LAMP (loop-mediated isothermal amplification) primer group for detecting moraxella bovis, detection method, kit and application of LAMP primer group

Abstract

The invention discloses an LAMP (Loop-Mediated Isothermal Amplification) primer group, a detection method and a kit for detecting moraxella nasi and application of the LAMP primer group, the detection method and the kit. The primer group comprises outer primers MN-F3 and MN-B3 and inner primers MN-FIP and MN-BIP, and sequences of the inner primers MN-FIP and MN-BIP are respectively shown as SEQ ID NO.1-NO.4. The invention further discloses a kit for detecting moraxella nasi. The LAMP visual detection method established by using the primer group can specifically detect moraxella bulbiferi, has no cross reaction with other 22 bacteria of moraxella and 33 common cattle bacteria, and has strong specificity. The established LAMP visual detection method can be used for detecting a recombinant plasmid standard substance pMD-MN-16S with the lowest concentration of 3.5 copies / [mu] L, and has relatively high sensitivity. The detection method or the kit can be used for rapid detection of moraxella bovis in a clinical bovine sample, can also be used for rapid identification of moraxella bovis, can be used for laboratory detection and can also be used for cattle field on-site detection, a reliable method is provided for visual detection of moraxella bovis infection, and good application and popularization value is achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of veterinary microbiological detection technology, and particularly relates to a LAMP primer set for detecting Moraxella nasibovis, a detection method, a kit and application thereof. BACKGROUND

[0002] Moraxella nasibovis is a new species of Moraxella classified and named by Li Fuxiang et al. in 2023 [1-2] This bacterium is a new pathogen of bovine respiratory disease [1] , which causes great economic losses to the cattle industry, therefore, establishing a rapid detection method for Moraxella nasibovis is of great significance for the prevention and control of Moraxella nasibovis infection in cattle. At present, there is no LAMP nucleic acid detection method and other nucleic acid detection methods for Moraxella nasibovis at home and abroad.

[0003] Loop-mediated isothermal amplification (LAMP) is a new type of isothermal nucleic acid amplification technology developed by Notomi et al. in Japan in 2000 [3] This technology has many advantages such as strong specificity, high sensitivity, rapidity (reaction is completed only in about 1h), no need for expensive equipment (only simple instruments and equipment such as water bath are needed), low detection cost, simple operation, simple reaction result determination (the detection result can be determined according to the color change of the reaction solution before and after the reaction), etc., and has become a hot spot in the research of rapid detection technology for livestock and poultry diseases. The application of LAMP indicator makes the determination of LAMP reaction result visualized (the LAMP reaction result can be determined according to the color change of the reaction solution before and after the reaction), and at present, the LAMP indicator mainly includes calcium green, SYBR Green I and hydroxyl naphthol blue (HNB) [4-5] . High concentration of SYBR Green I can inhibit LAMP reaction and reduce reaction efficiency, so it is often necessary to add it to the reaction product after the LAMP reaction is completed by opening the reaction tube, which increases the aerosol pollution of the reaction product and causes false positive results in the next LAMP detection, so the application of SYBR Green I is limited [5] Although hydroxyl naphthol blue is added to the reaction system before LAMP reaction, it greatly reduces the aerosol pollution caused by adding after the reaction is completed, which leads to false positive results in the next LAMP detection, but it has the shortcomings of high price and not obvious color change as LAMP reaction indicator, which limits its application [5]Calcium-chlorophyllin has the following advantages as an indicator for LAMP reaction: calcium-chlorophyllin is added to the reaction system before the LAMP reaction, which greatly reduces the possibility of aerosol pollution caused by adding after the reaction is completed; calcium-chlorophyllin as an indicator for LAMP reaction has more obvious color change, which is conducive to direct observation of color change of LAMP reaction solution by naked eye and accurate determination of detection results [4-5] .

[0004] References:

[0005] [1] Li F, Zhao W, Zhu P, et al. Moraxella nasibovis sp. nov., isolated from a cow with respiratory disease [J]. Curr Microbiol, 2023, 80(9): 305.

[0006] [2] Oren A, M. Validation List no. 214. Valid publication of new names and new combinations effectively published outside the IJSEM [J]. Int J Syst Evol Microbiol, 2023, 73(11).

[0007] [3] Notomi T, Okayama H, Masubuchi H, et al. Loop-mediated isothermal amplification of DNA [J]. Nucleic Acids Res, 2000, 28(12): E63.

[0008] [4] Hu Z, Shan X, He X, et al. Establishment of a rapid detection method based on loop-mediated isothermal amplification of the actA gene of Listeria monocytogenes [J]. Chinese Journal of Preventive Veterinary Medicine, 2022, 44(1): 47-52.

[0009] [5] Bai R, Bai L, Wang S, et al. Research progress in detection methods for LAMP amplification products and application of gene editing technology in them [J]. Acta Agric Biotec Sin, 2021, 29(10): 2016-2030. SUMMARY

[0010] The technical problem to be solved by the present application is to overcome the shortcomings of the LAMP nucleic acid detection method and other nucleic acid detection methods of Moraxella nasibovis at home and abroad, and to provide a LAMP primer set for detecting Moraxella nasibovis and a visual detection method thereof, which has the advantages of strong specificity, high sensitivity, simple operation, rapidness, low detection cost, and no need for expensive instruments and equipment.

[0011] The application downloads 16S rRNA gene sequences of all 23 species of Moraxella (Moraxella nasibovis, Moraxella atlantae, Moraxella boevre, Moraxella bovis, Moraxella bovoculi, Moraxella canis, Moraxella caprae, Moraxella catarrhalis, Moraxella caviae, Moraxella cuniculi, Moraxella equi, Moraxella lacunata, Moraxella lincolnii, Moraxella nasicaprae, Moraxella nasovis, Moraxella haemolytica, Moraxella nonliquefaciens, Moraxella oblonga, Moraxella osloensis, Moraxella ovis, Moraxella pluranimalium, Moraxella porci and Moraxella oculli) from the GenBank database, carries out gene homology alignment by using MegAlign software in the DNAstar software package, selects a specific gene fragment of Moraxella nasibovis, designs LAMP outer primers MN-F3 and MN-B3 and inner primers MN-FIP and MN-BIP by using online software PrimerExplorer V4 (http: / / primerexplorer.jp / elamp4.0.0 / index.html), optimizes a reaction system and a reaction procedure, and establishes a specific LAMP visual detection method for Moraxella nasibovis, which can determine a detection result by observing color change of a reaction solution after reaction by naked eyes. The established LAMP visual detection method can specifically detect Moraxella nasibovis, has no cross reaction with other 22 species of Moraxella and 33 common bacteria of cattle, and has good specificity. The established LAMP visual detection method can detect recombinant plasmid standard pMD-MN-16S with a lower limit of 3.5 copies / μL, and has high sensitivity.

[0012] According to the first aspect, the technical solution of the present application is:

[0013] A LAMP primer set for detecting Moraxella bovorum, the primer set comprising outer primers and inner primers, the outer primers being MN-F3 and MN-B3, and the inner primers being MN-FIP and MN-BIP; the sequence of the outer primer MN-F3 is AACGCGAAGAACCTTACCTG; the sequence of the outer primer MN-B3 is CCAGTTTGTCACTGGCAGTA; the sequence of the inner primer MN-FIP is CGACAGCCATGCAGCACCTGATCTTGCAGAGATGCGAGAG; and the sequence of the inner primer MN-BIP is CTCGTGTCGTGAGATGTTGGGTAGAGTTCCCGACCGAGTC.

[0014] According to the second aspect, the technical solution of the present application is:

[0015] A non-disease diagnosis purpose Moraxella bovorum specific LAMP visual detection method based on the above primer set, comprising the following steps:

[0016] Extracting bacterial genomic DNA of a bovine clinical tissue or a bacterial isolate sample as a template;

[0017] Using the above primers and calcein indicator to perform LAMP reaction;

[0018] After the reaction is completed, whether Moraxella bovorum exists in the bovine clinical tissue or the bacterial isolate sample is accurately determined by directly observing the color change of the reaction solution before and after the reaction with the naked eye.

[0019] Further, 25 μL of the reaction system comprises:

[0020] In the PCR tube, first add ddH2O 9.3 μL, 10×Isothermal amplification buffer 2.5 μL, dNTP (10 μmol / L) 1.5 μL, Betaine (5 mol / L) 2 μL, Bst 2.0 WarmStart DNA polymerase (8 U / μL) 1 μL, Calcein (200 μmol / L) 3 μL, outer primer MN-F3 (25 μmol / L) 0.3 μL, outer primer MN-B3 (25 μmol / L) 0.3 μL, inner primer MN-FIP (25 μmol / L) 0.9 μL and inner primer MN-BIP (25 μmol / L) 0.9 μL and mix well, then add MnCl2 (25 mmol / L) 0.8 μL and mix well, finally add MgSO4 (100 mmol / L) 1.5 μL and DNA sample 1 μL.

[0021] Further, the optimal reaction procedure is 64℃, 60 min.

[0022] Further, the detection result determination standard is: the color change of the reaction solution is observed directly by naked eyes to determine the detection result, the detected sample whose reaction solution color changes from orange yellow before reaction to yellow green after reaction is determined as M. bovis positive, and the detected sample whose reaction solution color remains unchanged as orange yellow before reaction is determined as M. bovis negative.

[0023] According to a third aspect, the present application further provides a kit for detecting M. bovis, which comprises the LAMP primer set, reaction system and reaction procedure for detecting M. bovis and the DNA positive control of M. bovis model strain ZY190618 and ddH2O negative control. T

[0024] According to a fourth aspect, the detection method or kit of the present application can be applied in rapid identification of M. bovis and visual detection of M. bovis in non-disease diagnosis purposes in clinical tissue samples of cattle. The M. bovis LAMP visual detection method can be not only used for laboratory detection, but also applied in on-site detection in cattle farms. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The reaction results under the optimal reaction system and reaction procedure.

[0026] Figure 2 The specificity evaluation results of the LAMP visual detection method or kit.

[0027] Figure 3 ​The result of evaluating the sensitivity of the LAMP visual detection method or kit.

[0028] Figure 4 The result of identifying Moraxella nasibovis by the LAMP visual detection method or kit.

[0029] Figure 5 The result of detecting the bovine clinical tissue sample by the LAMP visual detection method or kit. DETAILED DESCRIPTION

[0030] The following examples are further illustrations of the present application and are not intended to limit the present application. Unless otherwise indicated, the reagents, kits, consumables, and apparatuses used in the present application are routine reagents, kits, consumables, and apparatuses in the art.

[0031] Example 1 Design and synthesis of LAMP primers

[0032] The 16S rRNA gene sequences of all 23 species of Moraxella bacteria model strains were obtained from the GenBank database. The 16S rRNA gene accession numbers of the 23 species of Moraxella bacteria model strains and their 16S rRNA gene sequences are as follows: Moraxella nasibovis (gene accession number OL841892), Moraxella atlantae (gene accession number AB680638), Moraxella bovoculi (gene accession number DQ156147), Moraxella bovis (gene accession number AF005182), Moraxella bovoculi (gene accession number DQ153089), Moraxella canis (gene accession number AJ269511), Moraxella caprae (gene accession number DQ156148), Moraxella catarrhalis (gene accession number AF005185), Moraxella caviae (gene accession number AF005187), Moraxella cuniculi (gene accession number NR_041695.1), Moraxella equi (gene accession number AF005184), Moraxella lacunata (gene accession number AF005160), Moraxella lincolnii (gene accession number AJ417490), Moraxella nasalis (gene accession number OL841894), Moraxella nasalis (gene accession number OL841893), Moraxella haemolytica (gene accession number OL841891), Moraxella nonliquefaciens (gene accession number JN175343), Moraxella prolonga (gene accession number NR_104725), Moraxella osloensis (gene accession number MW578397), Moraxella ovis (gene accession number CP011158), Moraxella plurispecies (gene accession number AM884564), Moraxella porci (gene accession number FM872292), and Moraxella oculi (gene accession number ON062946).

[0033] The MegAlign software in the DNAstar (version number 7.0.1) software package was used for gene homology alignment, and the specific gene fragment of Moraxella bovorum was selected as the target sequence for primer design, and the sequence was as follows:

[0034] CCTGGCATCATACTGACACTGAGGTGCGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCAC

[0035] GCCGTAAACGATGTCTACCAGTCGTTGGGTCTCTTGAAGACTTAGTGACGCAGTTAACGCAATAAGTAGACCG

[0036] CCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGT

[0037] GGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATACTAAGAATCTTGCAGAGATGCGAGAGT

[0038] GCCTTCGGGAACTTAGATACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCC

[0039] GCAACGAGCGCAACCCTTTTCCTTAGTTACCAGCGACTCGGTCGGGAACTCTAAGGATACTGCCAGTGACAA

[0040] ACTGGAGGAAGGCGGGGACGACGTCAAGTCATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATG

[0041] GTTGGTACAAAGGGTTGCTACACAGCGATGTGATGCTAATCTCAAAAAGCCAATCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCT(As shown in SEQ ID No. 5).

[0042] The specific LAMP outer primers MN-F3 and MN-B3 and inner primers MN-FIP and MN-BIP for M. bovis were designed by using online software PrimerExplorer V4 (http: / / primerexplorer.jp / elamp4.0.0 / index.html).

[0043] The sequence of outer primer MN-F3 is:

[0044] AACGCGAAGAACCTTACCTG (as shown in SEQ ID NO. 1);

[0045] The sequence of outer primer MN-B3 is:

[0046] CCAGTTTGTCACTGGCAGTA (as shown in SEQ ID NO. 2);

[0047] The sequence of inner primer MN-FIP is:

[0048] CGACAGCCATGCAGCACCTGATCTTGCAGAGATGCGAGAG (as shown in SEQ ID NO. 3);

[0049] The sequence of inner primer MN-BIP is:

[0050] CTCGTGTCGTGAGATGTTGGGTAGAGTTCCCGACCGAGTC (as shown in SEQ ID NO. 4);

[0051] All primers were synthesized by Beijing Encke Biotechnology Co., Ltd.

[0052] Example 2 Optimization of LAMP visual detection method

[0053] 1. Strains

[0054] The M. bovis model strain was isolated and identified by Yunnan Academy of Animal Husbandry and Veterinary Science, and was also preserved in Yunnan Academy of Animal Husbandry and Veterinary Science / Yunnan Key Laboratory of Tropical and Subtropical Animal Virus Diseases (Preservation Number: ZY190618 T ), China Center for Type Culture Collection (Address: No. 299, Bayi Road, Wuchang District, Wuhan, Hubei Province, Preservation Number: CCTCC AB 2021472 T ) and Culture Collection University of Gothenburg (Preservation Number: CCUG 75921 T ).

[0055] 2. Extraction of genomic DNA of bacterial sample

[0056] The genomic DNA of Moraxella bovallis ZY190618 and Moraxella bovallis ZY190619 was extracted by using a bacterial genomic DNA extraction kit (purchased from Tiangeng Biochemical Technology (Beijing) Co., Ltd.) respectively as the template of LAMP reaction, and was stored at -20℃ for standby use. The method for extracting the genomic DNA of the bacterial sample is not particularly limited in the present application, and the DNA extraction can be performed by using a conventional method. T

[0057] 3. Optimization of LAMP reaction system

[0058] ​Under the condition of keeping the basic reaction program (water bath in 60 ℃ water bath pot for 60 min) unchanged, the square method was used to optimize the volume of outer primer MN-F3 (0.3 μL, 0.6 μL, 0.9 μL and 1.2 μL), outer primer MN-B3 (0.3 μL, 0.6 μL, 0.9 μL and 1.2 μL), inner primer MN-FIP (0.3 μL, 0.6 μL, 0.9 μL and 1.2 μL) and inner primer MN-BIP (0.3 μL, 0.6 μL, 0.9 μL and 1.2 μL) in 25 μL basic reaction system (firstly adding 10×Isothermal amplification buffer 2.5 μL, dNTP (10 μmol / L) 1.5 μL, Betaine (5 mol / L) 2 μL, Bst 2.0 WarmStart DNA polymerase (8 U / μL) 1 μL, Calcein (200 μmol / L) 3 μL, outer primer MN-F3 (25 μmol / L) 0.3 μL, outer primer MN-B3 (25 μmol / L) 0.3 μL, inner primer MN-FIP (25 μmol / L) 0.3 μL, inner primer MN-BIP (25 μmol / L) 0.3 μL, supplementing ddH2O to a total volume of 21.7 μL and mixing, then adding MnCl2 (25 mmol / L) 0.8 μL and mixing, finally adding MgSO4 (100 mmol / L) 1.5 μL and DNA sample 1 μL in PCR tube and mixing), and the optimal reaction system of 25 μL was obtained: firstly adding ddH2O 9.3 μL, 10×Isothermal amplification buffer 2.5 μL, dNTP (10 μmol / L) 1.5 μL, Betaine (5 mol / L) 2 μL, Bst 2.0 WarmStart DNA polymerase (8 U / μL) 1 μL, Calcein (200 μmol / L) 3 μL, MN-F3 (25 μmol / L) 0.3 μL, MN-B3 (25 μmol / L) 0.3 μL, MN-FIP (25 μmol / L) 0.9 μL and MN-BIP (25 μmol / L) 0.9 μL in PCR tube and mixing, then adding MnCl2 (25 mmol / L) 0.8 μL and mixing, finally adding MgSO4 (100 mmol / L) 1.5 μL and DNA sample 1 μL. ddH2O was selected as negative control in the optimization process of LAMP reaction system.The 10x Isothermal amplification buffer and Bst 2.0 WarmStart DNA polymerase are both purchased from NEB Company of the United States; MnCl2, MgSO4 and Betaine are all purchased from Beijing Solabio Technology Co., Ltd.; Calcein is purchased from Hefei BASF Biotechnology Co., Ltd.; dNTP is purchased from Baobi Engineering (Dalian) Co., Ltd.

[0059] 4. Optimization of LAMP reaction program

[0060] Under the condition that the above optimal 25 μL reaction system remains unchanged, the reaction temperature (56℃, 58℃, 60℃, 62℃, 64℃, 66℃ and 68℃) and reaction time (40 min, 50 min, 60 min, 70 min, 80 min and 90 min) are optimized respectively.

[0061] Through optimization, the optimal reaction temperature is 64℃, and the optimal reaction time is 60 min, that is:

[0062] The optimal reaction program is 64℃ for 60 min.

[0063] 5. Composition of the kit

[0064] The LAMP primer set described in Example 1 and the reagents in the above reaction system, the DNA positive control of the bovine Moraxella model strain ZY190618T and the ddH2O negative control are combined to obtain a LAMP kit for detecting bovine Moraxella.

[0065] 6. Detection result determination standard

[0066] See Figure 1 , Figure 1 : 1 - bovine Moraxella ZY190618 T ; NC - negative control (ddH2O); M - DNA molecular weight marker.

[0067] To verify the visualization effect of the established LAMP visual detection method, the LAMP detection results are determined by two result determination methods. The two result determination methods are:

[0068] (1) Directly observing the color change of the reaction solution by naked eye: the color of the reaction solution changes from orange yellow before reaction to yellow green after reaction, and the detected sample is determined as bovine Moraxella positive; the color of the reaction solution remains unchanged as orange yellow before reaction, and the detected sample is determined as bovine Moraxella negative;

[0069] (2) Agarose gel electrophoresis method for reaction products: The reaction products were subjected to 1.2% agarose gel electrophoresis (5 μL of the reaction product was mixed with 3 μL of loading buffer and then loaded). The test samples with LAMP-specific ladder-like bands were judged to be positive for Bovine Moraxella buergerianum, and the test samples without LAMP-specific ladder-like bands were judged to be negative for Bovine Moraxella buergerianum.

[0070] The LAMP reaction results, determined by direct visual observation, showed that it contained *Moraxella bognosus* ZY190618. T The color of the DNA reaction solution changed from orange-yellow before the reaction to yellow-green. Figure 1 A) was determined to be positive for *Moraxella bovis*; the reaction solution containing the negative control (ddH2O) retained its original orange-yellow color. Figure 1 A) was determined to be negative for Bovine Moraxella vesiculosus.

[0071] Agarose gel electrophoresis analysis of the LAMP reaction results showed that it contained *Morax bognosus* ZY190618. T The reaction products of DNA showed ladder-like electrophoretic bands on agarose gel electrophoresis. Figure 1 B), was determined to be positive for *Moraxella bovis*; the reaction product containing the negative control (ddH2O) did not show step-like bands on agarose gel electrophoresis. Figure 1 B) was determined to be negative for Bovine Moraxella vesiculosus.

[0072] The results of the two result determination methods are completely consistent. Figure 1 Therefore, the established LAMP visualization detection method has good visualization effect, and the detection results can be accurately determined by directly observing the color change of the reaction solution after the reaction is completed (eliminating the complicated process of agarose gel electrophoresis of reaction products and saving detection time).

[0073] Example 3: Performance Evaluation of LAMP Visualization Detection Method or Kit

[0074] 1. Specificity evaluation

[0075] (1)Strain

[0076] All 23 type strains of the genus *Moraxella* and 33 common bovine bacteria were preserved by the Yunnan Academy of Animal Science and Veterinary Medicine / Yunnan Key Laboratory of Tropical and Subtropical Animal Viral Diseases. The type strain of all 23 species of *Moraxella* (Bovine-nose *Moraxella* ZY190618) is mentioned. T Moraxella tarda NBRC 14588 T Moraxella bortaniformis ATCC 700022 T Moraxella tamariscina ATCC 10900 T Moraxella bubalana CCUG 52049 TMoraxella kansei LMG 11194 T Moraxella catarrhalis ATCC 700019 T Moraxella catarrhalis ATCC 25238 T Moraxella guinea pig CCUG 355 T Rabbit Moraxella salina CCUG 2154 T Mamorella ATCC25576 T Moraxella cladomastrae ATCC 17967 T Moraxella linnsis CCUG 9405 T Moraxella nasalis NBRC 115473 T Moraxella muttonis nausea (CCUG 75922) T Hemolytic Moraxella acicularis CCUG 75920 T Non-liquefied Moraxella catarrhalis NCTC 10464 T , Prolonged Moraxella brevicornu NBRC 102422 T Oslo Moraxella CCUG 350 T Moraxella muttonensis CCUG 354 T Moraxella 248-01 T Moraxella suis DSM 25326 T And Moraxella catarrhalis ATCC TSD-373 T Table 1 shows the sources of 33 common bovine bacteria and their GenBank accession numbers for 16S rRNA genes.

[0077] (2) Extraction of bacterial genomic DNA

[0078] Genomic DNA was extracted from all 23 species of Moraxella genus and 33 common bovine bacteria using the bacterial genomic DNA extraction method described in Example 2 as test samples, and stored at -20°C for later use.

[0079] (3) Specificity evaluation

[0080] See Figure 2 As shown, in Figure 2 middle:

[0081] 1-23 are respectively *Moraxella bognosus* ZY190618 T Moraxella tarda NBRC 14588 T Moraxella bortaniformis ATCC 700022 T Moraxella tamariscina ATCC 10900 T Moraxella bubalana CCUG 52049 T Moraxella kansei LMG 11194 TM. capricolum ATCC 700019 T M. catarrhalis ATCC 25238 T M. caviae CCUG 355 T M. cuniculi CCUG 2154 T M. equi ATCC 25576 T M. lacunata ATCC 17967 T M. lincolnii CCUG 9405 T M. nasalis NBRC 115473 T M. ovis CCUG 75922 T M. haemolyticum CCUG 75920 T M. nonliquefaciens NCTC 10464 T M. elongatum NBRC 102422 T M. osloensis CCUG 350 T M. ovis CCUG 354 T M. polyresis 248-01 T M. hyopneumoniae DSM 25326 T M. oculi ATCC TSD-373 T ;

[0082] 24-56 are M. blattae CCUG 74657 TM. haemolytica ASV17113, A. lwoffii 20083, A. pseudolwoffii 211144, M. equinum ASV210852, B. safensis ASV21102, B. algicola ASV21108, C. perfringens ASV200849, C. pseudotuberculosis ASV220610, E. faecalis ASV210624, E. coli ASV210723, E. fergusonii ASV220419, L. lactis ASV22069, L. monocytogenes DSM 20600, L. inokuti ASV201080, P. aerogenes ASV20097, P. multocida ASV201160, P. mirabilis ASV210740, P. aeruginosa 200921, R. typhi ASV210634, S. enterica ASV201158, S. flexneri ASV211126, S. aureus ASV201020, S. epidermidis ASV220412, S. chromogenes ASV210921, S. dysgalactiae ASV201139, S. paris ASV200845, S. suis ASV211143, S. zooepidemicus ASV210759, Y. enterocolitica ASV211212, B. abortus A19, U. urealyticum YN13077 and M. bovis YN13077;

[0083] NC: negative control (ddH2O);

[0084] M: DNA marker.

[0085] The genomic DNA samples of all 23 species of Moraxella bacteria model strains and 33 common bovine bacteria were detected by the LAMP visual detection method optimized in Example 2. The visual detection results showed that after the reaction, only the genomic DNA sample of the bovine Moraxella catarrhalis model strain ZY190618 T changed from orange yellow before the reaction to yellow green Figure 2 A, Table 1), and was determined to be positive for bovine Moraxella catarrhalis; the reaction solutions of the genomic DNA samples of the other 22 species of Moraxella bacteria model strains and 33 common bovine bacteria remained unchanged Figure 2 A, Table 1), and were determined to be negative for bovine Moraxella catarrhalis, indicating that the LAMP visual detection method optimized in Example 2 can specifically detect bovine Moraxella catarrhalis and has no cross-reaction with the 22 species of Moraxella bacteria model strains and 33 common bovine bacteria, and has strong specificity.

[0086] In addition, to further verify the visualization effect of the optimized LAMP visualization detection method obtained in Example 2, the agarose gel electrophoresis method described in Example 2 was used to determine the LAMP detection results. The results showed that the LAMP detection method contained the *Moraxella bognosum* model strain ZY190618. T The LAMP reaction products of DNA showed ladder-like bands on agarose gel electrophoresis. Figure 2 B) was determined to be positive for *Moraxella bovis*; no step-like bands were observed in the agarose gel electrophoresis of the reaction products containing genomic DNA from 22 other *Moraxella* species and 33 common bovine bacteria, as well as the negative control (ddH2O). Figure 2 B), all were determined to be negative for *Moraxella bovinenacea*. This result is completely consistent with the above visual detection results. Figure 2 This further demonstrates that the LAMP visualization detection method optimized in Example 2 has a good visualization effect, and the detection results can be accurately determined by directly observing the color change of the reaction solution after the reaction is completed with the naked eye.

[0087] Table 1. Strains used for specificity evaluation and specificity test results

[0088]

[0089]

[0090] 2. Sensitivity assessment

[0091] (1) Preparation of LAMP recombinant plasmid standards

[0092] The preparation method of LAMP recombinant plasmid standards is as follows:

[0093] ① PCR amplification of the target gene fragment: 50μL PCR reaction system: ddH2O 23μL, 2×EasyTaq PCRSuperMix 25μL, primer MN-F3 0.5μL, primer MN-B3 0.5μL, *Moraxella bovis* ZY190618 T 1 μL of DNA template was used. PCR reaction program: 94℃ pre-denaturation for 3 min; 94℃ for 30 s, 57℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 5 min. The PCR-amplified gene fragment was approximately 200 bp, consistent with the expected target gene fragment of 208 bp.

[0094] ② Gel recovery of target gene fragment: The above PCR products were subjected to 1.2% agarose gel electrophoresis, and the target gene fragment was recovered using an agarose gel DNA recovery kit (purchased from Tiangen Biotech (Beijing) Co., Ltd.).

[0095] ③ Connecting the target gene fragment with the vector: the target gene fragment was cloned in the pMD19-T vector to construct the pMD-MN-16S recombinant plasmid by using the pMD19-T vector cloning kit (purchased from the Bioengineering (Dalian) Co., Ltd.);

[0096] ④ Transforming the recombinant plasmid into E. coli: the obtained recombinant plasmid was transformed into the E. coli DH5a competent cells, and a single colony was picked and cultured to obtain a bacterial liquid;

[0097] ⑤ Extracting the recombinant plasmid: the recombinant plasmid in the bacterial liquid was extracted by using the plasmid extraction kit (purchased from the Beijing Tiangen Biochemical Technology Co., Ltd.);

[0098] ⑥ Sequencing identification of the recombinant plasmid

[0099] The recombinant plasmid pMD-MN-16S was sent to the Beijing Genesee Biological Technology Co., Ltd. for sequencing; the obtained inserted gene sequence is as follows:

[0100] AACGCGAAGAACCTTACCTGGTCTTGACATACTAAGAATCTTGCAGAGATGCGAGAGTGCCTTCGGGAACTTAGATACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTTTCCTTAGTTACCAGCGACTCGGTCGGGAACTCTAAGGATACTGCCAGTGACAAACTGG (SEQ ID NO.6);

[0101] The homology of the above inserted gene sequence and the target gene sequence AACGCGAAGAACCTTACCTGGTCTTGACATACTAAGAATCTTGCAGAGATGCGAGAGTGCCTTCGGGAACTTAGATACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTTTCCTTAGTTACCAGCGACTCGGTCGGGAACTCTAAGGATACTGCCAGTGACAAACTGG (as shown in SEQ ID NO.7) was 100% by using the MegAlign software, indicating that the recombinant plasmid pMD-MN-16S was successfully constructed;

[0102] ⑦Recombinant plasmid concentration determination and concentration conversion: the concentration of recombinant plasmid pMD-MN-16S was determined by UV spectrophotometry to be 11 ng / μL, and the mass concentration was converted to copy number concentration 3.5 x 10 23 copies / μL as the standard for LAMP using the formula (copies / μL) = (6.02 x 10 -9 ) x (mass concentration (ng / μL) x 10 9 ) / (DNA sequence size x 660).

[0103] The PCR amplification, target gene fragment gel recovery, target fragment and vector ligation, E. coli transformation, plasmid extraction, plasmid concentration determination and plasmid concentration conversion methods in this example are not particularly limited and can be performed using conventional methods.

[0104] (2) Sensitivity evaluation

[0105] Referring to Figure 3 .

[0106] In Figure 3 :

[0107] 1-10 are recombinant plasmid standards pMD-MN-16S with concentrations of 3.5 x 10 8 - 3.5 x 10 -1 copies / μL, respectively;

[0108] NC: negative control (ddH2O);

[0109] M: DNA molecular weight standard.

[0110] The recombinant plasmid standard pMD-MN-16S with a concentration of 3.5 x 10 9 copies / μL was diluted by a factor of 10 in 10 gradients, i.e., the concentrations of the standard obtained by dilution were 3.5 x 10 8 - 3.5 x 10 -1 copies / μL, respectively, and the LAMP method optimized in Example 2 was used for detection to determine the minimum concentration of pMD-MN-16S standard that was positive. The visual detection results showed that after the reaction, the reaction solution containing pMD-MN-16S standard with a concentration of 3.5 x 10 8 - 3.5 x 10 0 copies / μL all changed from the original orange-yellow color to yellow-green color Figure 3 A), and were all determined to be positive for M. bovis; the reaction solution containing pMD-MN-16S standard with a concentration of 3.5 x 10 -1 copies / μL and the negative control (ddH2O) remained the original orange-yellow color unchangedFigure 3 A), all were determined as Moraxella bovorum negative; therefore, the minimum concentration of the standard pMD-MN-16S detected as Moraxella bovorum positive was 3.5 copies / μL, indicating that the LAMP method established had high sensitivity.

[0111] In addition, to verify the visualization effect of the LAMP visual detection method optimized in Example 2 again, the LAMP detection results were determined by agarose gel electrophoresis as described in Example 2, and the results showed that the reaction products of the pMD-MN-16S standard containing 3.5 x 10 8 0 copies / μL appeared ladder-like electrophoresis bands Figure 3 B), all were determined as Moraxella bovorum positive; the reaction products of the pMD-MN-16S standard containing 10 -1 copies / μL and the negative control (ddH2O) did not appear ladder-like bands Figure 3 B), all were determined as Moraxella bovorum negative. This result was completely consistent with the above visual detection result Figure 3 ), again verifying that the visualization effect of the LAMP visual detection method optimized in Example 2 was good, and the detection result could be accurately determined by directly observing the color change of the reaction solution after the reaction by the naked eye.

[0112] Application of the LAMP visual detection method or kit in the rapid identification of Moraxella bovorum

[0113] 1. Strains

[0114] The bacterial isolates ASV211134, YN180721, YN240515 and 210445 were isolated from dairy cows, and the bacterial isolates ASV210756 and ASV170220 were isolated from sheep, all of which were isolated and preserved by the Yunnan Institute of Animal Husbandry and Veterinary Medicine / Yunnan Key Laboratory of Viral Diseases of Tropical and Subtropical Animals.

[0115] 2. Extraction of bacterial genomic DNA

[0116] The bacterial genomic DNA of the 6 bacterial isolates ASV211134, ASV210756, YN180721, ASV170220, YN240515 and 2104555 was extracted by the bacterial genomic DNA extraction method described in Example 2 as a detection sample, and was stored at -20℃ for standby use.

[0117] 3. Identification of Moraxella bovorum by LAMP visual detection method or kit

[0118] Referring to Figure 4 shown in​Figure 4 In the present application, the LAMP visual detection method is used to detect the DNA samples of the six bacterial isolates, and the visual detection results show that the color of the reaction solution containing the DNA sample of bacterial isolate 210445 changes from orange yellow before reaction to yellow green after reaction (A, Table 2), which is identified as M. bovis; the reaction solutions containing the DNA samples of bacterial isolates ASV211134, ASV210756, YN180721, ASV170220 and YN240515 all remain unchanged orange yellow before reaction (A, Table 2), which are all identified as non-M. bovis (other bacteria other than M. bovis).

[0119] 1-6 are bacterial isolates 210445, ASV211134, ASV210756, YN180721, ASV170220, YN240515, respectively;

[0120] PC: positive control (M. bovis ZY190618 T );

[0121] NC: negative control (ddH2O);

[0122] M: DNA molecular weight marker.

[0123] The LAMP visual detection method optimized in Example 2 is used to detect the DNA samples of the six bacterial isolates, and the visual detection results show that the color of the reaction solution containing the DNA sample of bacterial isolate 210445 changes from orange yellow before reaction to yellow green after reaction (A, Table 2), which is identified as M. bovis; the reaction solutions containing the DNA samples of bacterial isolates ASV211134, ASV210756, YN180721, ASV170220 and YN240515 all remain unchanged orange yellow before reaction (A, Table 2), which are all identified as non-M. bovis (other bacteria other than M. bovis). Figure 4 Figure 4 In addition, in order to verify again the visual effect of the LAMP visual detection method optimized in Example 2, the agarose gel electrophoresis method described in Example 2 is used to determine the LAMP detection results, and the results show that the reaction product agarose gel electrophoresis of the DNA sample containing bacterial isolate 210445 appears ladder-like electrophoresis bands (B), which is identified as M. bovis; the reaction product agarose gel electrophoresis of the DNA samples containing bacterial isolates ASV211134, ASV210756, YN180721, ASV170220 and YN240515 all do not appear ladder-like bands (B), which are all identified as non-M. bovis. This result is completely consistent with the above visual result (C), which again proves that the visual effect of the LAMP visual detection method optimized in Example 2 is good, and the detection results can be accurately determined by directly observing the color change of the reaction solution after reaction with the naked eye.

[0124] In addition, in order to verify again the visual effect of the LAMP visual detection method optimized in Example 2, the agarose gel electrophoresis method described in Example 2 is used to determine the LAMP detection results, and the results show that the reaction product agarose gel electrophoresis of the DNA sample containing bacterial isolate 210445 appears ladder-like electrophoresis bands (B), which is identified as M. bovis; the reaction product agarose gel electrophoresis of the DNA samples containing bacterial isolates ASV211134, ASV210756, YN180721, ASV170220 and YN240515 all do not appear ladder-like bands (B), which are all identified as non-M. bovis. This result is completely consistent with the above visual result (C), which again proves that the visual effect of the LAMP visual detection method optimized in Example 2 is good, and the detection results can be accurately determined by directly observing the color change of the reaction solution after reaction with the naked eye. Figure 4 Figure 4 Figure 4

[0125] ​​​​To verify the accuracy of the LAMP method for identifying M. bovis optimized in Example 2, this example compares the LAMP detection and identification method optimized in Example 2 with the traditional 16S rRNA gene homology alignment method for identifying bacteria. The specific steps of the 16S rRNA gene homology alignment method for identifying bacteria are as follows:

[0126] (1) The DNA of the above 6 bacterial isolates ASV211134, ASV210756, YN180721, ASV170220, YN240515 and 2104555, respectively, was used as a template to amplify the 16S rRNA gene by ordinary PCR with bacterial universal primers 27F and 1492 primers. The 50 μL PCR reaction system: ddH2O 23 μL, 2x EasyTaq PCR SuperMix 25 μL, primer 27F 0.5 μL, primer 1492R 0.5 μL, bacterial DNA template 1 μL. The PCR reaction program: 94°C pre-denaturation for 3 min; 94°C for 30 s, 57°C for 30 s, 72°C for 30 s, 35 cycles; 72°C for 5 min. The PCR amplified gene fragment is about 1500 bp, which is consistent with the expected target gene fragment 1500 bp;

[0127] (2) The PCR amplification products of the 6 bacterial isolates were sequenced to obtain the 16S rRNA gene sequence, and the gene sequence was submitted to GenBank database. The 16S rRNA gene GenBank accession numbers of the 6 bacterial isolates ASV211134, ASV210756, YN180721, ASV170220, YN240515 and 2104555 are ON715871, ON715872, MT516321, ON715870, PP859510 and PV211277, respectively (Table 2);

[0128] (3) The 16S rRNA gene sequences of the 6 bacterial isolates were respectively BLAST compared and identified in the NCBI database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The results showed that the bacterial isolate ASV211134 had the highest homology of 99.9% with Moraxella bovoculi 22581 strain (Table 2), and was identified as Moraxella bovoculi (non-bovine nasal Moraxella); the bacterial isolate ASV210756 had the highest homology of 99.4% with Moraxella ovis 199 / 55 strain (Table 2), and was identified as Moraxella ovis (non-bovine nasal Moraxella); the bacterial isolate YN180721 had the highest homology of 99.9% with Streptococcus parasuis FZ2 strain (Table 2), and was identified as Streptococcus parasuis (non-bovine nasal Moraxella); the bacterial isolate ASV170220 had the highest homology of 100% with Mannheimia haemolytica YL9 strain (Table 2), and was identified as Mannheimia haemolytica (non-bovine nasal Moraxella); the bacterial isolate YN240515 had the highest homology of 100% with Streptococcus ruminantium DTK320 strain (Table 2), and was identified as Streptococcus ruminantium (non-bovine nasal Moraxella); the bacterial isolate 210445 had the highest homology of 99.9% with Moraxella nasi bovis ZY190618 strain (Table 2), and was identified as Moraxella nasi bovis (non-bovine nasal Moraxella). T

[0129] The identification results of the LAMP detection method for identifying Moraxella nasi bovis optimized in Example 2 and the traditional 16S rRNA gene homology comparison method for identifying bacteria were consistent (i.e. both methods identified the bacterial isolate 210445 as Moraxella nasi bovis, and identified the bacterial isolates ASV211134, ASV210756, YN180721, ASV170220, YN240515 as non-bovine nasal Moraxella) (Table 2), indicating that the LAMP method for identifying Moraxella nasi bovis optimized in Example 2 had high accuracy. In addition, the 16S rRNA gene homology comparison method for identifying bacteria required about 24 h, while the LAMP method for identifying Moraxella nasi bovis optimized in Example 2 required about 2 h, so the LAMP method for identifying Moraxella nasi bovis optimized in Example 2 also had the advantage of being fast.

[0130] Table 2. Results of identification of strains by two methods

[0131]

[0132] ​Application of the LAMP visual detection method or kit in the detection of bovine clinical samples

[0133] 1. Clinical samples

[0134] Three lung samples YLL001, YLL002 and YLL003 and five nasal swab samples YLN004, YLN005, YLN006, YLN007 and YLN008 were obtained from a dairy farm in Yiliang County, Kunming City, Yunnan Province, which had respiratory disease. The eight bovine clinical tissue samples were stored in the Yunnan Institute of Animal Husbandry and Veterinary Medicine / Key Laboratory of Tropical and Subtropical Animal Virus Diseases in Yunnan Province.

[0135] 2. Extraction of genomic DNA from clinical samples

[0136] Before extracting the genomic DNA of the bovine clinical tissue samples, the bovine clinical tissue samples were pretreated:

[0137] (1) Pretreatment of lung samples: 1 g of lung sample was placed in a mortar, 5 mL of sterile physiological saline was added, and the lung tissue was ground into a slurry with a mortar rod. Then 5 mL of sterile physiological saline was added and mixed well. 1 mL of the slurry was centrifuged at 2000 rpm for 1 min, 0.5 mL of the supernatant was transferred to a new centrifuge tube, and centrifuged at 12000 rpm for 3 min. The supernatant was discarded and the precipitate was used for extraction of bacterial genomic DNA.

[0138] (2) Pretreatment of nasal swab samples: The cotton head of the nasal swab was broken and placed in a 2 mL centrifuge tube. 1.5 mL of sterile physiological saline was added and shaken vigorously for 5 min. After removing the cotton head, it was centrifuged at 12000 rpm for 3 min, and the supernatant was discarded and the precipitate was used for extraction of bacterial genomic DNA.

[0139] (3) The genomic DNA of the above eight bovine tissue samples was extracted using the bacterial genomic DNA extraction method described in Example 2 as the detection sample, and stored at -20°C for later use.

[0140] 3. Detection of M. bovis in bovine clinical samples by LAMP visual detection method or kit

[0141] Referring to Figure 5 , in Figure 5 :

[0142] 1-3 are bovine lung samples YLL001, YLL002 and YLL003, respectively;

[0143] 4-8: bovine nasal swab samples YLN004, YLN005, YLN006, YLN007 and YLN008, respectively;

[0144] PC: Positive control (Morax bovis ZY190618) T );

[0145] NC: Negative control (ddH2O);

[0146] M: DNA molecular weight standard.

[0147] The LAMP method optimized in Example 2 was used to detect the genomic DNA samples from the above 8 bovine tissue samples. The visualization results showed that the color of the reaction solution containing lung YLL002 and nasal swab YLN005 DNA samples changed from orange-yellow before the reaction to yellow-green after the reaction. Figure 5 A, Table 3), all were determined to be positive for *Moraxella bovis*; the reaction solution containing lung DNA samples YLL001 and YLL003, and nasal swab DNA samples YLN004, YLN006, YLN007, and YLN008, all retained their original orange-yellow color. Figure 5 A, Table 3), were all determined to be negative for Bovine Moraxella vesiculosus.

[0148] Table 3. Results of Clinical Tissue Sample Testing

[0149] Sample No. Source Disease Color of reaction solution after reaction ended Detection result YLL001 Lung of dairy cow Respiratory disease Orange yellow Negative YLL002 Lung of dairy cow Respiratory disease Yellow green Positive YLL003 Lung of dairy cow Respiratory disease Orange yellow Negative YLN004 Nasal swab of dairy cow Respiratory disease Orange yellow Negative YLN005 Nasal swab of dairy cow Respiratory disease Yellow green Positive YLN006 Nasal swab of dairy cow Respiratory disease Orange yellow Negative YLN007 Nasal swab of dairy cow Respiratory disease Orange yellow Negative YLN008 Nasal swab of dairy cow Respiratory disease Orange yellow Negative

[0150] In addition, to further verify the visualization effect of the optimized LAMP visualization detection method obtained in Example 2, the agarose gel electrophoresis method described in Example 2 was also used to determine the LAMP detection results. The results showed that the reaction products containing lung YLL002 and nasal swab YLN005 DNA samples all showed ladder-like electrophoretic bands on agarose gel electrophoresis. Figure 5 B), all were determined to be positive for *Moraxella bovinenacea*; no ladder-like bands were observed in the agarose gel electrophoresis of the reaction products of lung DNA samples YLL001 and YLL003 and nasal swab DNA samples YLN004, YLN006, YLN007 and YLN008. Figure 5 B) All were determined to be negative for Bovine Moraxella vesiculosus.

[0151] This result is completely consistent with the above visual detection results. Figure 5 This further demonstrates that the LAMP visualization detection method optimized in Example 2 has a good visualization effect, and the detection results can be accurately determined by directly observing the color change of the reaction solution after the reaction is completed with the naked eye.

[0152] Sequence list information:

[0153] DTD Version: V1_3

[0154] Filename: A LAMP primer set, detection method, kit and its application for detecting *Moraxella bovis*.xml

[0155] Software name: WIPO Sequence

[0156] Software version: 2.1.0

[0157] Date of creation: 2025-03-17

[0158] Basic information:

[0159] Current application / application file name: 125300004312022453

[0160] Applicant name or designation: Yunnan Academy of Animal Science and Veterinary Medicine

[0161] Applicant name or designation / language: zh

[0162] Applicant name or designation / Latin name: Yunnan Academy of Animal Science and Veterinary Medicine.

[0163] Invention title: A LAMP primer set for detecting Moraxella bovoculi, a detection method, a kit and its application (zh)

[0164] Total number of sequences: 7

[0165] Sequence:

[0166] Sequence number (ID): 1

[0167] Length: 20

[0168] Molecular type: DNA

[0169] Feature position / limit:

[0170] -source, 1..20

[0171] >mol_type, other DNA

[0172] >organism, synthetic construct

[0173] Residue:

[0174] aacgcgaaga accttacctg 20

[0175] Sequence number (ID): 2

[0176] Length: 20

[0177] Molecular type: DNA

[0178] Feature position / limit:

[0179] -source, 1..20

[0180] >mol_type, other DNA

[0181] >organism, synthetic construct

[0182] Residues:

[0183] cgagttcctg gatctgctg gatctgctg gatctgctg gatctgctg 20 SEQ ID NO: 1

[0184] Length: 40

[0185] Molecule Type: DNA

[0186] Feature Location / Qualifier:

[0187] -source, 1..40

[0188] >mol_type, other DNA

[0189] >organism, synthetic construct

[0190] Residues:

[0191] cgacagccat gcagcacctg atcttgcaga gatgcgagag 40 SEQ ID NO: 4

[0192] Length: 40

[0193] Molecule Type: DNA

[0194] Feature Location / Qualifier:

[0195] -source, 1..40

[0196] >mol_type, other DNA

[0197] >organism, synthetic construct

[0198] Residues:

[0199] ctcgtgtcgt gagatgttgg gtagagttcc cgaccgagtc 40 SEQ ID NO: 5

[0200] Length: 608

[0201] Molecule Type: DNA

[0202] Feature location / qualifier:

[0203] -source, 1..608

[0204] >mol_type, genomic DNA

[0205] >organism, Moraxella nasibovis

[0206]

[0207] Sequence number (ID): 6

[0208] Length: 208

[0209] Molecule type: DNA

[0210] Feature location / qualifier:

[0211] -source, 1..208

[0212] >mol_type, other DNA

[0213] >organism, synthetic construct

[0214]

[0215] Sequence number (ID): 7

[0216] Length: 208

[0217] Molecule type: DNA

[0218] Feature location / qualifier:

[0219] -source, 1..208

[0220] >mol_type, other DNA

[0221] >organism, synthetic construct

[0222] .

Claims

1. A LAMP primer set for detecting Moraxella nasibovis, characterized in that, It includes outer primers and inner primers; the outer primers are MN-F3 and MN-B3, whose sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively; the inner primers are MN-FIP and MN-BIP, whose sequences are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

2. A LAMP visualization detection method for the specific detection of Moraxella nasibovis for non-disease diagnostic purposes, established using a LAMP primer set for detecting Moraxella nasibovis according to claim 1, characterized in that, Includes the following steps: Bacterial genomic DNA was extracted from bovine clinical tissue or bacterial isolate samples as a template; LAMP reaction was performed using the above primers and calcein indicator; After the reaction is complete, the presence of *Morax bovis* in bovine clinical tissue or bacterial isolate samples is determined by visually observing the color changes of the reaction solution before and after the reaction.

3. The LAMP visualization detection method for specific detection of Moraxella nasibovis for non-disease diagnostic purposes according to claim 2, characterized in that, The reaction system for LAMP is as follows: ddH2O 9.3 μL, 10× Isothermal amplification buffer 2.5 μL, dNTP 1.5 μL (10 μmol / L), Betaine 2 μL (5 mol / L), Bst 2.0 (8 U / μL), WarmStart DNA polymerase 1 μL, Calcein 3 μL (200 μmol / L), outer primer MN-F3 0.3 μL (25 μmol / L), outer primer MN-B3 0.3 μL (25 μmol / L), inner primer MN-FIP 0.9 μL (25 μmol / L), inner primer MN-BIP 0.9 μL (25 μmol / L), MnCl2 0.8 μL (25 mmol / L), MgSO4 1.5 μL (100 mmol / L), and DNA sample 1 μL.

4. The LAMP visualization detection method for specific detection of Moraxella nasibovis for non-disease diagnostic purposes according to claim 3, characterized in that, The LAMP reaction procedure is as follows: The reaction temperature was 64℃ and the reaction time was 60 min.

5. The LAMP visualization detection method for specific detection of Moraxella nasibovis for non-disease diagnostic purposes according to claim 2, characterized in that, After the reaction, samples whose color changes from orange-yellow before the reaction to yellow-green after the reaction are determined to be positive for *Morax bognosus* by direct visual observation; samples whose color remains orange-yellow after the reaction are determined to be negative for *Morax bognosus*.

6. The LAMP visualization detection method for specific detection of Moraxella nasibovis for non-disease diagnostic purposes according to claim 3, characterized in that, The order of reagent addition is as follows: First, add all reagents except MnCl2 and MgSO4 to the PCR tube and mix well. Then add MnCl2 and mix well. Finally, add MgSO4 and mix well.

7. The LAMP visualization detection method for specific detection of Moraxella nasibovis for non-disease diagnostic purposes according to any one of claims 2-6, characterized in that, No cross-reaction was observed with other bacteria in the genus Moraxella and common bovine bacteria when specifically detected in bovine Moraxella. Other bacteria in the genus *Morax* include: Moraxella tarda, Moraxella bortani, Moraxella bovis, Moraxella bubovis, Moraxella canis, Moraxella goat, Moraxella catarrhalis, Moraxella guinea pig, Moraxella rabbit, Moraxella horse, Moraxella cavitaria, Moraxella lintonensis, Moraxella goat nasal cavity, Moraxella sheep nasal cavity, hemolytic Moraxella, non-liquefied Moraxella, Moraxella prolongata, Moraxella osloi, Moraxella sheep, Moraxella multianisella, Moraxella swine, and Moraxella ophthalmica; The common bovine bacteria mentioned include: Blackman's bacillus, hemolytic Mansonia, Acinetobacter rhusiopathiae, pseudoacinetobacter rhusiopathiae, *Bacillus simulans*, *Bacillus safras*, *Bretschneidera sinensis*, *Clostridium perfringens*, *Corynebacterium pseudotuberculosis*, *Enterococcus faecalis*, *Escherichia coli*, *Escherichia fergersen*, *Lactococcus lactis*, *Listeria monocytogenes*, *Listeria innocense*, *Pasteurella multocida*, *Pasteurella multocida*, *Proteus mirabilis*, *Pseudomonas aeruginosa*, *Roseidonella spp.*, *Salmonella enterica*, *Shigella fischeri*, *Staphylococcus aureus*, *Staphylococcus epidermidis*, *Staphylococcus chromogenic*, *Streptococcus galactiae*, *Streptococcus parviflora*, *Streptococcus parasuis*, *Streptococcus multifiliis*, *Yersinia enterocolitica*, *Brucella abortus*, *Cryptospirae*, and *Mycoplasma bovis*.

8. The LAMP visualization detection method for specific detection of Moraxella nasibovis for non-disease diagnostic purposes according to any one of claims 2-6, characterized in that, It can detect recombinant plasmid standard pMD-MN-16S with a minimum concentration of 3.5 copies / μL.

9. The LAMP visualization detection method for non-disease diagnostic purposes of Moraxella nasibovis according to any one of claims 2-8 is used in the rapid identification of Moraxella nasibovis and the visualization detection of non-disease diagnostic purposes of Moraxella nasibovis in bovine clinical tissue samples; wherein the visualization detection includes laboratory detection or on-site detection at a cattle farm.

10. A kit for detecting Moraxella nasibovis, characterized in that, The kit includes the LAMP primer set for detecting Moraxella nasibovis as described in claim 1.

Images