Design method of lamp primer set with self-shielding function, primer set, kit and application

Abstract

The application provides a design method, a primer set, a kit and an application of a LAMP primer set with a self-shielding function, and belongs to the field of loop-mediated isothermal amplification (LAMP) technology. The design method comprises the following steps: designing and screening an initial LAMP primer set of target bacteria according to target genes and SNP sites of the target bacteria; adding a first shielding sequence which is reverse complementary to 8-15 bases close to the 3' end of an F2 region of an initial inner primer FIP to the 5' end of the initial inner primer FIP; adding a second shielding sequence which is reverse complementary to 8-15 bases close to the 3' end of a B2 region of an initial inner primer BIP to the 5' end of the initial inner primer BIP; replacing the initial inner primer FIP with a new FIP and replacing the initial inner primer BIP with a new BIP to obtain a LAMP primer set with a self-shielding function. The LAMP primer set provided by the application can avoid cross-reaction, non-specific amplification and misjudgment.

Description

Technical Field

[0001] This invention belongs to the field of loop-mediated isothermal amplification (LAMP) technology, and particularly relates to the design method, primer set, kit, and application of LAMP primer sets with self-shielding function. Background Technology

[0002] In aquaculture, livestock and poultry farming, food hygiene, and clinical diagnosis and treatment, rapid and accurate detection of pathogenic microorganisms is crucial for early warning, early treatment, and minimizing losses from disease outbreaks. Currently, routine detection of pathogenic microorganisms still relies on traditional microbial isolation and culture combined with biochemical identification. While this method is a classic technique for pathogen detection, offering advantages such as intuitive results and the ability to obtain pure culture strains, it suffers from unavoidable core shortcomings: the detection process is cumbersome and time-consuming. The entire process of isolating, purifying, and biochemically validating a single strain typically takes 3-5 days, and for some difficult-to-culture or slow-growing pathogens, the detection cycle can take more than a week. In emergency scenarios such as disease outbreaks and public health emergencies, this technology cannot achieve rapid screening and accurate identification of pathogens, easily delaying optimal treatment and causing large-scale economic losses to the aquaculture industry or posing public health and safety risks.

[0003] To overcome the time-sensitivity limitations of traditional detection technologies, molecular detection technologies centered on polymerase chain reaction (PCR) and their derivatives are gradually being applied to pathogen detection. These technologies, relying on the principle of specific nucleic acid amplification, significantly shorten detection time, improve detection sensitivity, and enable rapid initial screening of pathogens. However, these technologies are highly dependent on sophisticated thermal cycling instruments and fluorescence detection equipment, and require stringent professional operational skills from testing personnel and stringent laboratory environment requirements. They also result in high testing costs and poor equipment portability. Especially in resource-constrained scenarios such as grassroots breeding sites, remote pastoral areas, and emergency field testing, large-scale promotion and routine application are difficult to achieve, failing to meet the urgent needs of frontline workers for "rapid on-site testing and immediate interpretation."

[0004] Loop-Mediated Isothermal Amplification (LAMP), a novel isothermal nucleic acid amplification technology, overcomes the temperature cycling limitations of traditional PCR. Utilizing 6-8 specific primers to recognize 8 independent regions on the target gene, it enables highly efficient nucleic acid amplification under isothermal conditions (60-65℃), offering core advantages such as speed, efficiency, high sensitivity, low equipment dependence, and easy result interpretation. This technology eliminates the need for expensive thermal cyclers, requiring only simple equipment such as a constant-temperature water bath or metal bath for detection. Amplified products can be directly interpreted through visual observation of turbidity, fluorescence development, and test strip bands. It perfectly meets the rapid detection needs of grassroots fieldwork and resource-scarce scenarios, and is widely recognized in the industry as an ideal technology for rapid on-site pathogen detection. It possesses extremely high application value and promotion potential in fields such as aquaculture pathogen screening, livestock and poultry disease monitoring, and foodborne pathogen detection.

[0005] Currently, LAMP technology is mostly used for qualitative detection at the genus and species level. However, in actual aquaculture and testing scenarios, multiple closely related species often exist within the same genus, with significant differences in pathogenicity, drug resistance, and pathogenic mechanisms (for example, Aeromonas hydrophila and Aeromonas vesiculosus, which are prevalent in aquaculture, belong to the same genus Aeromonas, have similar clinical symptoms and overlapping host ranges, but their virulence factors and drug resistance profiles differ greatly; accurate differentiation between them is crucial for precise disease treatment). Therefore, achieving accurate identification of closely related species within a genus is key to improving the practicality of pathogen detection and guiding scientific prevention and control.

[0006] However, applying LAMP technology to precise identification at the genus-level species level faces a core technological bottleneck that has yet to be overcome, severely restricting its precise application: the target gene sequences of closely related species within a genus are highly conserved and homologous, with only a few single nucleotide polymorphism (SNP) site differences between species, and no large fragments of specific sequences that can be used as primer design targets. Traditional LAMP primer design only focuses on specific matching of target sequences, without specific optimization design for trace SNP sites. Primers are prone to non-specific binding with homologous sequences of non-target species, triggering cross-amplification reactions, ultimately leading to false positives and species misidentification.

[0007] Furthermore, traditional LAMP primer systems have extremely weak ability to recognize single-base mismatches, making it impossible to accurately distinguish SNP differences between target and closely related species. Even with a single base mismatch between the primer and template, isothermal amplification can still be initiated, thus losing species-specific identification. This technical deficiency means that existing LAMP detection systems can only achieve general detection at the genus level, failing to accurately distinguish closely related species within the same genus. This makes it difficult to meet the needs of precise pathogen typing and treatment in scenarios such as aquaculture and clinical diagnosis, greatly limiting the application scenarios and industrial value of LAMP technology in high-end precision detection fields.

[0008] In summary, existing detection technologies are either time-consuming or difficult to apply in the field. LAMP technology, which is suitable for field testing, cannot overcome the technical barrier of accurate identification of SNP sites in closely related species, and has problems such as insufficient specificity, easy cross-reaction, and species misidentification. Summary of the Invention

[0009] In view of this, the purpose of this invention is to provide a design method, primer set, kit and application of LAMP primer set with self-shielding function. The self-shielding primers provided by this invention can accurately identify multiple bacterial species under the same harsh conditions, and can avoid cross-reaction, non-specific amplification and misjudgment during detection.

[0010] This invention provides a method for designing LAMP primer sets with self-shielding function, comprising the following steps: 1) Design several sets of LAMP primers for the target bacteria based on the target gene and the SNP sites within the gene sequence; 2) From the several sets of LAMP primers obtained in step 1), screen for LAMP primers that form intramolecular secondary structures with a free energy ΔG ≥ -11.2 kcal / mol and ≤ -6.0 kcal / mol, and then select the LAMP primer with the best specificity as the initial LAMP primer through specificity experiments. The initial LAMP primers include initial outer primers F3 and B3, initial inner primers FIP and BIP, and initial loop primers LF and LB; 3) A new FIP is obtained by adding a first shielding sequence to the 5' end of the initial inner primer FIP. The first shielding sequence is inversely complementary to 8-15 bases near the 3' end in the F2 region of the initial inner primer FIP. A new BIP is obtained by adding a second shielding sequence to the 5' end of the initial inner primer BIP. The second shielding sequence is inversely complementary to the 8-15 base sequence near the 3' end of the B2 region in the initial inner primer BIP. 4) Replace the initial inner primer FIP with the new FIP obtained in step 3), and replace the initial inner primer BIP with the new BIP to obtain a highly specific LAMP primer set with self-shielding function.

[0011] Preferably, the target bacteria are one or more strains within the same genus.

[0012] This invention provides a LAMP primer set for the precise identification of species within the genus Aeromonas, with target bacteria including Aeromonas hydrophila, Aeromonas vernix, Aeromonas guinea pig, Aeromonas schubert, and Aeromonas temperate. The LAMP primer set is shown in Table 2.

[0013] This invention provides a LAMP primer set for the precise identification of species within the genus Vibrio, targeting Vibrio harveyi, Vibrio parahaemolyticus, Vibrio anguillarum, and Vibrio alginolyticus; the LAMP primer set is shown in Table 5.

[0014] This invention provides the application of the LAMP primer set described above in the preparation of a kit for detecting multiple target bacteria.

[0015] Preferably, the plurality of target bacteria include different species within the same genus.

[0016] This invention provides an aquatic pathogen detection kit, comprising the aforementioned LAMP primer set, strand displacement DNA polymerase, dNTPs, buffer solution, and fluorescent indicator.

[0017] Preferably, the kit also includes a medication decision guide.

[0018] Compared with existing technologies, this invention has the following beneficial effects: The design method of LAMP primer sets with self-shielding function provided by this invention, by adding a screening step and adding a shielding sequence to the inner primers based on traditional LAMP primer design, enables the design of LAMP primer sets with self-shielding function to accurately identify multiple bacterial species under identical harsh conditions, especially different species within the same genus, with high specificity, avoiding cross-reaction, non-specific amplification, and misjudgment during detection. This invention provides a key technological breakthrough that can solve industry technical bottlenecks and achieve "accurate detection" from "undetectable". Detailed Implementation

[0019] This invention provides a method for designing LAMP primer sets with self-shielding function, specifically including the following steps: First, the present invention determines the target gene based on the species of the target bacteria. In the present invention, the target gene is preferably a housekeeping gene. The present invention does not have a special limitation on the species of the housekeeping gene, but determines it according to the taxonomic genus of the target bacteria. For example, when the target bacteria is a species of Aeromonas, the gyrB gene (DNA gyrase B subunit) is selected as the target gene. When the target bacteria is a species of Vibrio, the rpoB gene (RNA polymerase β subunit) is selected as the target gene.

[0020] This invention, after selecting a target gene, determines the SNP sites within that target gene. Specifically, this invention uses sequence alignment to identify the specific SNP sites within the target gene of different species within the same genus. Then, the sequence of the target gene and the corresponding SNP sites for each target bacterium are input into primer design software. Several sets of LAMP primers are then designed, each containing F3, B3, FIP (F1c + F2), and BIP (B1c + B2) primers, all of which are linear sequences.

[0021] This invention screens LAMP primers from several sets of obtained primers that have a free energy ΔG ≥ -11.2 kcal / mol and ≤6.0 kcal / mol for forming intramolecular secondary structures. Then, through specificity experiments, the LAMP primers with the best specificity are selected as the initial LAMP primers. The initial LAMP primers include initial outer primers F3 and B3, initial inner primers FIP and BIP, and initial loop primers LF and LB.

[0022] After obtaining the initial LAMP primers, this invention adds a first shielding sequence to the 5' end of the initial inner primer FIP to obtain a new FIP. The first shielding sequence is inversely complementary to 8-15 bases near the 3' end in the F2 region of the initial inner primer FIP. A second shielding sequence is added to the 5' end of the initial inner primer BIP to obtain a new BIP. The second shielding sequence is inversely complementary to 8-15 bases near the 3' end in the B2 region of the initial inner primer BIP. This invention replaces the initial inner primer FIP with a new FIP and replaces the initial inner primer BIP with a new BIP to obtain a highly specific LAMP primer set with self-shielding function; the LAMP primer set is synthesized according to the sequence of the obtained LAMP primer set; this invention does not have any special limitations on the synthesis, and conventional primer synthesis methods in the art can be used; in the specific implementation of this invention, a biotechnology company was commissioned to synthesize the above-mentioned LAMP primer set.

[0023] This invention provides a LAMP primer set for the precise identification of species within the genus Aeromonas, with target bacteria including Aeromonas hydrophila, Aeromonas vernix, Aeromonas guinea pig, Aeromonas schubert, and Aeromonas temperate. The LAMP primer set is shown in Table 2; the LAMP primer set was designed and prepared according to the above design method.

[0024] This invention provides a LAMP primer set for the precise identification of species within the genus Vibrio, targeting Vibrio harveyi, Vibrio parahaemolyticus, Vibrio anguillarum, and Vibrio alginolyticus; the LAMP primer set is shown in Table 5, and the LAMP primer set was designed and prepared according to the above design method.

[0025] This invention also provides the application of the LAMP primer set in the preparation of a kit for detecting multiple target bacteria. In this invention, the multiple target bacteria include different species within the same genus, or may simultaneously include different species within different genera.

[0026] The present invention also provides an aquatic pathogen detection kit, comprising the aforementioned LAMP primer set, strand displacement DNA polymerase, dNTPs, buffer solution, and fluorescent indicator.

[0027] In this invention, the kit further includes a medication decision guide, which contains a correspondence between pathogen species and recommended drugs, including: Aeromonas hydrophila (… Aeromonas hydrophila Corresponding to florfenicol or thiamphenicol; Aeromonas verrucosa ( Aeromonas veronii ) corresponds to sulfonamide drugs; Aeromonas vulgaris ( Aeromonas caviae ) corresponds to enrofloxacin or fluoroquinolone drugs; Aeromonas schuberis ( Aeromonas schubertii Corresponding cephalosporin antibiotics; Aeromonas hydrophila ( Aeromonas sobria ) corresponds to amoxicillin or β-lactam antibiotics; Vibrio harveyi ( Vibrio harveyi ) corresponds to doxycycline or tetracycline drugs; Vibrio parahaemolyticus ( Vibrio parahaemolyticus Corresponding to quinolone drugs; Vibrio anguillarum ( Vibrio anguillarum ) corresponds to sulfamethoxypyrimidine or florfenicol; Vibrio alginolyticus ( Vibrio alginolyticus This corresponds to chloramphenicol or furazolidone. The medication decision guideline provides a reference for medication after aquatic pathogen detection. This invention does not specifically limit the strand displacement DNA polymerase, dNTPs, buffer solution, and fluorescent indicator; commercially available products commonly used in the field can be used.

[0028] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0029] For specific experiments in the method of this invention, please refer to the methods disclosed in the book "Principles and Applications of PCR Technology".

[0030] Example 1

[0031] This embodiment uses the method of the present invention to design LAMP primers for common Aeromonas species: Step 1, download the whole genome sequences of the following strains of Aeromonas from the NCBI database: Aeromonas hydrophila (accession number: CP033097.1), Aeromonas veronii (accession number: CP038967.1), Aeromonas caviae (accession number: CP046654.1), Aeromonas schubertii (accession number: CP033102.1), and Aeromonas sobria (accession number: CP053214.1); use MEGA 11 software to perform multiple sequence alignment on the coding region of the gyrB (DNA gyrase B subunit) gene of the above strains to find single nucleotide polymorphism (SNP) sites that exist among different species and are suitable for high-specificity primer design.

[0032] Through comparative analysis, a species-specific SNP site was selected within the F2 primer-binding region of the gyrB gene for each species (the following site numbers are all relative to the gyrB gene coding region of the reference sequence A. hydrophila CP033097.1): Aeromonas hydrophila: A at position 742. Aeromonas veronii: G at position 742, Aeromonas caviae: T at position 808, Aeromonas schubertii: locus 721 is C, Aeromonas sobria: G at position 895.

[0033] This embodiment uses traditional LAMP primer design: Use PrimerExplorer V5 software (https: / / primerexplorer.jp / lampv5 / ). The target sequences of the gyrB gene and the SNP sites of the five Aeromonas species were input respectively. Free energies ΔG ≥ -11.2 kcal / mol and ≤ -6.0 kcal / mol for intramolecular secondary structures were screened. The software automatically generated five sets of conventional LAMP primers, each containing primers F3, B3, FIP (F1c + F2), and BIP (B1c + B2). All primers were linear sequences and synthesized by Sangon Biotech (Shanghai) Co., Ltd. The initial LAMP primer sets for each bacterium obtained in this example are shown in Table 1.

[0034] Table 1 LAMP primer sets obtained by traditional methods

[0035] Primer design with self-shielding function: Taking Aeromonas hydrophila as an example, the complete sequence of its initial FIP (see Table 1) is as follows: 5′-TGTGTTGTTCCAACCTTGAATTCGAAGTGACTGTGGGGC-3′ The FIP consists of F1c (5′ end portion) and F2 (3′ end portion), where the F2 region is the sequence closest to the 3′ end: 5′-ATTCGAAGTGACTGTGGGGC-3′ (20 nt) 12 bases are selected from the F2 region near the 3′ end: 5′-TGACTGTGGGGC-3′ Design a masking sequence that is inversely complementary to the above 12 bases: 5′-GCCCCACAGTCA-3′ The shielding sequence was directly added to the 5′ end of the initial FIP without any gap, forming a self-shielded FIP primer (Ah-FIP-shielded): 5′-GCCCCACAGTCA-TGTGTTGTTCCAACCTTGAATTCGAAGTGACTGTGGGGC-3′ The final optimized primer sequences with self-shielding function are shown in Table 2.

[0036] Using the same strategy, self-shielding FIP primers for Aeromonas vernix (Av-FIP-shielded), Aeromonas guinea pig (Ac-FIP-shielded), Aeromonas schubert (As-FIP-shielded), and Aeromonas temperate (Aso-FIP-shielded) were designed and synthesized. All modified primers were evaluated using the OligoAnalyzer Tool (IDT) online software, confirming that they all formed stable hairpin structures at 25°C (predicted ΔG values ​​were all less than -6.0 kcal / mol).

[0037] The optimized LAMP primer sets for each bacterium are shown in Table 2 (Table 2 only shows the self-shielding primers FIP and BIP; F3, B3, LF and BF are the same as in Table 1).

[0038] Table 2 LAMP primer sets with self-shielding function

[0039] Example 2

[0040] This example verifies the specificity of the primers designed in Example 1: Template DNA preparation: The five standard strains of Aeromonas (all five strains were purchased from Beijing Baina Biotechnology Co., Ltd., with strain numbers of Aeromonas hydrophila BNCC336453, Aeromonas vernix casei BNCC138468, Aeromonas guinea pig BNCC368412, Aeromonas schubert BNCC139096, and Aeromonas temperate BNCC337464) were inoculated into LB liquid medium and cultured overnight at 37°C with shaking at 200 rpm. 1 mL of the bacterial culture was taken, and genomic DNA was extracted using a bacterial genomic DNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd., DP302). The concentration and purity were determined using a NanoDrop One ultra-micro spectrophotometer, and the samples were uniformly diluted to 50 ng / μL for later use.

[0041] LAMP reaction system and procedure: Reaction system: 12.5 μL of 2× Isothermal Amplification Buffer (NEB), with additional MgSO4 added to a final concentration of 8 mM, 1.6 μM FIP / BIP, 0.4 μM F3 / B3, 1.4 mM dNTP Mix, and 1.2 μL of Bst 2.0 WarmStart® DNA Polymerase (NEB, M0538L). 2 μL (100 ng) of template DNA was added to 25 μL of nuclease-free water; the amount of primers corresponding to each bacterium in the reaction system was 1 μL (the amount of primers corresponding to each bacterium in the mixed primers was 1 μL).

[0042] Reaction procedure: Run on a QuantStudio 5 real-time quantitative PCR instrument (Applied Biosystems), react at 65°C for 40 minutes, and collect the fluorescence signal of the SYBR Green I channel every 30 seconds.

[0043] Validation of the specificity of self-shielding primers in a mixed template environment: To simulate common mixed infection scenarios in actual aquatic samples and to more rigorously verify the specificity of the self-shielding primers, mixed template detection was performed: five genomic DNAs (each at a concentration of 10 ng / μL) of Aeromonas hydrophila (Ah), Aeromonas vernix (Av), Aeromonas guinea pig (Ac), Aeromonas schubert (A. sch), and Aeromonas temperate (A. so) were mixed in equal volumes to prepare a mixed template with a total concentration of 10 ng / μL; Subsequently, LAMP reactions were performed with single templates and the above mixed templates using the five self-shielded primer sets shown in Table 2 (Ah-, Av-, Ac-, Asch-, Aso-shielded-set) and the traditional Aeromonas hydrophila linear primer set shown in Table 1 (Ah-linear-set), respectively.

[0044] The results of the self-shielding primer set detection are shown in Table 3. + indicates amplification, and the number in parentheses after + is the Ct value. In the mixed template containing genomic DNA of five Aeromonas species, each self-shielding primer set showed high specificity.

[0045] Table 3 Detection results in the mixed template

[0046] Table 3 shows that in the mixed template containing genomic DNA from five Aeromonas species, each set of self-shielding primers specifically amplified only its target pathogen, with Ct values ​​of the amplification curves being largely consistent with the single-template control, while showing no amplification signal for the other four non-target bacteria. This confirms that the self-shielding structure effectively prevents primers from erroneously binding to non-target sequences in a complex template background, solving the core interference problem in multiplex detection and mixed infection samples.

[0047] Traditional linear primer sets (Ah-linear-set) exhibited severe cross-reactivity in mixed templates, producing significant amplification signals not only for the target bacterium (Ah) but also for the other four non-target bacteria. This indicates that traditional primers are completely incapable of accurate identification in the context of mixed infections, and their detection results can seriously mislead medication decisions.

[0048] For example, Ah-linear-set's target template ( A.h Strong amplification occurred (Ct = 15.5). However, for the non-target Aeromonas verrucosa (…),… A.v Aeromonas vaginalis ( ), A.c Aeromonas schubert ( A.sch ) and Aeromonas hydrophila ( A.so The template also produced obvious amplification curves (Ct values ​​of 18.3, 19.7, 22.1, and 20.8, respectively), indicating severe cross-reactivity. This result proves that traditional linear primers are completely unsuitable for the accurate identification of species within the genus Aeromonas.

[0049] Example 3

[0050] This embodiment uses the method of the present invention to design primers for Vibrio-related bacteria.

[0051] The highly conserved rpoB (RNA polymerase β subunit) gene from the genus *Vibrio* was selected as the target. Comparison with *Vibrio harveyi* (… Vibrio harveyi ), Vibrio parahaemolyticus ( Vibrio. parahaemolyticu s), Vibrio anguillarum ( Vibrio anguillarum ) and Vibrio alginolyticus ( Vibrio alginolyticus The rpoB gene sequence of each of the four Vibrio species was determined to identify their respective specific SNP sites. Self-shielding FIP primers (Vh-FIP-shielded, Vp-FIP-shielded, Va-FIP-shielded, Val-FIP-shielded) were designed and synthesized in strict accordance with the "self-shielding" design strategy described in Example 1.

[0052] The free energy (ΔG) of the self-shielding FIP primers for the four Vibrio species was evaluated using the OligoAnalyzer Tool. At 25°C, the predicted ΔG values ​​for all Vibrio self-shielding FIP primers were in the range of -7.9 kcal / mol to -10.5 kcal / mol, also meeting the screening criterion of ΔG < -5 kcal / mol. This result demonstrates that the self-shielding primer design strategy of this invention is universally applicable to pathogens of different genera (Aeromonas and Vibrio), and can stably form the desired self-shielding structure. These selected primers were used in subsequent specificity verification experiments.

[0053] The primer set designed using the conventional method in this embodiment is shown in Table 4.

[0054] Table 4 LAMP primer sets designed using traditional methods

[0055] The optimized LAMP primer sets for each bacterium are shown in Table 5 (Table 5 only shows the self-shielding primers FIP and BIP; F3, B3, LF and BF are consistent with Table 4).

[0056] Table 5 LAMP primer sets with self-shielding function

[0057] Example 4

[0058] This embodiment validates the specificity of the optimized primers designed in Examples 1 and 3. To comprehensively evaluate the specificity of each primer set, a fully crossover design was used in this experiment. Nine self-shielded primer sets were paired with nine pathogen DNA templates, resulting in 81 detection combinations (9 primer sets × 9 templates). Each combination included triple technical replicates.

[0059] The standard strains of the above four Vibrio species used genomic DNA (prepared using the same method as in Example 1). Vibrio harveyi BNCC336937, Vibrio parahaemolyticus BNCC138541, Vibrio anguillarum BNCC354817, and Vibrio alginolyticus were all purchased from Beijing Beina Chuanglian Biotechnology Research Institute.

[0060] Template DNA preparation: Single template: Aeromonas hydrophila were prepared separately ( A.h Aeromonas versicolor ( A.v Aeromonas vaginalis ( ), A.c Aeromonas schubert ( A.sch Aeromonas hydrophila ( ), Aeromonas hydrophila A.so ), Vibrio harveyi ( V.h ), Vibrio parahaemolyticus ( V.p ), Vibrio anguillarum ( V.a ) and Vibrio alginolyticus ( V.al The genomic DNA of a total of 9 pathogens was collected, and the concentration of each was adjusted to 10 ng / μL.

[0061] Nine mixed templates: Equal volumes of DNA from the above nine pathogens were mixed to prepare a mixed template solution containing genomic DNA from all bacterial species. The total DNA concentration of this mixed template was 10 ng / μL, meaning the DNA concentration of each bacterial species was approximately 1.11 ng / μL.

[0062] 9×9 complete cross-validation: Nine sets of self-shielding primers were paired with nine single pathogen DNA templates, forming 81 detection combinations to verify the specificity of each primer set at the baseline level. The reaction system and procedure were the same as in Example 2. The results are expressed as the Ct value (threshold cycle number) of the amplification curve. + indicates the presence of amplification, and the number in parentheses after + is the Ct value; no amplification signal is marked as "-". The summary results are shown in Table 6 below.

[0063] Multiplex detection with mixed template: Nine sets of self-shielding primers were mixed at a 1:1 molar ratio and placed in the same LAMP reaction tube for single-tube multiplex amplification using a mixed DNA template from nine pathogens; the reaction system and procedure were the same as in Example 2. The results are shown in Table 7.

[0064] Table 6 Summary of 9×9 Cross-Validation Experiment Results

[0065] As shown in Table 6, among all 81 detection combinations, each set of self-shielding primers only produced a clear amplification signal (Ct value <20) for its corresponding target pathogen DNA template, while no detectable amplification signal was produced for the templates of the other 8 non-target pathogens (including closely related species within the same genus).

[0066] This embodiment, through rigorous 9×9 complete cross-validation, fully demonstrates that the self-shielding primers designed in this invention completely solve the cross-reactivity problem in intrageneric interspecific identification using LAMP technology. Even for closely related species with highly homologous sequences and only a few SNP differences (such as *Aeromonas hydrophila* and *Aeromonas vesiculosus*), accurate differentiation can be achieved. This self-shielding primer design strategy has been successful on multiple pathogens in different genera (*Aeromonas* and *Vibrio*), indicating that this method is a universal and reliable solution that can be widely applied to the accurate identification of pathogens in aquaculture and other fields. This lays a solid and reliable technical foundation for subsequent precision control based on "medication decision guidelines."

[0067] Table 7 Results of Nine-fold Mixed Detection

[0068] As shown in Table 7, in the extremely complex nine-fold mixed reaction system with both primers and templates, each set of self-shielding primers still specifically amplifies only its target pathogen, with no significant delay in Ct value compared to single-fold detection, and the amplification efficiency is not significantly affected. Simultaneously, no detectable amplification signal ("-") is generated for any non-target pathogens. This fully demonstrates that the self-shielding design of this invention completely solves the core technical problem of primer interference and non-specific amplification in multiplex LAMP detection, enabling accurate and rapid simultaneous identification of multiple pathogens under complex conditions closest to real-world applications.

[0069] Comparative Example 1

[0070] The comparative samples were tested under exactly the same conditions using primers designed using conventional methods (Tables 1 and 4).

[0071] Template and experimental protocol: The template used was the same as in Example 4, consisting of a mixture of nine pathogen DNA (total concentration 10 ng / μL). Ninefold mixed LAMP amplification was performed, with the reaction system and procedure identical to Example 4. The results are shown in Table 8 below.

[0072] Table 8 Results of traditional primer nine-fold mixture detection

[0073] The results are shown in Table 8. The amplification signals of all target pathogens were extremely weak and severely delayed (Ct values ​​were all >27), indicating that a large number of primer dimers and non-specific pairings consumed the reaction components, making effective amplification difficult.

[0074] Each primer set generated amplification signals for all nine pathogen templates, indicating that in a highly complex mixture of primers and templates, traditional primers cannot distinguish between target and non-target sequences, and the cross-reactivity reaches its limit, making the results completely unusable for differential diagnosis.

[0075] This comparative example fully demonstrates that traditional linear primers completely fail in complex multiplex detection scenarios. Their inherent interprimer interference and interspecies cross-reactivity problems are drastically amplified, resulting in results with absolutely no specificity and providing no valuable diagnostic information whatsoever. This result stands in stark contrast to the performance of the self-shielding primers of this invention, which still achieve accurate identification under the same stringent conditions (see Table 7). This powerfully proves that what this invention provides is not a simple improvement, but a key technological breakthrough that can solve industry technical bottlenecks and achieve "accurate detection" from "undetectable" to "precise detection."

[0076] Example 5

[0077] This embodiment simulates a real-world application scenario in aquaculture, demonstrating the complete process from sample collection, nucleic acid extraction, rapid multiplex LAMP detection, to final medication decision-making, and validating the self-shielding primers of this invention.

[0078] Its ability to distinguish between real-world samples in complex contexts is consistent with standard methods.

[0079] 1. Sample Collection and Preprocessing

[0080] Three diseased tilapia (15-20 cm in length) exhibiting typical hemorrhagic ulcer symptoms were collected from a tilapia farm.

[0081] Under sterile operating conditions, approximately 0.1 g of liver tissue from each fish was collected and combined in a 1.5 mL sterile centrifuge tube. 1 mL of phosphate-buffered saline (PBS, pH 7.4) was added, and the mixture was thoroughly homogenized using a tissue homogenizer. The homogenate was centrifuged at 12,000 rpm for 5 minutes, and 200 μL of the supernatant was collected for later use.

[0082] 2. Gold standard method for pathogen identification (control method)

[0083] To confirm the actual types of pathogens present in the sample, traditional microbial isolation and culture methods were employed.

[0084] We conducted a parallel analysis of chemical identification, the "gold standard".

[0085] Bacterial isolation and culture: Take 100 μL of the above supernatant and spread it on nutrient agar (NA) plates and thiosulfate-citrate-cholesterol-sucrose (TCBS) agar plates respectively, and incubate at 30°C for 24-48 hours.

[0086] Colony observation and purification: Two dominant colonies were observed on NA plates: one was a round, smooth, grayish-white colony (suspected to be Aeromonas); the other was a round, yellow colony (suspected to be Vibrio). Numerous green colonies (suspected to be Vibrio alginolyticus) and a small number of yellow colonies were observed on TCBS plates. Typical single colonies with different morphologies were picked from each plate for streaking purification.

[0087] Biochemical identification and molecular verification: The purified strain was preliminarily identified by Gram staining and oxidase test, and then sent to a commercial sequencing company for full-length 16S rRNA gene sequencing.

[0088] The sequencing results, after comparison with NCBI BLAST, confirmed the species as Aeromonas hydrophila (…). Aeromonas hydrophila Aeromonas versicolor ( Aeromonas veronii ) and Vibrio alginolyticus ( Vibrio alginolyticus This result serves as the "gold standard" for evaluating the accuracy of the method of this invention.

[0089] 3. Multiplex LAMP detection based on the self-shielding primers of this invention

[0090] Rapid nucleic acid extraction: Take the remaining 100 μL of supernatant and use the commercially available bacterial genomic DNA rapid extraction kit (TIANamp Marine Animals DNA Kit) to obtain 50 μL of DNA elution buffer.

[0091] Construction of Multiplex LAMP Reaction System: For common pathogens in livestock farms, five sets of self-shielded primers (sequences shown in Table 5) designed in the above examples were selected for multiplex detection of Aeromonas hydrophila, Aeromonas vesiculosus, Vibrio harveyi, Vibrio parahaemolyticus, and Vibrio alginolyticus. The FIP, BIP, F3, B3, LF, and LB dry powders from each primer set were pre-alimited into five independent reaction wells of a five-tube PCR tube according to the optimized concentration ratios determined in Example 2 (e.g., final FIP / BIP concentration 1.6 μM, final F3 / B3 concentration 0.4 μM, final LF / LB concentration 0.8 μM).

[0092] Reaction system preparation: Prepare a 125 μL LAMP premix with the following composition: 62.5 μL 2×Isothermal Amplification Buffer, 2 μL MgSO4 (100 mM) (final concentration 8 mM), 3.5 μL dNTPMix (10 mM each) (final concentration 1.4 mM), 6 μL Bst 2.0 WarmStart® DNA Polymerase, 41 μL nuclease-free water, and 10 μL template DNA (i.e., DNA extract from diseased fish tissue). Gently mix the premix and aliquot 25 μL into each of the five wells of a quintuple PCR tube, resuspending the primer powder.

[0093] Amplification reaction: Place the reaction tube in a portable real-time PCR instrument (or a constant-temperature fluorescence detector), set the program to incubate at 65°C for 40 minutes, and collect the fluorescence signal of the SYBR Green I channel every 30 seconds.

[0094] After the reaction, the instrument software automatically generated real-time amplification curves. Results: Well 1 (Aeromonas hydrophila primers): A clear "S"-shaped amplification curve appeared at approximately 16.5 minutes, with a Ct value of 16.5, indicating a positive result (+). Well 2 (Aeromonas vesiculosus primers): A clear "S"-shaped amplification curve appeared at approximately 17.1 minutes, with a Ct value of 17.1, indicating a positive result (+).

[0095] Well 3 (Vibrio harveyi primer): No amplification curve, judged as negative (-).

[0096] Well 4 (Vibrio parahaemolyticus primer): No amplification curve, judged as negative (-).

[0097] Well 5 (Vibrio alginolyticus primer): A clear "S"-shaped amplification curve appeared at approximately 18.3 minutes, with a Ct value of 18.3, indicating a positive result (+).

[0098] LAMP assay results: The diseased tilapia sample showed a mixed infection of Aeromonas hydrophila, Aeromonas verrucosa, and Vibrio alginolyticus. This result is completely consistent with the aforementioned "gold standard" isolation, culture, and sequencing identification results.

[0099] 4. Comparative Experiment of Multiplex LAMP Detection Based on Traditional Linear Primers

[0100] To objectively and rigorously evaluate the advancements of the self-shielding primers of this invention compared to existing technologies, this comparative example was designed. This experiment compares the detection performance of traditional linear primers and the self-shielding primers of this invention in parallel under identical complex sample conditions, aiming to reveal the inherent limitations of traditional methods in practical applications.

[0101] 4.1 Experimental Materials and Methods

[0102] Test sample: The same mixed genomic DNA extracted from diseased tilapia tissue as the template used in Section 3 of this embodiment was used. This sample was confirmed by the gold standard method to be a mixed infection of Aeromonas hydrophila, Aeromonas vernix, and Vibrio alginolyticus.

[0103] Control primer set: Primers obtained according to the traditional LAMP primer design principles were used. Specifically, primer sets targeting the above three pathogens were selected, and their sequences correspond to Table 1 (traditional Aeromonas LAMP primers) and Table 4 (traditional Vibrio LAMP primers) of this specification, respectively.

[0104] Experimental design: A single-tube multiplex detection method was adopted. The three sets of conventional primers were mixed according to the final concentration ratio determined in Example 2 and placed in the same reaction tube.

[0105] Reaction system and procedure: Except for the use of traditional mixed primers, the other conditions are exactly the same as those of the self-shielding primer multiplex detection system described in Section 3 of this embodiment, including: reaction volume, buffer composition, Mg²⁺ + The concentrations of dNTPs, Bst DNA polymerase, reaction temperature (65℃), and real-time fluorescence monitoring duration (40 minutes) were all controlled to ensure fairness and adherence to the principle of a single variable in the experimental comparison.

[0106] 4.2 Test Results

[0107] Single-tube multiplex LAMP assays were performed on mixed-infection samples using traditional mixed primers to obtain...

[0108] The following results were obtained: Amplification curve characteristics: The reaction system exhibits a rapid, non-specific fluorescence rise in the early stages (approximately 10-12 minutes), followed by a plateau in the fluorescence signal. The entire amplification process does not present a typical "S"-shaped curve with a clear exponential growth phase targeting a specific target; instead, it shows an irregular upward trajectory with an undetermined threshold.

[0109] Result Interpretation: Due to the abnormal amplification curve morphology and the inability to obtain a valid Ct value, based on the standard LAMP result interpretation criteria, this test cannot definitively determine the presence of any specific target pathogen in the sample. The result is "invalid" or "indeterminate".

[0110] 4.3 Analysis and Conclusion

[0111] The comparative results demonstrate that, when faced with mixed infections commonly found in actual aquatic samples, traditional primers perform well in multiplex detection systems.

[0112] Complete failure. Clear, specific, and interpretable results were obtained by using the self-shielding primers of this invention to perform multiple detections on the same sample (successfully identifying three pathogens and providing accurate Ct values).

[0113] In summary, traditional LAMP primer design methods face insurmountable technical bottlenecks when addressing the core need for intrageneric species identification and mixed infection detection. The "self-shielding" primer design method provided by this invention, by endowing primers with the ability to self-close before the reaction, fundamentally solves the problem of non-specific interactions between primers and between primers and non-target sequences. This achieves a key technological breakthrough from "undetectable" to "precise, rapid, and multiplex detection," providing the only feasible solution for rapid on-site diagnosis and precision medicine in this field.

[0114] Furthermore, according to the "Aquatic Pathogen Drug Use Decision Guidelines" built into the kit, the recommended drug use for the test samples in this example is as follows: Aeromonas hydrophila -> Recommended drug: Florfenicol or thiamphenicol. Aeromonas vesiculosus -> Recommended drug: Sulfonamides. Vibrio alginolyticus -> Recommended drug: Chloramphenicol or furazolidone.

[0115] Comprehensive Decision-Making Approach: Given that the sample is a mixed infection of three pathogens, to avoid the localized effects of single-drug therapy...

[0116] To limit limitations and reduce the risk of drug resistance, the following combination therapy regimens are recommended: "Flofenicol (15 mg / kg fish body weight) + sulfamethoxypyrimidine (50 mg / kg fish body weight)" is mixed with feed and administered once daily for 5-7 consecutive days. This regimen covers both detected Aeromonas species. Furthermore, given that Vibrio alginolyticus is also sensitive to some sulfonamides, this combined regimen has synergistic therapeutic effects. Therapeutic effect. It is recommended to closely observe the condition of the fish during the medication period, and the dosage can be adjusted according to the situation after 3 days.

[0117] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for designing a LAMP primer set with self-shielding function, characterized in that, Includes the following steps: 1) Design several sets of LAMP primers for the target bacteria based on the target gene and the SNP sites within the gene sequence; 2) From the several sets of LAMP primers obtained in step 1), screen for LAMP primers that form intramolecular secondary structures with a free energy ΔG ≥ -11.2 kcal / mol and ≤ -6.0 kcal / mol, and then select the LAMP primer with the best specificity as the initial LAMP primer through specificity experiments. The initial LAMP primers include initial outer primers F3 and B3, initial inner primers FIP and BIP, and initial loop primers LF and LB; 3) A new FIP is obtained by adding a first shielding sequence to the 5' end of the initial inner primer FIP. The first shielding sequence is inversely complementary to 8-15 bases near the 3' end in the F2 region of the initial inner primer FIP. A new BIP is obtained by adding a second shielding sequence to the 5' end of the initial inner primer BIP. The second shielding sequence is inversely complementary to the 8-15 base sequence near the 3' end of the B2 region in the initial inner primer BIP. 4) Replace the initial inner primer FIP with the new FIP obtained in step 3), and replace the initial inner primer BIP with the new BIP to obtain a highly specific LAMP primer set with self-shielding function.

2. The design method according to claim 1, characterized in that, The target bacteria are one or more strains within the same genus.

3. A LAMP primer set for precise identification of species within the genus Aeromonas, characterized in that, The target bacteria include Aeromonas hydrophila, Aeromonas vernix, Aeromonas guinea pig, Aeromonas schubert, and Aeromonas temperate. The LAMP primer set is as follows: 。 4. A LAMP primer set for precise identification of species within the genus Vibrio, characterized in that, The target bacteria include Vibrio harveyi, Vibrio parahaemolyticus, Vibrio anguillarum, and Vibrio alginolyticus; the LAMP primer set is as follows: 。 5. The use of the LAMP primer set according to claim 3 or 4 in the preparation of a kit for detecting multiple target bacteria.

6. The application according to claim 5, characterized in that, The target bacteria include different species within the same genus.

7. A reagent kit for detecting aquatic pathogens, characterized in that, It includes the LAMP primer set, strand displacement DNA polymerase, dNTPs, buffer, and fluorescent indicator as described in claims 3 and / or 4.

8. The aquatic pathogen detection kit according to claim 5, characterized in that, The kit also includes a medication decision guide.

Images