A specific detection method for fish nocardia based on its sequence analysis

Abstract

The application relates to the technical field of molecular detection of aquatic animal pathogenic microorganisms, and discloses a fish Nocardia specific detection method based on ITS sequence analysis, which comprises the following steps: extracting genomic DNA of fresh culture of a to-be-detected strain as template DNA; taking the obtained template DNA as a template, performing PCR amplification by using a specific primer pair, wherein the specific primer pair comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 2; and finally performing gel electrophoresis detection on the amplification product, and determining whether the fish Nocardia is contained according to a target band. The specific primer designed based on the conservative region of the Nocardia ITS sequence can synchronously recognize star-shaped Nocardia, Nocardia salmonis and Nocardia shachii, the cross-reaction interference of common aquatic pathogenic bacteria is excluded, the specific primer has the characteristics of high specificity, wide coverage and high sensitivity, and is suitable for rapid screening of aquatic clinical pathogens.

Description

Technical Field

[0001] This invention relates to the field of molecular detection technology for pathogenic microorganisms in aquatic animals, specifically a method for the specific detection of Nocardia in fish based on ITS sequence analysis. Background Technology

[0002] Currently, nocardiosis in fish is a significant bacterial disease limiting the profitability of aquaculture. The disease is primarily caused by *Nocardia amberjack*, *Nocardia sand salmon*, and *Nocardia asteroides*. Infected fish often exhibit chronic granulomas on their body surface or internal organs. The disease affects a variety of high-value economic fish species, including largemouth bass and pomfret. Once the pathogen spreads, it often leads to high mortality rates, posing a threat to the aquaculture industry.

[0003] To address the detection needs of the aforementioned pathogens, traditional pathogen isolation, culture, and biochemical identification techniques are commonly used in clinical practice. Operators need to extract lesion tissue and inoculate it onto specific culture media such as BHI. After several weeks of constant-temperature incubation and colony growth, the bacterial species is determined through morphological observation and physiological and biochemical assays. In addition, some protocols utilize bacterial 16S rRNA gene sequences for PCR amplification. This process relies on the conservation of ribosomal RNA genes to screen for pathogens, and the amplification products are detected by electrophoresis.

[0004] Existing detection technologies have shortcomings in terms of application logic and effectiveness. Traditional culture methods are time-consuming and cannot meet the needs of early and rapid diagnosis of emerging epidemics. The 16S rRNA gene sequence is highly homologous within the Nocardia genus, and conventional primers are insufficient to accurately distinguish between pathogenic and closely related non-pathogenic species, easily leading to cross-reactions and limited specificity. Existing molecular detection schemes mostly focus on single bacterial species. When multiple Nocardia species are mixed infecting water bodies, single-target detection is prone to missed detection. In addition, existing systems have low capture efficiency for low-load pathogens, and their sensitivity is insufficient to support early warning.

[0005] Therefore, this invention provides a fish-specific detection method for Nocardia based on ITS sequence analysis to address the shortcomings of existing technologies. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a fish-specific detection method for Nocardia based on ITS sequence analysis. This method solves the problem that existing detection methods are difficult to distinguish between multiple pathogenic Nocardia species in fish while maintaining broad-spectrum detection, and that they are prone to cross-reaction with common aquatic pathogens.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A method for the specific detection of Nocardia in fish based on ITS sequence analysis includes the following steps:

[0009] Genomic DNA was extracted from fresh cultures of the test strain as template DNA;

[0010] Using the obtained template DNA as a template, PCR amplification was performed using specific primer pairs to obtain amplification products. The specific primer pairs included upstream primer Nocardia F3 and downstream primer Nocardia R3. The nucleotide sequence of upstream primer Nocardia F3 is shown in SEQ ID NO.1, and the nucleotide sequence of downstream primer Nocardia R3 is shown in SEQ ID NO.2.

[0011] The amplification products were tested to determine whether the test strain contained Nocardia ferruginosa.

[0012] By employing the above technical solution, and using primer pairs designed for the conserved region of ITS for targeted nucleic acid amplification, a pathogen detection effect with high specificity and coverage of multiple strains is achieved. The specific amplification reaction steps are as follows:

[0013] Step 1: Template unwinding. The reaction system is heated, disrupting the double helix structure of the template DNA, breaking hydrogen bonds, and forming a single-stranded DNA template.

[0014] Step 2: Primer annealing. The reaction system is cooled, and the upstream primer Nocardia F3 and the downstream primer Nocardia R3 undergo complementary base pairing with the target sequence on the single-stranded DNA template.

[0015] Step 3: Strand extension. Catalyzed by Taq DNA polymerase, using single-stranded DNA as a template, free deoxyribonucleotides are linked to the 3' end of the primer according to the base complementarity pairing principle, synthesizing a new complementary DNA strand.

[0016] Since the primer sequences mentioned above are derived from the highly homogeneous ITS sequence region of Nocardia fish, which is precisely located by multiple sequence alignment, this reaction process can stably cover the conserved regions of Nocardia asteroides, Nocardia sand salmon, and Nocardia yellowtail, avoiding interference from polymorphic variation regions of non-pathogenic bacteria or closely related species, eliminating cross-reactions with common aquatic pathogens, and taking into account both the specificity and applicability of the detection system.

[0017] Preferably, PCR amplification uses a standard reaction system, which includes: Taq DNA polymerase, upstream primer Nocardia F3, downstream primer Nocardia R3, template DNA, and sterile double-distilled water. The standard reaction system has a volume of 25 μL and consists of the following components: 11 μL of Taq DNA polymerase at a concentration of 5 U / μL; 1 μL of upstream primer Nocardia F3 at a concentration of 10 μM; 1 μL of downstream primer Nocardia R3 at a concentration of 10 μM; 2 μL of template DNA at a concentration of 100 ng / μL; and 10 μL of sterile double-distilled water.

[0018] By adopting the above technical solution, the ratio of enzyme, primer and template in the amplification reaction system is limited, ensuring the binding efficiency of primers in a micro-target nucleic acid environment, and improving the amplification efficiency and sensitivity of the detection method.

[0019] Preferably, the PCR amplification reaction conditions are set as follows: pre-denaturation at 94℃ for 4 min; denaturation at 94℃ for 45 s, annealing at 55 to 68℃ for 45 s, extension at 72℃ for 70 s, for 30 cycles; final extension at 72℃ for 10 min, and storage at 4℃.

[0020] By adopting the above technical solution, thermal cycling parameters adapted to the above primers are provided, ensuring sufficient denaturation and specific binding of nucleic acids. The appropriate annealing temperature range removes non-specific amplification products and maintains stable accumulation of the target fragment.

[0021] Preferably, the specific method for detecting the obtained amplification products is as follows: gel electrophoresis detection. The theoretical target product length for amplification is 302 bp.

[0022] By adopting the above technical solution, the amplification products are separated by the difference in the migration rate of nucleic acid molecules in an electric field. Combined with the theoretical target length of 302bp determined by the primers, the target band can be obtained intuitively, which facilitates the rapid determination of the presence of the target strain.

[0023] Preferably, the fish nocardia include one or a combination of several of Nocardia asteroides, Nocardia sand salmon, and Nocardia yellowtail.

[0024] By adopting the above technical solution, it was clarified that the method is applicable to Nocardia, the main pathogenic bacterium that causes nodular disease in fish, thus enhancing the practical application value of the solution in the screening of clinical diseases in aquatic products.

[0025] Preferably, before extracting genomic DNA from fresh cultures of the test strain as template DNA, the test Nocardia strain is pretreated as follows: the test Nocardia strain is inoculated into BHI medium and incubated at 28°C for 5 to 7 days until the colony diameter reaches 1 to 2 mm, and then the fresh culture is collected. The specific method for extracting genomic DNA from fresh cultures of the test strain is as follows: Genomic DNA is extracted using a bacterial genomic DNA extraction kit according to the manufacturer's instructions.

[0026] By adopting the above technical solution, the pretreatment of the test samples and the nucleic acid preparation process were standardized, potential amplification inhibitors in the culture medium and cell lysis debris were removed, high-purity DNA templates were obtained, and the stability of subsequent PCR reactions was ensured.

[0027] Preferably, both the upstream primer Nocardia F3 and the downstream primer Nocardia R3 have 23 bases.

[0028] By adopting the above technical solution, the primer length is limited to the appropriate length for conventional amplification, so that the primer has a reasonable melting temperature, reducing the probability of secondary structure or primer dimer formation and improving reaction specificity.

[0029] This invention provides a method for the specific detection of Nocardia in fish based on ITS sequence analysis. It has the following beneficial effects:

[0030] 1. This invention extracts conserved sequences common to target strains by comparing and analyzing the ITS sequences of various pathogenic Nocardia species in fish, and designs Nocardia F3 and Nocardia R3 specific primer pairs accordingly. Using these primer pairs as targeted recognition tools in the amplification step, this method can simultaneously cover major pathogenic strains such as Nocardia asteroides, Nocardia sand salmon, and Nocardia yellowtail, while avoiding sequence interference from other non-pathogenic bacteria and common pathogens in the aquatic environment. This approach prevents cross-reactions while ensuring the applicability of the detection system.

[0031] 2. This invention provides a suitable annealing environment for primer-target sequence binding by defining the ratio of DNA polymerase, primers, and template in a standardized reaction system and matching specific thermal cycling parameters. This system ensures stable annealing and strand extension of primers under trace amounts of nucleic acid template, eliminates non-specific amplification of non-target fragments, and enables the detection method to have good detection sensitivity, making it suitable for the early diagnosis of low-load pathogens in aquaculture clinical samples.

[0032] 3. The detection process of this invention relies on genome extraction, in vitro PCR amplification, and gel electrophoresis separation to obtain results. Compared with traditional bacterial isolation, culture, and biochemical identification methods, this detection step does not require complex culture conditions and reduces reliance on the morphological identification experience of the testing personnel. Routine molecular biology operations replace the time-consuming culture process, shortening the overall cycle of disease diagnosis and facilitating rapid screening of aquatic pathogens in grassroots laboratories. Attached Figure Description

[0033] Figure 1 This is an electrophoresis diagram of the specific PCR amplification results of this invention;

[0034] Figure 2 This is an electrophoresis diagram of the conventional PCR sensitivity detection of the present invention;

[0035] Figure 3 This is a fluorescence amplification curve for qPCR sensitivity detection according to the present invention. Detailed Implementation

[0036] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] The experimental strains involved in this invention include Nocardia seriolae, Streptococcus agalactiae, Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus, Lactococcus garvieae, Photobacterium damselae subsp. piscicida, Nocardia salmonicida, and Nocardia asteroides.

[0038] The culture media involved in this invention include brain heart infusion agar medium, 2216E seawater medium, Luria-Bertani medium, and nutrient agar medium. The 2216E seawater medium, Luria-Bertani medium, and nutrient agar medium are routinely prepared in the laboratory.

[0039] The biological and chemical reagents involved in this invention include a bacterial genomic DNA extraction kit (DP302), an agarose gel DNA recovery kit, Taq DNA polymerase (concentration 5 U / μL), pMD19-T vector (concentration 50 ng / μL), ligase buffer, and *E. coli* DH5α competent cells. Reagents used for electrophoresis detection include agarose (CAS No. 9012-36-6), GelRed nucleic acid dye (concentration 0.5 μg / mL), and DL2000 standard molecular weight marker. Chemical reagents used for blue-white screening include ampicillin (CAS No. 69-52-3, concentration 100 μg / mL), 5-bromo-4-chloro-3-indole-β-D-galactoside (CAS No. 92-31-9), and isopropyl-β-D-thiogalactoside (CAS No. 367-93-1). All of the above reagents and sterile double-distilled water can be obtained through conventional commercial channels.

[0040] The nucleic acid primers involved in this invention include Nocardia-specific primers, universal amplification primers for the transcribed spacer region of ribosomal RNA genes, and sequencing primers. These primers were synthesized using high-performance liquid chromatography (HPLC) purification technology, achieving a synthesis scale with an optical density greater than 10 and a purity greater than 90%. The primers were dissolved in sterile double-distilled water to a storage concentration of 100 μM and stored at -20°C. Before use, they were diluted to a working concentration of 10 μM. The specific nucleotide sequences of the primers are detailed in the sequence identifier numbers at the end of the instruction manual, which are used instead of directly stating the specific nucleotide sequences.

[0041] Preparation Examples 1-5:

[0042] Preparation Example 1:

[0043] This preparation example provides a method for preparing bacterial strain culture and template DNA extraction, including the following steps:

[0044] Nocardia strains were inoculated on BHI medium and incubated at 28°C for 5 to 7 days until the colony diameter reached 1 to 2 mm; Streptococcus, Lactococcus, and luminobacterium were inoculated on BHI medium and incubated at 28°C for 24 to 48 hours; Vibrio strains were inoculated on 2216E seawater medium and incubated at 28°C for 18 to 24 hours; Aeromonas strains were inoculated on LB medium and incubated at 28°C for 18 to 24 hours; Staphylococcus strains were inoculated on nutrient agar medium and incubated at 37°C for 18 to 24 hours.

[0045] Fresh cultures of the above-mentioned strains were collected, and genomic DNA was extracted from each strain strictly according to the instructions using a bacterial genomic DNA extraction kit, which served as templates for subsequent nucleic acid amplification.

[0046] To obtain the complete ITS sequence of Nocardia, PCR amplification was performed using universal primer pairs p1208f and Myco23S-r. The nucleotide sequence of the upstream primer p1208f is shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer Myco23S-r is shown in SEQ ID NO.4.

[0047] The standard reaction system consisted of 25 μL: 11 μL of 5 U / μL Taq DNA polymerase, 1 μL of 10 μM upstream primer p1208f, 1 μL of 10 μM downstream primer Myco23S-r, 2 μL of 100 ng / μL template DNA, and 10 μL of sterile double-distilled water.

[0048] Preparation Example 2:

[0049] This preparation example provides a method for preparing a universal ITS sequence amplification and cloning, including the following steps:

[0050] Using the 25 μL system from Preparation Example 1, the PCR reaction conditions were set as follows: pre-denaturation at 94 °C for 3 min; denaturation at 94 °C for 30 s, annealing at 50 °C for 30 s, extension at 72 °C for 60 s, for 30 cycles; final extension at 72 °C for 5 min.

[0051] Electrophoresis revealed clear amplified bands for *Nocardia asteroides*, *Nocardia sandfish*, and *Nocardia amberjack*. After gel purification, the products were ligated into the pMD19-T vector, incubated overnight at 16°C, and transformed into DH5α competent cells. Colony PCR was then performed for verification.

[0052] Preparation Example 3:

[0053] This preparation example provides a method for preparing a universal ITS sequence amplification and cloning, including the following steps:

[0054] Using the 25 μL system from Preparation Example 1, the PCR reaction conditions were set as follows: pre-denaturation at 94 °C for 4 min; denaturation at 94 °C for 45 s, annealing at 55 °C for 45 s, extension at 72 °C for 70 s, for 30 cycles; final extension at 72 °C for 10 min.

[0055] The amplification was successfully detected by electrophoresis. Subsequent purification, ligation, transformation and verification steps were the same as in preparation example 2.

[0056] Preparation Example 4:

[0057] This preparation example provides a method for preparing a universal ITS sequence amplification and cloning, including the following steps:

[0058] Using the 25 μL system from Preparation Example 1, the PCR reaction conditions were set as follows: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 60 s, annealing at 60 °C for 60 s, extension at 72 °C for 90 s, for 30 cycles; final extension at 72 °C for 15 min.

[0059] The amplification was successfully detected by electrophoresis. Subsequent purification, ligation, transformation and verification steps were the same as in preparation example 2.

[0060] Preparation Example 5:

[0061] This preparation example provides a method for sequence polymorphism analysis and specific primer design, including the following steps:

[0062] Positive clones obtained from the above preparation examples were selected for Sanger bidirectional sequencing. The bidirectional sequencing results of each strain were then assembled and corrected using CodonCode software.

[0063] Furthermore, the assembled sequences were submitted to NCBI GenBank for BLAST alignment and multiple sequence alignment analysis was performed using SnapGene software. The alignment results showed that the Nocardia ferruginosa ITS sequence exhibited high consistency in specific regions (conserved regions) while displaying significant polymorphism in other regions (specific regions). The sequences exhibited highly conserved characteristics of the Nocardia genus, while also showing specific variations that distinguish it from other non-pathogenic bacteria.

[0064] Based on the precise division of the conserved and specific regions mentioned above, a pair of primers specifically designed for the detection of pathogenic nocardiosis in fish was developed using Oligo7 software, targeting the conserved regions of Nocardia asteroides, Nocardia sand salmon, and Nocardia yellowtail.

[0065] The nucleotide sequence of the upstream primer Nocardia F3 is shown in SEQ ID NO.1.

[0066] The nucleotide sequence of the downstream primer Nocardia R3 is shown in SEQ ID NO.2.

[0067] The primer pair contains 23 bases each, and the theoretical amplification target product length is 302 bp.

[0068] Example 1:

[0069] This embodiment provides a method for the specific detection of Nocardia in fish based on ITS sequence analysis, including the following steps:

[0070] A standard 25 μL reaction mixture was used: 11 μL of 5 U / μL Taq DNA polymerase, 1 μL of 10 μM upstream primer Nocardia F3, 1 μL of 10 μM downstream primer Nocardia R3, 2 μL of 100 ng / μL template DNA, and 10 μL of sterile double-distilled water. The nucleotide sequence of the upstream primer Nocardia F3 is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer Nocardia R3 is shown in SEQ ID NO.2.

[0071] The PCR reaction conditions were set as follows: pre-denaturation at 94℃ for 4 min; denaturation at 94℃ for 45 s, annealing at 68℃ for 45 s, extension at 72℃ for 70 s, for 30 cycles; final extension at 72℃ for 10 min, and storage at 4℃.

[0072] Verification showed that the amplification specificity was strongest and the gel electrophoresis bands were clearest and free of impurities when the annealing temperature was 68℃.

[0073] Example 2:

[0074] This embodiment provides a method for the specific detection of Nocardia in fish based on ITS sequence analysis, including the following steps:

[0075] A standard 25 μL reaction mixture was used: 11 μL of 5 U / μL Taq DNA polymerase, 1 μL of 10 μM upstream primer Nocardia F3, 1 μL of 10 μM downstream primer Nocardia R3, 2 μL of 100 ng / μL template DNA, and 10 μL of sterile double-distilled water. The nucleotide sequence of the upstream primer Nocardia F3 is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer Nocardia R3 is shown in SEQ ID NO.2.

[0076] The PCR reaction conditions were set as follows: pre-denaturation at 94℃ for 4 min; denaturation at 94℃ for 45 s, annealing at 55℃ for 45 s, extension at 72℃ for 70 s, for 30 cycles; final extension at 72℃ for 10 min, and storage at 4℃.

[0077] Example 3:

[0078] This embodiment provides a method for the specific detection of Nocardia in fish based on ITS sequence analysis, including the following steps:

[0079] A standard 25 μL reaction mixture was used: 11 μL of 5 U / μL Taq DNA polymerase, 1 μL of 10 μM upstream primer Nocardia F3, 1 μL of 10 μM downstream primer Nocardia R3, 2 μL of 100 ng / μL template DNA, and 10 μL of sterile double-distilled water. The nucleotide sequence of the upstream primer Nocardia F3 is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer Nocardia R3 is shown in SEQ ID NO.2.

[0080] The PCR reaction conditions were set as follows: pre-denaturation at 94℃ for 4 min; denaturation at 94℃ for 45 s, annealing at 61℃ for 45 s, extension at 72℃ for 70 s, for 30 cycles; final extension at 72℃ for 10 min, and storage at 4℃.

[0081] Comparative Examples 1-3:

[0082] Comparative Example 1:

[0083] Compared with Example 1, the difference is that universal primers for the 16S rRNA gene sequence of common bacteria are used for amplification and detection, while the rest are the same.

[0084] Comparative Example 2:

[0085] Compared with Example 1, the difference is that PCR amplification was performed using primers specifically targeting only a single species of Nocardia amberjack as reported in existing literature; all other aspects are the same.

[0086] Comparative Example 3:

[0087] Compared with Example 1, the difference is that traditional bacterial isolation, culture, and morphological and biochemical identification methods were used for detection, while the rest are the same.

[0088] Test Example 1-3:

[0089] Test Example 1: Specificity and Broad-spectrum Verification

[0090] Test steps:

[0091] To verify the specificity and broad-spectrum coverage of the detection method in this scheme, the method optimized in Example 1 was used for amplification detection, and comparative analysis was conducted in conjunction with Comparative Examples 1 to 3.

[0092] The genomic DNA of Nocardia amberjack, Nocardia asteroides, Nocardia sand salmon, and control strains Streptococcus agalactiae, Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus, Lactococcus gasseri, and Bryophyte melanogaster subsp. fish-killing bacteria extracted in Preparation Example 1 was used as templates.

[0093] PCR reaction solution was prepared and amplified using the system and reaction conditions of Example 1. The upstream primer shown in SEQ ID NO.1 and the downstream primer shown in SEQ ID NO.2 were added to this reaction system.

[0094] Take 5 μL of the amplification product and place it in a 1.0% agarose gel containing nucleic acid dye. Perform electrophoresis at 120V and 200mA for 15 minutes. Then observe and record the amplification bands under a gel imaging system.

[0095] Test data:

[0096] like Figure 1 As shown. Figure 1 The first lane on the left is the standard molecular weight marker (M), with the DNA fragment size clearly marked in bp increments on the left vertical axis, from top to bottom as 2000, 1000, 750, 500, 250, and 100. Lanes to the right of lane M are designated as test lanes 1 through 9. Lanes 1 through 3 correspond to templates of *Nocardia amberjack*, *Nocardia asteroides*, and *Nocardia sandfish*, respectively. Lanes 4 through 9 correspond to templates of *Vibrio alginolyticus*, *Bacillus famosa* subsp. *famosa*, *Vibrio vulnificus*, *Streptococcus agalactiae*, *Vibrio parahaemolyticus*, and *Lactococcus gasseri*, respectively.

[0097] in conclusion:

[0098] according to Figure 1 The data showed that between 250bp and 500bp on the marker scale (i.e., close to 250bp, corresponding to the theoretical length of the target product of 302bp), lanes 1 to 3 exhibited bright, clear, and single specific amplification bands; while lanes 4 to 9 showed no amplification bands in the target region. This protocol, through multiple alignments of ITS sequences of various fish pathogenic Nocardia species, defined conserved and specific regions, and designed primers as shown in SEQ ID NO. 1 and SEQ ID NO. 2 for the specific regions common to the target strains. These primers can accurately identify the target sequences of Nocardia amberjack, Nocardia asteroides, and Nocardia sand salmon, and effectively avoid sequence interference from non-target pathogens. Comparative Example 1 used a universal 16S rRNA amplification method with high target sequence homology, but this method had the problem of cross-reaction with non-pathogenic bacteria or closely related species; Comparative Example 2 used a single primer method, which was limited by the target sequence selection and could only amplify a specific single species; Comparative Example 3 used a traditional isolation, culture, and identification method with a long cycle. This method achieves simultaneous and specific amplification of pathogenic Nocardia in major fish species, completely eliminating cross-reaction interference from common aquatic pathogens, and effectively balancing the specificity and coverage of the detection system.

[0099] Test Example 2: Sensitivity Test of Conventional PCR Methods

[0100] Test steps:

[0101] To verify the sensitivity of the detection method proposed in this protocol, PCR amplification tests were performed on Nocardia genomic DNA at known concentrations using optimized reaction parameters.

[0102] Obtain a Nocardia genomic DNA template of known concentration, and perform 10-fold serial dilutions of the template DNA to prepare concentrations of 2×10⁻⁶. -7 ng / μL, 2×10 -8 ng / μL, 2×10 -9 ng / μL, 2×10 -10 ng / μL, 2×10 -11 A series of dilution templates in ng / μL.

[0103] Using the DNA at the above-mentioned gradient concentrations as templates, PCR reaction systems were prepared. The upstream primer shown in SEQ ID NO.1 and the downstream primer shown in SEQ ID NO.2 were added to the reaction system, and conventional PCR amplification was performed using the amplification conditions of Example 1.

[0104] After the amplification reaction was completed, the PCR products were placed in an agarose gel for electrophoresis detection, and the amplification bands were observed and recorded under a gel imaging system.

[0105] Test data:

[0106] like Figure 2 As shown. Figure 2 The first lane on the left is the standard molecular weight marker (M), with molecular weight scales of 2000, 1000, 750, 500, 250, and 100 bp clearly marked from top to bottom to its left. The amplification product lanes to the right of lane M correspond to template DNA concentrations of 2 × 10⁻⁶ from left to right. -7 2×10 -8 2×10 -9 2×10 -10 2×10 -11 ng / μL.

[0107] in conclusion:

[0108] according to Figure 2 The data, between 250 bp and 500 bp on the marker scale (corresponding to the theoretical length of the target product at 302 bp), shows the first three high concentration gradients (2 × 10⁻⁶). -7 2×10 -8 2×10 -9Clear, specific target amplification bands were observed in all lanes containing template DNA (ng / μL), and the brightness of the target amplification bands decreased with decreasing template DNA concentration; when the template DNA concentration was further reduced to 2×10... -10 ng / μL and 2×10 -11 At a concentration of ng / μL, the target amplification band completely disappeared within the corresponding lane. This protocol utilizes specific primers designed based on ITS conserved region targets precisely anchored by multiplex sequence alignment. Combined with optimized annealing temperatures, this ensures efficient and specific binding and stable extension of the primers in low-volume target nucleic acid environments. This conventional PCR detection system achieves a minimum detection concentration of 2 × 10⁻⁶ for Nocardia fowleri genomic DNA. -9 With a detection capacity of ng / μL, it possesses high detection sensitivity and can meet the clinical detection and early diagnosis needs of low-load pathogens in aquaculture.

[0109] Test Example 3: Sensitivity and Time Consumption Test of qPCR Method

[0110] Test steps:

[0111] To further verify the application potential of the primers in the real-time PCR detection system, the sensitivity and detection time of this quantitative detection method were tested.

[0112] Obtain a Nocardia genomic DNA template of known concentration, and perform 10-fold serial dilutions of the template to prepare concentrations of 2×10⁻⁶. -1 ng / μL to 2×10 -14 A series of gradient dilution templates in ng / μL.

[0113] Using the DNA at the above-mentioned gradient concentrations as templates, a real-time PCR reaction system was prepared. A fluorescent dye was added to the reaction system, along with the upstream primer shown in SEQ ID NO.1 and the downstream primer shown in SEQ ID NO.2.

[0114] The prepared reaction system was placed in a real-time PCR instrument for amplification, and the total operation time from the start of system preparation to the final detection result obtained by the instrument was recorded.

[0115] After the reaction is complete, export the fluorescence amplification curve data corresponding to each concentration gradient automatically generated by the instrument.

[0116] Test data:

[0117] like Figure 3 As shown. Figure 3The horizontal axis represents the amplification cycle number (Cycle), and the vertical axis represents the fluorescence signal intensity (Rn). The red horizontal line represents the set fluorescence signal threshold. The figure records the S-shaped amplification curves generated by amplification with different template concentrations. The template DNA concentration decreases sequentially according to the order of the curve peaks (from left to right). The figure marks the high concentration range (corresponding to 2×10⁻⁶). -1 Up to 2×10 -6 ng / μL) and extremely low concentration range (corresponding to 2×10 -13 Up to 2×10 -14 The distribution location of the amplification curve (ng / μL).

[0118] in conclusion:

[0119] according to Figure 3 The data showed that as the template DNA concentration decreased, the cycle number (Ct value) of the fluorescence amplification curve shifted accordingly, with the highest template concentration crossing the threshold peak at the 24th to 25th cycle. When the template DNA concentration decreased to 2 × 10⁻⁶, the peak value was lowered. -14 At a concentration of ng / μL, the amplification curve crossed the threshold between cycles 29 and 31, and the detection system still generated a typical S-shaped amplification curve. This protocol uses primers designed based on conserved regions precisely located by ITS sequence polymorphism analysis, ensuring stable binding affinity and high-efficiency amplification of trace amounts of target nucleic acids in the quantitative PCR system. The minimum detection concentration of this qPCR detection system reaches 2 × 10⁻⁶. -14 The test results showed that the qPCR detection method took 1.2 hours in total. Compared with the traditional isolation and culture identification method in Comparative Example 3, which took 2 to 4 weeks, this quantitative fluorescence detection method effectively shortens the diagnostic cycle while ensuring high sensitivity, and is suitable for early rapid diagnosis and quantitative screening of nocardiosis in aquaculture.

[0120] Appendix:

[0121] SEQ ID NO.1 (Nocardia F3, upstream primer for specific detection):

[0122] CGTCAAGTCATCATGCCCCTTAT

[0123] SEQ ID NO.2 (Nocardia R3, downstream primer for specific detection):

[0124] ACCCTAACCGCTAACCCTGCTTC

[0125] SEQ ID NO.3 (p1208f, ITS universal amplification upstream primer):

[0126] GACGTCAAGTCATCC

[0127] SEQ ID NO.4 (Myco 23S-r, ITS universal amplification downstream primer):

[0128] GACTCGCAGGCTCATTCT

Claims

1. A method for the specific detection of Nocardia in fish based on ITS sequence analysis, characterized in that, Includes the following steps: Genomic DNA was extracted from fresh cultures of the test strain as template DNA; Using the obtained template DNA as a template, PCR amplification was performed using a specific primer pair to obtain the amplification product; the specific primer pair includes an upstream primer Nocardia F3 and a downstream primer Nocardia R3, the nucleotide sequence of the upstream primer Nocardia F3 is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer Nocardia R3 is shown in SEQ ID NO.2; The amplification products were tested to determine whether the test strain contained Nocardia ferruginosa.

2. The method for specific detection of Nocardia in fish based on ITS sequence analysis according to claim 1, characterized in that, The PCR amplification uses a standard reaction system, which includes: Taq DNA polymerase, upstream primer Nocardia F3, downstream primer Nocardia R3, template DNA, and sterile double-distilled water.

3. The method for specific detection of Nocardia in fish based on ITS sequence analysis according to claim 2, characterized in that, The standard reaction system has a volume of 25 μL and consists of the following components: 11 μL of Taq DNA polymerase at a concentration of 5 U / μL; 1 μL of the upstream primer Nocardia F3 at a concentration of 10 μM; 1 μL of 10 μM downstream primer Nocardia R3; 2 μL of template DNA at a concentration of 100 ng / μL; 10 μL of sterile double-distilled water.

4. The method for specific detection of Nocardia in fish based on ITS sequence analysis according to claim 1, characterized in that, The reaction conditions for the PCR amplification were set as follows: Pre-denaturation at 94℃ for 4 min; The process involves denaturation at 94℃ for 45 seconds, annealing at 55–68℃ for 45 seconds, and extension at 72℃ for 70 seconds, for a total of 30 cycles. Final extension at 72℃ for 10 min, then store at 4℃.

5. The method for specific detection of Nocardia in fish based on ITS sequence analysis according to claim 1, characterized in that, The specific method for detecting the obtained amplification products is as follows: gel electrophoresis detection is used.

6. The method for specific detection of Nocardia in fish based on ITS sequence analysis according to claim 5, characterized in that, The theoretical target product length for the amplification product is 302 bp.

7. The method for specific detection of Nocardia in fish based on ITS sequence analysis according to claim 1, characterized in that, The fish nocardia include one or a combination of several of Nocardia asteroides, Nocardia sandfish, and Nocardia yellowtail.

8. The method for specific detection of Nocardia in fish based on ITS sequence analysis according to claim 1, characterized in that, Before extracting genomic DNA from fresh cultures of the test strain as template DNA, the Nocardia strain to be tested was pretreated as follows: The Nocardia spp. strain to be tested was inoculated into BHI medium and incubated at 28°C for 5 to 7 days until the colony diameter reached 1 to 2 mm. Fresh culture was then collected.

9. The method for specific detection of Nocardia in fish based on ITS sequence analysis according to claim 1, characterized in that, The specific method for extracting genomic DNA from fresh cultures of the test strain is as follows: extract the DNA using a bacterial genomic DNA extraction kit according to the instructions.

10. The method for specific detection of Nocardia in fish based on ITS sequence analysis according to claim 1, characterized in that, The upstream primer Nocardia F3 and the downstream primer Nocardia R3 both have 23 bases.

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