A primer set, kit, and application for LAMP detection of Morganella morganii from crocodilian lizards.

Abstract

This invention discloses a LAMP detection primer set, kit, and application of *Morganella morganii*. This invention targets *Morganella morganii*. I have A LAMP primer set was designed for the conserved gene region, including outer primers F3 / B3 and inner primers FIP / BIP, with nucleotide sequences shown in SEQ ID NO. 2-5. This primer set exhibits high specificity, showing no cross-reactivity with common commensal bacteria and opportunistic pathogens of the Chinese alligator lizard; it has a sensitivity of up to 0.245 copies / µL, capable of detecting single-copy targets; the reaction is rapid (completed in 50 minutes), and results can be interpreted by gel electrophoresis or visual observation of turbidity changes. The primer set provided by this invention can be used to prepare a kit for detecting *Morganella morganii* in Chinese alligator lizards, suitable for rapid on-site detection in grassroots units such as Chinese alligator lizard breeding bases and wildlife conservation stations, and is of great significance for the early prevention and control of *Morganella morganii* disease in Chinese alligator lizards.

Description

Technical Field

[0001] This invention belongs to the field of microbial molecular diagnostic technology, specifically relating to a strain of Morganella morganii derived from crocodile lizards based on loop-mediated isothermal amplification (LAMP) technology. Morganella morganii This involves detection primer sets, reagent kits, and their applications, particularly a method using... nagk A four-primer isothermal amplification system that targets genes and achieves single-copy-level sensitivity without using circular primers LF / LB. Background Technology

[0002] 1. Current Status of Crocodile Lizard Conservation and Disease Threats Crocodile lizard ( Shinisaurus crocodilurus The Chinese crocodile lizard (Syngonius spp.) is a rare reptile endemic to China, belonging to the genus Syngonius in the family Syngonidae. It is a Class I protected wild animal in China and is listed as an endangered (EN) species on the IUCN Red List. This species is found only in localized mountain stream habitats in Guangxi Zhuang Autonomous Region and Guangdong Province, with a very small wild population. Artificial breeding is the core strategy for maintaining its population. However, in captivity, the Chinese crocodile lizard is susceptible to opportunistic pathogens, including Morganella morganii (Morganella spp.). Morganella morganii Morganella morganii is a newly discovered important pathogen threatening the health of Chinese alligator lizards in recent years. In a Chinese alligator lizard mortality incident that occurred in the Dagui Mountain National Nature Reserve in Guangxi Zhuang Autonomous Region in 2021, Morganella morganii was isolated and identified from the liver tissue of the diseased individuals. This bacterium can cause serious diseases such as septicemia and liver necrosis in Chinese alligator lizards, posing a significant threat to the ex-situ conservation population.

[0003] 2. Pathogenic characteristics of Morganella morganii Morganella morganii is a Gram-negative facultative anaerobic bacillus belonging to the genus Morganella in the family Enterobacteriaceae. Based on the ANI analysis of 1027 global isolates, the genus Morganella has been reclassified into 6 species, with Morganella morganii as the type species, comprising two subspecies: Morganella morganii subsp. morganii (…). M. morganii subsp. morganii ) and Morganella morganii intermediate subspecies ( M. morganii subsp. intermedius The genome of this bacterium is approximately 3.8-4.2 Mb in size and carries multiple virulence factors, including hemolysin, protease, lipopolysaccharide, and drug resistance genes. It exhibits natural or acquired resistance to various antibiotics, including β-lactams and aminoglycosides, posing a significant challenge to clinical treatment. The complete genome sequence of Morganella morganii strain DG56-16, isolated from the Chinese crocodile lizard, has been submitted to GenBank (accession number: NZ_CP032295.1). Its virulence characteristics and drug resistance lineage are not yet fully elucidated. Early and rapid diagnosis is crucial for timely isolation of infected individuals and preventing the horizontal spread of the disease within breeding populations.

[0004] 3. Limitations of existing detection technologies and the need for invention Currently, the detection of Morganella morganii mainly relies on traditional culture methods and molecular biology approaches. While traditional culture methods can serve as a fundamental means of pathogen identification, they typically require a long culture period (24-48 hours) and demand high standards in experimental conditions and operator skills, making them unsuitable for rapid on-site detection needs at the grassroots level. Conventional PCR technology, although shortening the detection cycle compared to traditional culture methods, still relies on thermal cycling and gel electrophoresis analysis, resulting in high equipment costs, complex operation, and the potential for cross-contamination of amplified products. Real-time quantitative PCR (qPCR) offers high sensitivity and specificity, but its high instrument and reagent costs hinder its widespread application in field monitoring, grassroots conservation stations, and breeding bases. Loop-mediated isothermal amplification (LAMP) technology can achieve efficient amplification of target sequences under isothermal conditions (60℃-65℃) using strand displacement DNA polymerase. It offers advantages such as ease of operation, rapid reaction, and easy result interpretation, and has been widely used in on-site detection of pathogenic microorganisms.

[0005] However, existing technologies have the following obvious drawbacks: First, there are no reported LAMP detection methods for Morganella morganii derived from crocodile lizards. Existing Morganella detection methods mostly focus on clinical human strains or food-derived strains, and their target selection, primer design, and reaction systems do not take into account the specific interference of reptile sample matrices (such as cloacal swabs, liver tissue, and blood).

[0006] Second, existing LAMP detection techniques for Morganella morganii rely heavily on a six-primer system (F3 / B3 / FIP / BIP / LF / LB). Primer design is complex and synthesis costs are high. Although the introduction of the circular primers LF / LB can shorten the reaction time, it also increases the risk of non-specific amplification and the probability of primer dimer formation, which is particularly unfavorable for low-concentration DNA samples such as those from rare wild animals.

[0007] Third, existing technologies lack the ability to... nagk A Morganella morganii detection protocol targeting a gene (N-acetylglutamate kinase gene, which is a key enzyme encoding the bacterial arginine biosynthesis pathway). nagk Genes are highly conserved in *Morganella molluscum*, but less so in closely related species (*Morganella siberianis*). M. sibonii ), cold-resistant Morganella morganii ( M. psychrotolerans With a sequence similarity of less than 85% and a similarity of less than 70% with common reptile symbiotic bacteria (Aeromonas hydrophila, Pseudomonas, etc.), it possesses genus-specific recognition capabilities and is an ideal target for LAMP detection.

[0008] Therefore, there is an urgent need to develop a method targeting Morganella morganii from crocodile lizards, using... nagkThe LAMP method, which targets genes, uses a minimal four-primer system (without LF / LB), and is suitable for rapid on-site detection at the grassroots level, aims to fill the existing technological gap and improve the early screening capability for crocodile lizard diseases. Summary of the Invention

[0009] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a LAMP detection primer set, kit, and application for *Morganella morganii* from the crocodile lizard. This invention is based on strain DG56-16 of *Morganella morganii* derived from the crocodile lizard (GenBank: NZ_CP032295.1). nagk By comparing and verifying the conserved gene regions through bioinformatics, a set of specific four primers was designed, which enabled single-copy, highly specific, rapid isothermal detection of Morganella morganii from the Chinese lizard without the use of loop primers LF / LB.

[0010] The first objective of this invention is to provide a LAMP primer set for detecting Morganella morganii, the primer set consisting of outer primers F3 and B3 and inner primers FIP and BIP, wherein the nucleotide sequence of outer primer F3 is shown in SEQ ID NO.2, the nucleotide sequence of outer primer B3 is shown in SEQ ID NO.3, the nucleotide sequence of inner primer FIP is shown in SEQ ID NO.4, and the nucleotide sequence of inner primer BIP is shown in SEQ ID NO.5.

[0011] The second objective of this invention is to provide a LAMP detection kit for Morganella morganii, characterized in that it comprises the LAMP primer set and LAMP amplification reagent as described in claim 1.

[0012] Preferably, the LAMP amplification reagent includes 10×ThermoPol buffer, Bst DNA polymerase, dNTP mixture, MgSO4 solution, betaine, and nuclease-free water; During LAMP amplification, the MgSO4 solution was diluted to a final concentration of 10 mM, and the betaine was diluted to a final concentration of 0.8 M.

[0013] Preferably, it further comprises a positive control; said positive control is a nucleotide sequence as shown in SEQ ID NO.1. nagk Recombinant plasmids of gene fragments.

[0014] Preferably, the nucleotide sequence is as shown in SEQ ID NO.1. nagk The recombinant plasmid for the gene fragment was pUC57-M.morganii-nagk, with a concentration of 2.45 × 10⁻⁶. 4 copies / μL.

[0015] A third objective of this invention is to provide the application of the described LAMP primer set in the preparation of a detection kit for Morganella morganii from the crocodile lizard.

[0016] The fourth objective of this invention is to provide a method for detecting Morganella morganii in crocodilians for non-diagnostic purposes, comprising the following steps: S1. Extract DNA from the sample to be tested; S2. Using the DNA obtained in step S1 as a template, perform LAMP amplification using the primer set described above to obtain the reaction solution; S3. Determine whether the sample contains Morganella morganii by performing agarose gel electrophoresis on the reaction solution or by observing whether the reaction solution is turbid; When determining whether a sample contains *Morganella sarcodactylis* by performing agarose gel electrophoresis on the reaction solution, if a characteristic ladder-shaped band appears in the range of 200 bp-2000 bp, the sample is considered to contain *Morganella sarcodactylis*, i.e., positive; if no ladder-shaped band appears, the sample is considered to not contain *Morganella sarcodactylis*, i.e., negative. When determining whether a sample contains Morganella morganii by observing whether the reaction solution is cloudy, if the reaction solution is cloudy, the sample is considered to contain Morganella morganii, i.e., positive; if the reaction solution remains clear, the sample is considered to not contain Morganella morganii, i.e., negative.

[0017] Preferably, the LAMP amplification reaction system is as follows (based on a 25 μL volume): 2.5 μL 10×ThermoPol buffer, 1.0 μL 8 U / μL Bst DNA polymerase, 2.5 μL 10 mM dNTP mixture, 2.5 μL 100 mM MgSO4 solution, 4.0 μL 5 M betaine solution, 1.0 μL 10 μM outer primer F3, 1.0 μL 10 μM outer primer B3, 1.0 μL 40 μM inner primer FIP, 1.0 μL 40 μM inner primer BIP, 1.0 μL DNA template, with the remainder being nuclease-free water. The LAMP amplification reaction procedure is as follows: amplification at 61℃-65℃ for 40-50 minutes, followed by inactivation at 80℃ for 5-10 minutes to terminate the reaction.

[0018] Preferably, the LAMP amplification reaction procedure is as follows: amplification at 61℃ for 50 minutes, followed by inactivation at 80℃ for 5-10 minutes to terminate the reaction.

[0019] Preferably, the sample to be tested is a cloacal swab, liver tissue, blood, or a sample from the environment.

[0020] Compared with the prior art, the present invention has the following significant technical advantages and innovations: (1) Target innovation: the first time with nagk A LAMP detection system for Morganella morganii was established using genes as targets. This technology has not been publicly utilized. nagk This invention presents a protocol for isothermal amplification and detection of Morganella morganii genes. Through large-scale sequence alignment, it confirms... nagk The gene is highly conserved in Morganella morganii, while its similarity to closely related species and common reptile pathogens is less than 85%, giving it excellent intragenus-specific recognition capabilities and providing a novel high-confidence target for primer design.

[0021] (2) Primer System Innovation: A simplified four-primer system (F3 / B3 / FIP / BIP) was adopted. Without using circular primers LF / LB, good amplification efficiency was achieved through a precise six-region recognition structure and optimized reaction system. This design not only reduces primer synthesis costs (compared to the six-primer system) and batch-to-batch variation risks, but also avoids the non-specific amplification problems introduced by circular primers, offering unique advantages for detecting low-concentration DNA samples from rare wild animals. The designed primers can be prepared into detection kits, significantly reducing primer synthesis costs and non-specific amplification risks. They can be applied to the epidemiological monitoring of Morganella morganii in crocodilians, pathogen screening in breeding environments, or detection in germplasm resource conservation.

[0022] (3) Breakthrough in sensitivity: The detection limit of this invention is as low as 2.45 × 10⁻⁶. -1 The sensitivity is 1000 times higher than that of conventional PCR (detection limit 2.45×10² copies / μL) and 100 times higher than that of real-time quantitative PCR (detection limit 2.45×10¹ copies / μL). It can detect early infection or low viral load carrier status, providing technical possibility for screening during the incubation period of the disease.

[0023] (4) Application scenario innovation: For the first time, a rapid on-site detection scheme for Morganella morganii was established for the Chinese alligator lizard, a national first-class protected animal. The sample types cover cloacal swabs, liver tissue and blood. The sample processing procedure is highly compatible with the LAMP reaction system. It does not require expensive thermal cyclers or fluorescence quantitative PCR instruments. Only a constant temperature water bath or metal bath is needed to complete the detection. The results can be directly interpreted by the naked eye. It is particularly suitable for grassroots protection stations, breeding bases and field monitoring sites.

[0024] (5) Excellent specificity: Cross-reactivity tests showed that the primer set of this invention did not cross-react with Aeromonas hydrophila, Dermophila tectoris, Escherichia coli, Salmonella, or Staphylococcus aureus, with a specificity of 100%. It can effectively distinguish different species within the Morganella genus (Morganella siberiana, Morganella psychrophila, etc.), ensuring accurate identification in complex microbial communities. Attached Figure Description

[0025] Figure 1 Morganella morganii, a fungus originating from crocodilians, is located in... nagk Sequence alignment diagram of LAMP primer design target regions in conserved gene regions; the red-marked areas are the LAMP primer design target regions determined through screening. nagk This is the reference sequence.

[0026] Figure 2 The recombinant plasmid pUC57-M.morganii- nagk PCR identification results; M: DNA molecular weight standard (DL2000), 1: negative control (water), 2-3: inserted DNA nagk Recombinant plasmids containing gene fragments (approximately 900 bp).

[0027] Figure 3 The results show the optimized LAMP reaction temperature; M: DNA molecular weight standard (DL2000), 1: Electrophoretic bands of the reaction product obtained by reacting at 60℃ for 60 minutes with nuclease-free water as a template (negative control), lanes 2-7: Electrophoretic bands of the reaction product obtained at reaction temperatures of 60℃, 61℃, 62℃, 63℃, 64℃, and 65℃ respectively for a reaction time of 60 minutes.

[0028] Figure 4 The results show the optimized LAMP reaction time; M: DNA molecular weight standard (DL2000), 1: negative control (nuclease-free water template, reaction at 61℃ for 50 minutes), lanes 2-6: electrophoretic bands of amplification products at reaction times of 10, 20, 30, 40, and 50 minutes, respectively.

[0029] Figure 5 The results are for LAMP primer set specificity detection; M: DNA molecular weight standard (DL5000); 1: negative control (sterile enzyme water), 2: Morganella morganii positive control, 3: Aeromonas hydrophila, 4: Dermophilic bacteria, 5: Escherichia coli, 6: Salmonella; 7: Staphylococcus aureus.

[0030] Figure 6 The results are for LAMP method sensitivity detection; M: DNA molecular weight standard (DL2000); 1: negative control (sterile enzyme water); 2-8: template concentrations of 2.45 × 10⁻⁸ respectively. 42.45×10³, 2.45×10², 2.45×10¹, 2.45×10 0 2.45×10 - ¹、2.45×10 - ² copies / μL. Detailed Implementation

[0031] The following embodiments are further illustrations of the present invention, but not limitations thereof. Unless otherwise specified, the reagents used in the embodiments of the present invention are all conventional reagents in the art and are commercially available.

[0032] Example 1: LAMP primer design and target gene validation 1.1 Target gene selection and sequence analysis Based on the whole genome sequence of Morganella morganii reference strain DG56-16 from crocodile lizards (GenBank accession number: NZ_CP032295.1, isolated from liver tissue of diseased crocodile lizards in Dagui Mountain National Nature Reserve, Guangxi Zhuang Autonomous Region in 2021), bioinformatics comparative analysis was used to screen... nagk The gene (N-acetylglutamate kinase, GenBank accession number: WP_064483654.1) was used as the detection target. This gene is located at positions 14171-15082 of the genome, with a full length of 912 bp, and its nucleotide sequence is shown in SEQ ID NO.1.

[0033] choose nagk The core basis for using genes as targets lies in: (1) Conservation: By comparing the 12 strains of Morganella morganii (including CP032295_1, CP086203_1, CP134699_1, CP043955_1, CP139988_1, CP064828_1, CP069157_1, CP159228_1, CP059986_1, CP166048_1, CP168464_1, CP132554_1) and 5 non-target bacteria (Vibrio parahaemolyticus) shown in Table 1, conservation was determined. Vibrio parahaemolyticus CP102434.1, Escherichia coli ( Escherichia coli CP076318.1 or CP053787.1, Mycobacterium rijalinum ( Mycobacterium riyadhensis CP045092.1, Intracellular Mycobacterium ( Mycobacterium intracellulare The genome sequence of CP076378.1 was compared with the results of the alignment. Figure 1 Confirmed nagk The gene is 100% conserved among Morganella morganii strains, while its sequence similarity with closely related species Morganella siberianis and Morganella psychrophila is less than 85%. nagkThis is a gene specific to Morganella morganii, not found in common bacteria such as Aeromonas hydrophila and Pseudomonas, but only in Escherichia coli. It exhibits excellent genus-specificity.

[0034] Table 1 nagk Gene comparison of different selected strains (2) Functionality: nagk The gene encodes N-acetylglutamate kinase, a key rate-limiting enzyme in the bacterial arginine biosynthesis pathway. It is an essential gene for bacteria to maintain basal metabolism and is not easily mutated or lost in the host, ensuring the long-term stability of the detection target.

[0035] 1.2 Design of four primer sets Using the LAMP primer design software PrimerExplorer V5 (https: / / primerexplorer.jp / e / ) for... nagk Primer sets were designed for conserved gene regions. Design parameters: primer Tm value 58℃-65℃, GC content 40%-60%, amplification product length 200 bp-2000 bp. Unlike the commonly used six-primer system (including loop primers LF / LB) in existing technologies, a simplified four-primer system was adopted based on the following considerations: First, nagk The length and structure of the conserved gene region are suitable for four-primer recognition. Sequence analysis revealed six consecutive and highly distinguishable recognition sites within the target region. F3 / B3 recognize the outer region, and FIP / BIP recognize the inner region, forming a complete strand substitution amplification initiation structure. Highly efficient amplification can be completed within 40-50 minutes without the assistance of loop primers.

[0036] Second, reduce the risk of nonspecific amplification. While loop primers LF / LB can shorten reaction time, their binding sites with the template are located between the FIP / BIP recognition regions. Improper design can easily lead to mismatch extensions, resulting in false positives. For samples like those from crocodile lizards, which contain low concentrations of templates that may contain PCR inhibitors, reducing the number of primers can significantly reduce the probability of nonspecific amplification.

[0037] Third, reduce testing costs. Funding for the protection of rare and endangered wildlife is limited. Reducing the synthesis of two primers can significantly lower the production cost of the reagent kit, which is beneficial for its promotion at grassroots conservation stations.

[0038] The primer sequences were determined after manual optimization, as shown in Table 2.

[0039] Table 2. Targeting Morganella morganii nagk Genetically designed LAMP primers Note: The M. Morganii-FIP primer consists of F1c (nucleotide sequence as shown in SEQ ID NO.4, 5' end 20 nt) and F2 (nucleotide sequence as shown in SEQ ID NO.4, 3' end 19 nt); the M. Morganii-BIP primer consists of B1c (nucleotide sequence as shown in SEQ ID NO.5, 5' end 22 nt) and B2 (nucleotide sequence as shown in SEQ ID NO.5, 3' end 16 nt).

[0040] 1.3 Construction and Validation of Recombinant Plasmids Using the genomic DNA of Morganella morganii reference strain DG56-16 as a template, high-fidelity PCR enzymes were used to amplify... nagk Gene fragment (approximately 900 bp). Primer pair: nagk-F (5'-ATGTATTACGGTTTTGAT-3', SEQ ID NO.6) and nagk-R (5'-ATTGAGACAGGCTGCGCC-3', SEQ ID NO.7). PCR conditions: 95℃ for 5 min; 95℃ for 30 s, 58℃ for 30 s, 72℃ for 1 min, 35 cycles; 72℃ for 10 min. The amplified product was purified and cloned into the pUC57 vector (Beijing Qingke Biotechnology), and transformed into *E. coli* DH5α competent cells. Positive clones were picked, expanded, and plasmids were extracted and amplified by PCR. The results are shown below. Figure 2 As shown, the recombinant plasmid band numbered 3 was specific and of accurate size, and was further sent for sequencing verification. Sequencing verified the sequence accuracy, and the recombinant plasmid was named pUC57-M.morganii-nagk, with a concentration of 2.45 × 10⁻⁶. 4 Copies / μL were stored at -80°C as a positive control.

[0041] Example 2: Optimization of LAMP Reaction System 2.1 Optimization of reaction temperature Reaction system (25 μL): 2.5 μL 10×ThermoPol buffer, 1.0 μL 8 U / μL Bst 3.0 DNA polymerase, 2.5 μL 10 mM dNTP mixture, 2.5 μL 100 mM MgSO4, 4.0 μL 5 M betaine, 1.0 μL 10 μM outer primer F3, 1.0 μL 10 μM outer primer B3, 1.0 μL 40 μM inner primer FIP, 1.0 μL 40 μM inner primer BIP, 1.0 μL DNA template (recombinant plasmid pUC57-M.morganii-nagk (2.45×10⁻⁶)). 4(copies / μL), with the remainder being nuclease-free water.

[0042] Reaction procedure: Set a temperature gradient within the range of 60℃-65℃ (60℃, 61℃, 62℃, 63℃, 64℃, 65℃ respectively), with a fixed reaction time of 60 minutes, followed by inactivation at 80℃ for 5-10 minutes to terminate the reaction.

[0043] The results were verified by electrophoresis. 5 μL of the product was subjected to 2.0% agarose gel electrophoresis (100 V, 30 min). Positive reactions showed characteristic trapezoidal bands (200 bp-2000 bp), while negative reactions showed no bands.

[0044] result( Figure 3 The results showed that amplification efficiency was better and band brightness was higher within the range of 61℃-63℃, with the best amplification effect observed at a reaction temperature of 61℃; the bands were clear, the background was clean, and there was no primer dimer interference. Therefore, the optimal reaction temperature was determined to be 61℃. This temperature is lower than the commonly used temperature for conventional LAMP (63℃-65℃), which helps reduce the probability of mismatch extension of Bst 3.0 polymerase at non-specific sites, while also reducing the risk of degradation of low-concentration templates by high temperatures.

[0045] 2.2 Optimization of Reaction Time The reaction system and result determination method are the same as in 2.1.

[0046] Reaction procedure: React at 61°C for 10, 20, 30, 40 or 50 minutes respectively, followed by inactivation at 80°C for 10 minutes to terminate the reaction.

[0047] result( Figure 4 The results showed that a faint trapezoidal band was visible after 10 minutes of reaction, indicating that the four-primer system started up rapidly. The band brightness gradually increased with increasing reaction time, reaching its peak at 50 minutes, with no background amplification in the negative control. Further extending the reaction time to 60 minutes did not significantly increase the band brightness, but the nonspecific background slightly increased. Therefore, the optimal reaction time was determined to be 50 minutes. Under these conditions, the LAMP reaction exhibits higher amplification yield and lower nonspecific risk compared to the six-primer system, making it of significant value for the accurate detection of rare and endangered wildlife samples.

[0048] Example 3: Specificity Detection 3.1 Cross-reactivity test The following strains were detected using the four primer sets shown in Table 2, the optimal LAMP detection system and procedure optimized in Example 2 (reaction at 61°C for 50 minutes): recombinant plasmid pUC57-M. morganii-nagk (positive control), Aeromonas hydrophila (… Aeromonas hydrophila ), Escherichia coli ( Escherichia coliATCC 25922), Salmonella from crocodile lizards ( Salmonella spp. LK18-19), Staphylococcus aureus ( Staphylococcus aureus (ATCC 25923), with sterile enzyme water as the negative control. The above-mentioned strains are all common symbiotic or opportunistic pathogens of the crocodile lizard, covering the main disruptive groups in the crocodile lizard's microbial community.

[0049] result( Figure 5 The results showed that only the Morganella morganii positive control exhibited a positive ladder-shaped band (lane 2), while Aeromonas hydrophila, Dermophilic dermatophytes, Escherichia coli, Salmonella, Staphylococcus aureus, and the negative control all showed no amplification bands. This result confirms that the four-primer set used is highly specific, showing no cross-reactivity with the five non-target strains tested, achieving 100% specificity. This specificity stems from two aspects: firstly... nagk The gene itself exhibits significant sequence differences within and outside the Morganella genus; secondly, the six-region recognition structure requires simultaneous matching of six sites, and any mismatch in any region will block the cascade reaction of strand substitution amplification, thus ensuring recognition fidelity from a mechanistic perspective.

[0050] Example 4: Sensitivity Detection 4.1 Determination of detection limit The recombinant plasmid pUC57-M.morganii-nagk was serially diluted 10-fold to obtain a concentration of 2.45 × 10⁻⁶. 4 copies / µL up to 2.45×10 -2 Samples were prepared in copies / µL, with three replicates for each concentration, and LAMP amplification was performed using the optimal LAMP detection system and optimal reaction procedure obtained in Example 2.

[0051] result( Figure 6 The display showed a concentration as low as 2.45 × 10⁻⁶. -1 Clear trapezoidal bands were still detectable at 2.45 × 10⁻⁶ copies / µL. - No amplification band was observed at 2 copies / µL. The calculated limit of detection (LOD) was 2.45 × 10⁻⁶. -1 The detection limit was calculated at copies / µL, and statistical probability analysis showed that the detection rate at the single-molecule level was >95%.

[0052] 4.2 Comparison with PCR method The samples obtained from the above 10-fold serial dilutions were detected in parallel using conventional PCR (primers nagk-F / R, nucleotide sequences as shown in SEQ ID NO.6 and SEQ ID NO.7, respectively) and real-time quantitative PCR (primers nagk-F / R).

[0053] Results: The detection limit for conventional PCR was 2.45 × 10² copies / µL, and the detection limit for qPCR was 2.45 × 10¹ copies / µL. The LAMP method of this invention has a sensitivity that is 1000 times higher than that of conventional PCR and 100 times higher than that of qPCR.

[0054] In summary, a LAMP detection system for Morganella morganii infection in crocodile lizards can be established, suitable for rapid on-site detection in grassroots units such as crocodile lizard breeding bases and wildlife conservation stations. This system has significant application value for the early prevention and control of Morganella morganii infection in crocodile lizards and the protection of endangered populations. The specific method steps are as follows: Collect cloacal swabs, liver tissue (approximately 25 mg), blood samples (50-100 μL), or environmental samples from the crocodile lizard. Tissue samples were ground in liquid nitrogen, and total DNA was extracted using a commercial bacterial genomic DNA extraction kit. Swab samples were directly immersed in 500 μL PBS buffer, vortexed, and the supernatant was collected for DNA extraction. The DNA extract was stored at -20°C for later use. Environmental samples were directly immersed in 500 μL PBS buffer, vortexed, and the supernatant was collected for DNA extraction. The DNA extract was stored at -20°C for later use.

[0055] The extracted DNA was used as a template to prepare the following reaction mixture (25 μL): 2.5 μL 10×ThermoPol buffer, 1.0 μL 8 U / μL Bst 3.0 DNA polymerase, 2.5 μL 10 mM dNTP mixture, 2.5 μL 100 mM MgSO4, 4.0 μL 5 M betaine, 1.0 μL 10 μM outer primer F3, 1.0 μL 10 μM outer primer B3, 1.0 μL 40 μM inner primer FIP, 1.0 μL 40 μM inner primer BIP, and 1.0 μL DNA template (recombinant plasmid pUC57-M.morganii-nagk (2.45×10⁻⁶)). 4 The reaction mixture consists of copies / μL, with the remainder being nuclease-free water. The reaction procedure is as follows: react at a constant temperature of 61℃-65℃ for 40-50 minutes (preferably 61℃ for 50 minutes), followed by inactivation at 80℃ for 5-10 minutes to terminate the reaction.

[0056] After the reaction, the resulting products were subjected to gel electrophoresis to detect the presence of *Morganella morganii* in the samples. Specifically, samples showing characteristic ladder-shaped bands in the 200-2000 bp range were considered positive for *Morganella morganii*, while samples without bands were considered negative. Furthermore, the presence of *Morganella morganii* could be determined by changes in the turbidity of the electrophoresis reaction solution; positive samples would become turbid after the reaction due to the formation of a white magnesium pyrophosphate precipitate, while negative samples would remain clear.

Claims

1. A LAMP primer set for detecting Morganella morganii in crocodilians, characterized in that, The primer set consists of outer primers F3 and B3, and inner primers FIP and BIP. The nucleotide sequence of the outer primer F3 is shown in SEQ ID NO.2, the nucleotide sequence of the outer primer B3 is shown in SEQ ID NO.3, the nucleotide sequence of the inner primer FIP is shown in SEQ ID NO.4, and the nucleotide sequence of the inner primer BIP is shown in SEQ ID NO.

5.

2. A LAMP detection kit for Morganella morganii from the Chinese crocodile lizard, characterized in that, It includes the LAMP primer set and LAMP amplification reagent as described in claim 1.

3. The reagent kit according to claim 2, characterized in that, The LAMP amplification reagents include 10×ThermoPol buffer, Bst DNA polymerase, dNTP mixture, MgSO4 solution, betaine, and nuclease-free water; During LAMP amplification, the MgSO4 solution was diluted to a final concentration of 10 mM, and the betaine was diluted to a final concentration of 0.8 M.

4. The reagent kit according to claim 2, characterized in that, It also includes a positive control; the positive control is a nucleotide sequence as shown in SEQ ID NO.

1. nagk Recombinant plasmids of gene fragments.

5. The reagent kit according to claim 4, characterized in that, The nucleotide sequence described is as shown in SEQ ID NO.

1. nagk The recombinant plasmid for the gene fragment was pUC57-M.morganii-nagk, with a concentration of 2.45 × 10⁻⁶. 4 copies / μL.

6. The use of the LAMP primer set according to claim 1 in the preparation of a detection kit for Morganella morganii from crocodilians.

7. A method for detecting Morganella morganii in crocodilians for non-diagnostic purposes, characterized in that, Includes the following steps: S1. Extract DNA from the sample to be tested; S2. Using the DNA obtained in step S1 as a template, perform LAMP amplification reaction using the primer set described in claim 1 to obtain a reaction solution; S3. Determine whether the sample contains Morganella morganii by performing agarose gel electrophoresis on the reaction solution or by observing whether the reaction solution is turbid; When determining whether a sample contains Morganella morganii by performing agarose gel electrophoresis on the reaction solution, if a characteristic ladder-shaped band appears in the range of 200 bp-2000 bp, the sample is considered to contain Morganella morganii, i.e., positive. If there is no trapezoidal band, the sample is determined to be free of Morganella morganii, i.e., negative. When determining whether a sample contains Morganella morganii by observing whether the reaction solution is cloudy, if the reaction solution is cloudy, the sample is considered to contain Morganella morganii, i.e., positive; if the reaction solution remains clear, the sample is considered to not contain Morganella morganii, i.e., negative.

8. The method according to claim 7, characterized in that, The LAMP amplification reaction system is as follows (based on a 25 μL volume): 2.5 μL 10×ThermoPol buffer, 1.0 μL 8 U / μL Bst DNA polymerase, 2.5 μL 10 mM dNTP mixture, 2.5 μL 100 mM MgSO4 solution, 4.0 μL 5 M betaine solution, 1.0 μL 10 μM outer primer F3, 1.0 μL 10 μM outer primer B3, 1.0 μL 40 μM inner primer FIP, 1.0 μL 40 μM inner primer BIP, 1.0 μL DNA template, with the remainder being nuclease-free water. The LAMP amplification reaction procedure is as follows: amplification at 61℃-65℃ for 40-50 minutes, followed by inactivation at 80℃ for 5-10 minutes to terminate the reaction.

9. The method according to claim 8, characterized in that, The LAMP amplification reaction procedure is as follows: amplification at 61℃ for 50 minutes, followed by inactivation at 80℃ for 5-10 minutes to terminate the reaction.

10. The method according to claim 8, characterized in that, The samples to be tested are cloacal swabs, liver tissue, blood, or environmental samples.

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