Isothermal amplification detection primer for photobacterium damsonii subspecies and application of isothermal amplification detection primer

Abstract

The invention belongs to the technical field of microbiological detection, and particularly relates to an isothermal amplification detection primer of photobacterium damsonii subspecies and application of the isothermal amplification detection primer. The primer for loop-mediated isothermal amplification detection of the strain is designed aiming at the ureC gene of the photobacterium damsela subspecies, a quantitative analysis result can be provided by detection, and the primer has the advantages of rapidness, specificity and on-site detection.

Description

Technical Field

[0001] This invention belongs to the field of microbial detection technology, specifically relating to isothermal amplification detection primers for *Bacillus mermaidus* subsp. *mermaidus* and their applications. Background Technology

[0002] Mermaid bioluminescent bacteria, mermaid subspecies ( Photobacterium damselae subsp. damselae ☐ is one of the important pathogenic bacteria in current marine aquaculture, and can infect farmed species including leopard gill spiny perch (Scophagus leopardus). Plectropomus leopardus Pearl Giant Grouper ( Epinephelus fuscoguttatus♀×E.lanceolatus♂ ), large yellow croaker ( Larimichthys crocea ), Yellow-striped amberjack ( Seriola lalandi ), Xu's flatfish ( Sebastes schlegeli ) and tiger prawn ( Penaeus monodon Furthermore, there are reports of severe and life-threatening cases after infection with this pathogen. Currently, there are many molecular detection methods for *Bacillus mermaidus* subspecies *Mermaidus*, including PCR, PCR-denaturing gradient gel electrophoresis, random amplification of polymorphic DNA, and microfluidic quantitative PCR. However, these methods cannot yet meet the actual needs for rapid on-site detection of pathogens in aquaculture and aquatic product safety testing. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention provides isothermal amplification detection primers for *Bacillus mermaidina* subsp. *mermaidina*, specifically detection primers based on loop-mediated isothermal amplification (LAMP). This invention also provides its application method, which offers high and rapid detection specificity and allows for on-site sample detection under simple conditions (such as water baths or metal baths that provide isothermal conditions).

[0004] The isothermal amplification detection primers for *Bacillus mermaidus* subspecies *Mermaid* described in this invention include three sets of isothermal amplification detection primers, the nucleotide sequences of which are as follows: The nucleotide sequence of the first set of isothermal amplification detection primers: 1) Primer 1: Phdd _F3: 5'-ATCTGTCCTCAACAAGCC-3', as shown in SEQ ID NO: 1; 2) Primer 2: Phdd _B3: 5'-GCTTCAATTTGCTCTCGTAAT-3', as shown in SEQ ID NO: 2; 3) Primer 3: Phdd _FIP: 5'-CGGTTGTTGCATTGGAACCCGCTTTTGAAGAAGGATTATGTTCTGGC-3', as shown in SEQ ID NO: 3; 4) Primer 4: Phdd _BIP: 5'-GGTCTGGAATATGTCACGAATGCTTTTTCTGACACAACCTTTACCAAAT-3', as shown in SEQ ID NO: 4; 5) Primer 5: Phdd _LF:5'-CCGCCACCGATAAAGGTTGTTAA-3', as shown in SEQ ID NO:5; 6) Primer 6: Phdd _LB: 5'-GGAAGCCGTTGATGACTTACC-3', as shown in SEQ ID NO: 6; Nucleotide sequences of the second set of isothermal amplification detection primers: 1) Primer 1: Phdd _F3: 5'-CCCGGTACAGAAGTTGTGG-3', as shown in SEQ ID NO: 7; 2) Primer 2: Phdd _B3: 5'-GCTTCCAGCATTCGTGACAT-3', as shown in SEQ ID NO: 8; 3) Primer 3: Phdd _FIP: 5'-CCTTCTTCGGCTTGTTGAGGACATTTTCAGGCGAAGGCAAGATCGT-3', as shown in SEQ ID NO: 9; 4) Primer 4: Phdd _BIP: 5'-GGTGGCGGAACCGGTCCAGTTTTTATTCCAGACCCCTGGTGT-3', as shown in SEQ ID NO: 10; 5) Primer 5: Phdd _LF:5'-AGTATCGATTCCTCCAGCTGTT-3', as shown in SEQ ID NO:11; 6) Primer 6: Phdd _LB: 5'-GGCGGGTTCTAATGCAACA-3', as shown in SEQ ID NO: 12; Nucleotide sequences of the third set of isothermal amplification detection primers: 1) Primer 1: Phdd_F3: 5'-GATACTCACATTCATTTTATCTGTC-3', as shown in SEQ ID NO: 13; 2) Primer 2: Phdd _B3: 5'-GTCATCAACGGCTTCCAG-3', as shown in SEQ ID NO: 14; 3) Primer 3: Phdd _FIP: 5'-CCGGTTCCTCCACCGATAAAGTTTTCTCAACAAGCCGAAGAAGG-3', as shown in SEQ ID NO: 15; 4) Primer 4: Phdd _BIP: 5'-TCCAGTGGCGGGTTCTAATGTTTTATTCGTGACATATTCCAGACC-3', as shown in SEQ ID NO: 16.

[0005] The isothermal amplification detection primers for *Bacillus mermaidus* subsp. *mermaidus* were designed based on the conserved nucleotide sequence of the *ureC* gene of *Bacillus mermaidus* subsp. *mermaidus*.

[0006] The isothermal amplification detection primers for *Proteobacterium melanogaster* subsp. *mermaidum* described in this invention are applied to the detection and quantitative analysis of *Proteobacterium melanogaster* subsp. *mermaidum* for non-disease diagnosis and treatment purposes, including for the preparation of detection reagents or kit products.

[0007] The following primer combinations can be used for detection: Phdd _F3、 Phdd _B3、 Phdd _FIP and Phdd _BIP; It can also be Phdd _F3、 Phdd _B3、 Phdd _FIP、 Phdd _BIP and Phdd _LF; It can also be Phdd _F3、 Phdd _B3、 Phdd _FIP、 Phdd _BIP and Phdd _LB; It can also be used for Phdd _F3、 Phdd _B3、 Phdd _FIP、 Phdd _BIP、 Phdd _LF and Phdd_LB.

[0008] LAMP detection procedure: reaction temperature 60.0℃-66.0℃, reaction time 40 min.

[0009] LAMP assay system: 2.5 μL of 10×isothermal amplification buffer, MgSO4 at a final concentration of 4.0–12.0 mM, betaine at a final concentration of 0.1–1.4 M, dNTPs at a final concentration of 0.8–1.8 mM, and 20 μM... Phdd _FIP / Phdd _BIP 2μL, 10μM Phdd _F3 / Phdd _B3 0.5μL, 20μM Phdd _LF / Phdd 1 μL of LB yields a final concentration of 0.128-0.576 U / μL. Bst 2.0 WarmStart ® DNA polymerase (8000 U / mL, New England BioLabs), 1 μL DNA template, and 0.5-2.5 μM of 20× EvaGreen can also be added to the above system. ® Add 1 μL of Dye (Biotium) to the detection system and finally add water to bring the total volume to 25 μL.

[0010] Analysis of test results: (1) Adding EvaGreen ® During dyeing, the detection was performed using a quantitative PCR instrument, and positive samples showed an amplification curve. (2) No EvaGreen was added. ® During the reaction, the detection can be performed in a conventional PCR instrument or in a simple constant temperature environment (such as a metal bath or water bath). When the reaction products are electrophoresed, the positive sample products show a ladder-like electrophoretic band. After adding chimeric nucleic acid fluorescent dye or self-luminous fluorescent dye to the reaction products, the positive sample tubes show obvious color changes compared with the negative control or blank control tubes.

[0011] Compared with the prior art, the present invention has the following beneficial effects: 1. On-site testing can be performed under simplified conditions. The detection can be performed under simple constant temperature conditions such as a water bath, and a chimeric fluorescent dye such as SYBR Green or GeneFinder is added to the product tube. TM By adding self-luminescent fluorescent dyes such as calcein or hydroxynaphthol blue, positive sample tubes show a significant color change compared to negative or blank control tubes. This color change enables rapid on-site visual detection of samples.

[0012] 2. Can complete detection quickly and specifically. Using 6-8 primers in the test can effectively ensure the specificity of the test results, and can be used to discover new infected hosts of *Bacillus mermaidus* subspecies *Mermaidus*, including crustaceans; the test time is short, and can be completed within 15-40 minutes. Attached Figure Description

[0013] Figure 1 The first set of isothermal amplification detection primers of this invention is located in the ureC gene (front); Figure 2 The location of the first set of isothermal amplification detection primers of this invention in the ureC gene (below); Figure 3 Amplification curves for LAMP detection specificity assessment of *Bacillus mermaidus* subspecies *Mermaid*; Figure 4 Colorimetric images of samples from *LAMP* subsp. *mermaidensis*, obtained through on-site testing. Detailed Implementation

[0014] To further illustrate the method and effects of the present invention, the following embodiments are provided for further explanation. Unless otherwise specified, all pharmaceuticals and materials used in the following embodiments are commercially available.

[0015] Example 1: Primer design for the collection and isothermal amplification detection of *Bacillus mermaidus* subspecies *Mermaid*. (1) Strain collection From 2015 to 2022, we studied large yellow croaker and spotted sea bream. Oplegnathus punctatus ) and Litopenaeus vannamei, Litopenaeus vannamei ( Fenneropenaeus chinensis ) and three-spotted swimming crab ( Portunus trituberculatus Seventeen strains of *Bacillus mermaidus* subsp. *mermaidus* were isolated from the sample, and their specific sources are shown in Table 1.

[0016] Table 1. Origin of *Bacillus mermaidus* subspecies from *Mermaid*

[0017] (2) Design of primers for isothermal amplification detection Primers ureC_F (5'-TCCAGCCTAATGTTGATA-3', as shown in SEQ ID NO: 17) and ureC_R (5'-ATCTGCCACATTCATACA-3', as shown in SEQ ID NO: 18) were designed to target the ureC gene of *Bacillus mermaidina* subspecies *Mermaidina*. Amplification was performed using conventional PCR methods. The PCR system consisted of 25 μL of: 12.5 μL of 2×Premix Ex Taq Mix (TaKaRa, Dalian), 0.5 μL each of 10 μmol / L ureC_F and ureC_R, 1 μL of bacterial genomic DNA, and 10.5 μL of sterile water. The PCR amplification program was: 95℃ for 5 min; 94℃ for 30 s, 58℃ for 30 s, 72℃ for 30 s, for a total of 35 cycles; extension at 72℃ for 8 min. Determination of conserved nucleotide sequence: Based on the sequencing results of PCR products of ureC_F / ureC_R of the 17 strains of this strain collected in Table 1, and the comparison analysis results of the corresponding nucleotide sequences of 38 strains currently published by NCBI, the conserved nucleotide sequence of this gene was determined, and the three sets of isothermal amplification detection primers described in this invention were designed.

[0018] Taking the first set of isothermal amplification detection primers as an example, its sequence position in the ureC gene is shown below. Figure 1 and Figure 2 , Figure 1 End B1c connection Figure 2 The first term is B1c, yellow is A, red is T, blue is C, and green is G. Phdd _FIP consists of F1c+TTTT+F2, Phdd _BIP consists of B1c + TTTT + B2, Phdd _F3、 Phdd _B3、 Phdd _LF and Phdd _LB corresponds to F3, B3, LF, and LB in the figure, respectively. The corresponding sequences of the 17 collected strains in the figure are all published for the first time.

[0019] Example 2: pMD18_ Phdd Construction of _U The 401bp PCR product obtained in Example 1 was purified by gel extraction and ligated into the pMD18-T vector (TaKaRa, Dalian), and then transformed into Escherichia coli (E. coli). Escherichia coli Recombinant plasmid pMD18 was extracted from DH5α competent cells (TaKaRa, Dalian). Phdd Store at -20℃ for later use.

[0020] Example 3: Evaluation of Specificity of Isothermal Amplification Detection This embodiment uses the first set of isothermal amplification detection primers as an example. The LAMP detection of *Bacillus mermaidina* subsp. *mermaidensis* was performed using a real-time quantitative PCR instrument (Bio-Rad CFX Opus 96, USA). The 25 μL LAMP reaction system contained 2.5 μL of 10×Isothermal amplification buffer (containing 2.0 mM Mg). 2+ ), 100mM MgSO41.5μL, 5.0M Betaine 5.0μL, 10mM dNTPs 3.5μL, 20 μM Phdd _FIP / BIP 2.0μL, 10μM Phdd _F3 / B3 0.5μL, 20μM Phdd _LF / Phdd 1.0 μL of LB, 1.0 μL of 8000 U / mL Bst 2.0 WarmStart® DNA polymerase (New England BioLabs, USA), 0.75 μL of 20×EvaGreen® Dye (25 μM, Biotium, USA), and 1.0 μL of DNA template were added, with RNase-free water added to a final volume of 25 μL. For the assay, the DNA template from the sample, denatured at 95°C, was added to the reaction system. The reaction temperature was 65°C, and the assay time was set to 40 min. After the assay, the system was incubated at 80°C for 8 min to terminate the reaction.

[0021] To kill Pseudomonas aeruginosa ( Pseudomonas plecoglossicida Using DNA from various pathogens of 15 aquatic animals, or DNA extracted from diseased tissues, as templates (Table 2), and with nucleic acids from healthy large yellow croaker and healthy leopard-gill sea bass tissues as negative controls, and sterile water as a blank control, the detection specificity analysis of the first LAMP set of this invention was performed. The detection was repeated three times. The detection results are as follows: Figure 3 The results showed that the present invention has excellent detection specificity. Except for *Bacillus mermaidina* subsp. *mercantileveria* and its recombinant plasmid standard, which were positive, the detection results for the other 15 pathogens were negative. Figure 3In the table, 1-3 correspond to nucleic acid numbers 20221229001-2, 20150731003-2, and 20200724005-2 respectively; 4: plasmid standard; 5-13 correspond to nucleic acid numbers 20151115002-2, 20190611022, 20151114010-1, 20150606032-2, 20150919005-2, 20200703003-1, 20210817003-2, 20151115018-1, and 20170902203-1 respectively; 14-28 correspond to... Nucleic acid numbers in Table 2: 20250511001, 20250515001, 20250828-85, 20250511003, 20230505, 20240905001, 20211220002-1, 2021_GD_1, 20250711006, 20221021-36, 20250722, 20200908005, 20211220009, 20201125016, and 20250711007; 29: Nucleic acid from liver tissue of healthy large yellow croaker; 30: Nucleic acid from tissue of healthy leopard-gill spiny perch; 31: Blank control.

[0022] Table 2. Pathogens used for specific detection

[0023] Example 4: Field Detection Application of Isothermal Amplification The detection was performed under constant temperature conditions (metal bath). After the detection was completed, the nucleic acid dye GeneFinder was added to the amplification product at a volume ratio of 2%. TM The negative control and negative samples turned orange-yellow after the dye was added. The product tubes that tested positive for *Bacillus mermaidus* subsp. *mermaidensis* changed from orange-yellow to fluorescent green. Figure 4 , Figure 4 In the table, 1: negative control; 2: nucleic acid from healthy large yellow croaker tissue; 3: pMD18_ Phdd _U; 4: Large yellow croaker tissue nucleic acid 1; 5: Large yellow croaker tissue nucleic acid 2; 6: Golden tiger hybrid grouper tissue nucleic acid 1; 7: Golden tiger hybrid grouper tissue nucleic acid 2; 8: Strain 20221229001-2 nucleic acid. During the testing, the tissue sample of the Golden Tiger Hybrid Grouper showed positive detection of *Bacillus mermaidus* subspecies *Mermaidus* for the first time.

Claims

1. A primer for isothermal amplification detection of *Bacillus mermaidus* subspecies *Mermaidus*, characterized in that: It includes three sets of isothermal amplification detection primers, the nucleotide sequences of which are as follows: The nucleotide sequence of the first set of isothermal amplification detection primers: 1) Primer 1: Phdd _F3:5'-ATCTGTCCTCAACAAGCC-3', 2) Primer 2: Phdd _B3:5'-GCTTCAATTTGCTCTCGTAAT-3', 3) Primer 3: Phdd _FIP: 5'-CGGTTGTTGCATTGGAACCCGCTTTTGAAGAAGGATTATGTTCTGGC-3', 4) Primer 4: Phdd _BIP: 5'-GGTCTGGAATATGTCACGAATGCTTTTTCTGACACAACCTTTACCAAAT-3', 5) Primer 5: Phdd _LF: 5’-CCGCCACCGATAAAGGTTGTTAA-3’, 6) Primer 6: Phdd _LB: 5'-GGAAGCCGTTGATGACTTACC-3'; Nucleotide sequences of the second set of isothermal amplification detection primers: 1) Primer 1: Phdd _F3:5'-CCCGGTACAGAAGTTGTGG-3', 2) Primer 2: Phdd _B3:5'-GCTTCCAGCATTCGTGACAT-3', 3) Primer 3: Phdd _FIP: 5'-CCTTCTTCGGCTTGTTGAGGACATTTTCAGGCGAAGGCAAGATCGT-3', 4) Primer 4: Phdd _BIP: 5'-GGTGGCGGAACCGGTCCAGTTTTTATTCCAGACCCCTGGTGT-3', 5) Primer 5: Phdd _LF: 5'-AGTATCGATTCCTCCAGCTGTT-3', 6) Primer 6: Phdd _LB: 5'-GGCGGGTTCTAATGCAACA-3'; Nucleotide sequences of the third set of isothermal amplification detection primers: 1) Primer 1: Phdd _F3: 5'-GATACTCACATTCATTTTATCTGTC-3', 2) Primer 2: Phdd _B3: 5'-GTCATCAACGGCTTCCAG-3', 3) Primer 3: Phdd _FIP: 5'-CCGGTTTCCTCCACCGATAAAGTTTTCTCAACAAGCCGAAGAAGG-3', 4) Primer 4: Phdd _BIP: 5'-TCCAGTGGCGGGTTCTAATGTTTTATTCGTGACATATTCCAGACC-3'.

2. The isothermal amplification detection primers for *Bacillus mermaidus* subspecies *Mermaid* as described in claim 1, characterized in that: The detection primers were designed using the conserved nucleotide sequence of the ureC gene from *Bacillus mermaidus* subsp. *mermaidus*.

3. The application of the isothermal amplification detection primers for *Proteobacterium melanogaster* subsp. *mermaidum* as described in claim 1 in the detection and quantitative analysis of *Proteobacterium melanogaster* subsp. *mermaidum* for non-disease diagnosis and treatment purposes.

4. The application as described in claim 3, characterized in that: During the detection, primers were used as follows: Phdd _F3、 Phdd _B3、 Phdd _FIP and Phdd _BIP, or Phdd _F3、 Phdd _B3、 Phdd _FIP、 Phdd _BIP and Phdd _LF, or Phdd _F3、 Phdd _B3、 Phdd _FIP、 Phdd _BIP and Phdd _LB, or Phdd _F3、 Phdd _B3、 Phdd_ FIP, Phdd _BIP、 Phdd _LF and Phdd _LB.

5. The application as described in claim 3, characterized in that: This includes products used to prepare diagnostic reagents or kits.

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