-free Water, that is, the reagents / kit includes: 2×PCR Hero TM Mix(dye), 16S rRNA F, 16S rRNA R, Secretary A1 F, Secretary A1 R and DNase / RNase-free Water.

[0021] The fourth object of the present invention is to use the above-mentioned dual PCR primers or reagents or kits for detecting pathogens of fish gill nodule disease.

[0022] A fifth object of the present invention is to provide a method for detecting the pathogenic bacteria of fish gill nodule disease, comprising the following steps:

[0023] The genomic DNA of the sample to be tested was extracted, and PCR amplification was performed using the genomic DNA as a template with the above-mentioned dual PCR primers. The amplified products were detected by electrophoresis. If electrophoresis bands appeared at 263 bp and 593 bp, it indicated that the sample to be tested was infected with the pathogen of gill nodule disease.

[0024] Furthermore, the reaction system for PCR amplification is: 2×PCR Hero TM Mix (dye) 10 μL, DNA template 2 μL, primer pair 1 1.2 μL, primer pair 2 0.4 μL, DNase / RNase-free water to 20 μL.

[0025] Furthermore, the reaction conditions for PCR amplification were as follows: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 10 s, annealing at 60°C for 10 s, extension at 72°C for 20 s, 30 cycles, and finally extension at 72°C for 5 min.

[0026] The present invention has the following beneficial effects:

[0027] This application designed a pair of specific primers based on the highly conserved regions of 16S rRNA and SecA1 of Nocardia aurea, optimized the duplex PCR system and reaction conditions, and established a duplex PCR method that can specifically detect Nocardia aurea. The minimum detection value of this method is 1.8764×10 -1 ng / μL, with high sensitivity, and according to the results of specificity experiments and repeatability experiments, it is shown that the method of the present application has good specificity and repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The electrophoresis results after single PCR amplification in Example 1; wherein, M: DL2000 DNA Marker; 1-4: 16S primers; 5-8: SecA1 primers; 9-10: negative control.

[0029] Figure 2 The results of primer ratio optimization in Example 1 are as follows; wherein, M: DL2000 DNA Marker; 1: negative control; 2: the volume ratio of 16S and SecA1 is 1:1; 3: the volume ratio of 16S and SecA1 is 2:1; 4: the volume ratio of 16S and SecA1 is 1:2; 5: the volume ratio of 16S and SecA1 is 1:3; and 6: the volume ratio of 16S and SecA1 is 3:1.

[0030] Figure 3The annealing temperature optimization results in Example 1 are shown. M: DL2000 DNA Marker; 1: 58.3°C; 2: 59.1°C; 3: 60.2°C; 4: 61.3°C; 5: 62.3°C; and 6: 63°C.

[0031] Figure 4 The results of cycle number optimization in Example 1 are shown; wherein, M: DL2000 DNA Marker; 1: 25 cycles; 2: 30 cycles; 3: 35 cycles; 4: 40 cycles.

[0032] Figure 5 The results are specific for the duplex PCR method; M: DL2000 DNA Marker; 1: negative control; 2-3: Nocardia sphenocardiae; 4: Aeromonas hydrophila; 5: Citrobacter freundii; 6: Elizabethella; 7-8: Aeromonas versii; 9-10: Flavobacterium succinate.

[0033] Figure 6 The sensitivity test results of the double PCR method are shown in Figure 2. M: DL2000 DNA Marker; 1-9: The initial concentration of Nocardia sphenotype genomic DNA was 187.64 ng / μL, and the dilution gradients were 10 0 , 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 ; 10: negative control.

[0034] Figure 7 The figure shows the repeatability test results of the duplex PCR method; M: DL2000 DNA Marker; 1: negative control; 2-5: duplex PCR detection.

[0035] Figure 8 These are the histological (H&E) observation results of the California sea bass samples submitted for examination; AB: hyperplasia of the gill lamellae, forming granulomas (indicated by red arrows); C: inflammatory cell infiltration of the glomeruli, with an increase in the number of melanin-producing macrophage centers (indicated by red arrows); D: fibrosis and vacuolation of the liver (indicated by red arrows); E: necrosis of splenic parenchymal cells (indicated by red boxes); F: necrosis of the head kidney cells, with numerous and large melanin-producing macrophage centers (indicated by red arrows).

[0036] Figure 9 These are the results of conventional PCR testing of the California sea bass sample tissues submitted for testing; where M: Marker; Blank: negative control; Yang: positive control; 1-11: California sea bass submitted for testing.

[0037] Figure 10 These are the results of double PCR testing of California sea bass sample tissue DNA; where M: Marker; Blank: negative control; Yang: positive control; 1-11: California sea bass sample tissue DNA. DETAILED DESCRIPTION

[0038] The following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, where specific conditions are not specified, the experiments were performed under conventional conditions or those recommended by the manufacturer. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0039] Example 1: Designing primers

[0040] In this example, the 16S rRNA gene sequence of Nocardia aurea (hereinafter referred to as 16S) and the essential secretory protein SecA1 gene (hereinafter referred to as SecA1) were used as target gene sequences, and two pairs of specific primers were designed (Table 1). After single PCR amplification (amplification system and procedure are shown in Table 2), the results were detected by 1% gel electrophoresis. Figure 1 As shown, the sizes are 263 bp (16S) and 593 bp (SecA1), respectively.

[0041] Table 1 Duplex PCR specific primers

[0042]

[0043] Table 2 Single PCR amplification system and procedure

[0044]

[0045] In order to simultaneously amplify two clear and specific bands, the primer ratio and PCR amplification process were optimized as follows:

[0046] 1. Primer ratio optimization

[0047] In order to select the best primer ratio that can simultaneously amplify two clear specific bands, under the condition that the total volume of the above single PCR reaction system remains unchanged, the primer concentrations are adjusted, that is, the volume ratios of 16S primer and SecA1 primer are 1:1, 2:1, 1:2, 1:3, and 3:1, and then the two target genes are amplified. The amplification results are shown in Figure 2. Figure 2 .

[0048] Depend on Figure 2 It can be seen that when the volume ratio of 16S primer to SecA1 primer is 1:3, that is, 2×PCR Hero is added to the 20μL reaction system. TMMix (dye) 10 μL, template DNA 2 μL, 16S primer upstream and downstream 0.4 μL each, SecA1 primer upstream and downstream 1.2 μL each, and finally add DNase / RNase-free water to 20 μL. The resulting duplex PCR amplification bands were brightest and clear. Therefore, the optimal primer ratio of 16S primer to SecA1 primer volume ratio of 1:3 was used for subsequent experiments.

[0049] 2. Annealing temperature optimization

[0050] In order to determine the optimal annealing temperature for the dual PCR detection system, the present invention sets six annealing temperature gradients between 58-63°C, and finally screens the best annealing temperature suitable for this system based on the brightness of the gel electrophoresis bands. The results are shown in Figure 3 .

[0051] Depend on Figure 3 It can be seen that at each set annealing temperature, specific target bands with relatively high brightness appeared at 263 bp and 593 bp, and there were few non-specific amplification bands. Therefore, 60°C was finally selected as the annealing temperature for the dual PCR detection system in the present invention.

[0052] 3. Program loop parameter optimization

[0053] The amplification program was set to 25, 30, 35, and 40 cycles, and the optimal cycle parameters for the dual PCR detection system were screened by the brightness of the gel electrophoresis bands. Figure 4 .

[0054] Depend on Figure 4 It can be seen that when the number of cycles is set to 30, the bands at 263 bp and 593 bp are both brightest. Therefore, the number of cycles finally selected for the dual PCR detection method of the present invention is 30.

[0055] Example 2: Duplex PCR method specificity, sensitivity and repeatability test

[0056] 1. Specificity evaluation

[0057] DNA from different strains (Nocardia aurea, Aeromonas hydrophila, Citrobacter freundii, Elizabethella sp., Aeromonas versii, and Flavobacterium succinate) were amplified by duplex PCR, with sterile distilled water or DNase / RNase-free water as negative controls.

[0058] In the above PCR amplification process, the reaction system is: 2×PCR Hero TMMix (dye) 10 μL, DNA template 2 μL, SecA1 primer upstream and downstream 1.2 μL each, 16S primer upstream and downstream 0.4 μL each, DNase / RNase-free water to 20 μL.

[0059] The reaction conditions for PCR amplification were as follows: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 10 s, annealing at 60°C for 10 s, extension at 72°C for 20 s, 30 cycles, and finally extension at 72°C for 5 min.

[0060] The above PCR amplification products were detected by 1% gel electrophoresis. Figure 5 .

[0061] Depend on Figure 5 It can be seen that two clear positive target bands were amplified from the DNA of Nocardia spp. strains, while no specific bands were amplified from the DNA of other strains and the negative control, indicating that the dual PCR detection method established in this study has good specificity.

[0062] 2. Sensitivity evaluation

[0063] The genomic DNA of Nocardia sphenotype was extracted and diluted 10-fold to 10 -8 The sensitivity of the detection method was evaluated by PCR amplification of each gradient dilution at the same time, with sterile distilled water as blank control. The results are shown in Figure 6 .

[0064] Depend on Figure 6 It can be seen that the initial concentration of the extracted Nocardia seriola genomic DNA was 187.64 ng / μL measured on the nucleic acid protein analyzer. The sensitivity results showed that at 1.8764×10 -1 ng / μL, the target bands of 16S and SecA1 could still be detected. -2 ng / μL, only the 16S target band can be detected, so the sensitivity of this method can reach 1.8764×10 -1 ng / μL, with good sensitivity.

[0065] 3. Repeatability evaluation

[0066] The established duplex PCR detection method was used to test 4 times at different times and under different conditions (operators, instruments) to observe the repeatability. The test results are shown in Figure 7 .

[0067] Depend on Figure 7It can be seen that the duplex PCR experiments were performed by different operators, at different times, and with different instruments, and the repeatability of the system was demonstrated by gel electrophoresis bands. It was found that the duplex PCR detection method established by the present invention had consistent results in four tests, indicating that it is not affected by factors such as operation, environment, and instrumentation, and can ensure the stability and reliability of subsequent application experiments.

[0068] Example 3: Comparison of Duplex PCR Detection Method with Other Methods

[0069] Eleven California bass (denoted as F1-F11) with unknown pathogens were tested using different detection methods: clinical observation, bacterial isolation and identification, histopathology, conventional PCR and duplex PCR. The speed and accuracy of the methods were compared. The test results are shown in Figure 8-10 and Table 3.

[0070] Clinical observations revealed no symptoms in any of the samples, and no lesions could be detected with the naked eye.

[0071] Through bacterial isolation and identification, after two weeks, it was identified that all three pathogens were Nocardia spp.

[0072] Histopathological examination of each fish revealed that F5, F6, and F9-F11 showed no pathological changes and no bacteria were isolated. The remaining samples were observed by histological HE staining, showing small-diameter granulomatous lesions with hyperplasia of gill epithelial cells, mild inflammatory cell infiltration in the renal interstitium, fibrosis and vacuolation in the liver, and necrotic granulomatous lesions in the spleen and head kidney. Figure 8 ), which is a characteristic symptom of nocardiosis infection.

[0073] The DNA of the California bass sample was used as a template and a conventional PCR test was performed using bacterial universal primers. The universal primers used were 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 5) and 1429R: 5'-GGCTACCTTGTTACGACTT-3' (SEQ ID NO. 6); the amplification system was: 2×PCR Hero TM Mix (dye) 12.5μL, DNA template 2μL, 1μL each of upstream and downstream primers, DNase / RNase-free water to 25μL; amplification program: pre-denaturation at 94℃ for 3min, denaturation at 94℃ for 30s, annealing at 55℃ for 20s, extension at 72℃ for 20s, 35 cycles, and finally extension at 72℃ for 5min. Comparison with positive controls and sequencing revealed that only one sample, F8, had characteristic symptoms of nocardia infection ( Figure 9 ).

[0074] The double PCR test was performed using California bass sample tissue DNA as template. The results are shown in Figure 10 .Depend on Figure 10 It can be seen that except for the head kidney tissues numbered F6 and F11, which showed no positive bands, the DNA of the other extracted head kidney tissues could detect positive bands. Some bands were brighter, indicating that the amount of bacteria was less, while the negative control had no bands ( Figure 10 ).

[0075] Table 3 Comparison of different methods for detecting Nocardia infection in amberjack

[0076]

[0077]

[0078] Note: "+" indicates symptoms, bacterial isolation and identification of Nocardia spp., positive results of duplex PCR, or tissue lesions; "-" indicates no symptoms, no bacteria isolated, negative results of duplex PCR, negative results of conventional PCR, and no tissue lesions.

[0079] From the above results, it can be seen that the dual PCR method is more sensitive, rapid, simple, and accurate than the other four methods. That is, the dual PCR detection method can be used to detect the DNA of gill tissues of fish samples infected with nocardiosis, which can quickly and accurately detect the pathogen Nocardia seriola, especially in the early stage of infection (histopathology shows no pathological changes or mild changes), which has obvious advantages over other methods.

[0080] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A dual PCR primer for detecting pathogenic bacteria of fish gill nodule disease, characterized in that: include: Primer pair 1: Secretory A1 F: 5'-TGCATTACATGGCACCGAGT-3'; Secretory A1 R: 5'-GCTACTGGATTCGGAGACCG-3'; Primer pair 2: 16S rRNA F: 5'-TGAGTAGTGGCGAGCGAAAG-3'; 16S rRNA R: 5'-GGGTGGTCCCGACAGATT-3'; The pathogen of fish gill tuberculosis is Nocardia spheniscidae.

2. Use of the dual PCR primers according to claim 1 in preparing a reagent or a kit for detecting pathogenic bacteria of fish gill nodule disease; wherein, The pathogen of fish gill tuberculosis is Nocardia spheniscidae.

3. A kit for detecting pathogenic bacteria of fish gill nodule disease, characterized in that: The kit comprises the dual PCR primers according to claim 1.

4. The kit for detecting pathogenic bacteria of fish gill nodule disease according to claim 3, characterized in that: Also includes: 2×PCR Hero TM Mix and DNase / RNase-free Water.

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