LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) PRIMER SETS FOR DETECTING PORCINE SUSCEPTIBILITY-RELATED PATHOGENIC BACTERIA, AND KIT, LAMP CHIP AND USE BASED ON THE SAME

Abstract

The present disclosure belongs to the technical field of pathogen detection, in particular to loop-mediated isothermal amplification (LAMP) primer sets for detecting porcine susceptibility-related pathogenic bacteria, and a kit, a LAMP chip and use based on the same. The LAMP primer sets for detecting porcine susceptibility-related pathogenic bacteria include an Actinobacillus pleuropneumoniae primer set, a Haemophilus parasuis primer set, a Salmonella choleraesuis primer set, a Bordetella bronchiseptica primer set, a Pasteurella multocida primer set, a Streptococcus suis primer set, and an Erysipelothrix rhusiopathiae primer set.

Claims

1. Loop-mediated isothermal amplification (LAMP) primer sets for detecting porcine susceptibility-related pathogenic bacteria, wherein the porcine susceptibility-related pathogenic bacteria comprise: Actinobacillus pleuropneumoniae, Haemophilus parasuis, Salmonella choleraesuis, Bordetella bronchiseptica, Pasteurella multocida, Streptococcus suis, and Erysipelothrix rhusiopathiae; and the LAMP primer sets comprise an Actinobacillus pleuropneumoniae primer set, an H. parasuis primer set, an S. choleraesuis primer set, a B. bronchiseptica primer set, a P. multocida primer set, an S. suis primer set, and an E. rhusiopathiae primer set; the Actinobacillus pleuropneumoniae primer set comprises a forward outer primer F3 with the nucleotide sequence set forth in SEQ ID NO: 1, a reverse outer primer B3 with the nucleotide sequence set forth in SEQ ID NO: 2, a forward inner primer FIP with the nucleotide sequence set forth in SEQ ID NO: 3, and a reverse inner primer BIP with the nucleotide sequence set forth in SEQ ID NO: 4; the H. parasuis primer set comprises a forward outer primer F3 with the nucleotide sequence set forth in SEQ ID NO: 5, a reverse outer primer B3 with the nucleotide sequence set forth in SEQ ID NO: 6, a forward inner primer FIP with the nucleotide sequence set forth in SEQ ID NO: 7, and a reverse inner primer BIP with the nucleotide sequence set forth in SEQ ID NO: 8; the S. choleraesuis primer set comprises a forward outer primer F3 with the nucleotide sequence set forth in SEQ ID NO: 9, a reverse outer primer B3 with the nucleotide sequence set forth in SEQ ID NO: 10, a forward inner primer FIP with the nucleotide sequence set forth in SEQ ID NO: 11, and a reverse inner primer BIP with the nucleotide sequence set forth in SEQ ID NO: 12; the B. bronchiseptica primer set comprises a forward outer primer F3 with the nucleotide sequence set forth in SEQ ID NO: 13, a reverse outer primer B3 with the nucleotide sequence set forth in SEQ ID NO: 14, a forward inner primer FIP with the nucleotide sequence set forth in SEQ ID NO: 15, and a reverse inner primer BIP with the nucleotide sequence set forth in SEQ ID NO: 16; the P. multocida primer set comprises a forward outer primer F3 with the nucleotide sequence set forth in SEQ ID NO: 17, a reverse outer primer B3 with the nucleotide sequence set forth in SEQ ID NO: 18, a forward inner primer FIP with the nucleotide sequence set forth in SEQ ID NO: 19, and a reverse inner primer BIP with the nucleotide sequence set forth in SEQ ID NO: 20; the S. suis primer set comprises a forward outer primer F3 with the nucleotide sequence set forth in SEQ ID NO: 21, a reverse outer primer B3 with the nucleotide sequence set forth in SEQ ID NO: 22, a forward inner primer FIP with the nucleotide sequence set forth in SEQ ID NO: 23, and a reverse inner primer BIP with the nucleotide sequence set forth in SEQ ID NO: 24; and the E. rhusiopathiae primer set comprises a forward outer primer F3 with the nucleotide sequence set forth in SEQ ID NO: 25, a reverse outer primer B3 with the nucleotide sequence set forth in SEQ ID NO: 26, a forward inner primer FIP with the nucleotide sequence set forth in SEQ ID NO: 27, and a reverse inner primer BIP with the nucleotide sequence set forth in SEQ ID NO: 28.

2. A kit of porcine susceptibility-related pathogenic bacteria, comprising the LAMP primer sets according to claim 1 and a reaction buffer.

3. The kit according to claim 2, wherein the reaction buffer comprises Bst DNA Polymerase, a 10×Isothermal Amplification Reaction Buffer, BSA-A, dNTP, an MgSO.sub.4 aqueous solution, and a fluorescent dye.

4. A LAMP chip for detecting porcine susceptibility-related pathogenic bacteria, comprising the LAMP primer sets according to claim 1, a reaction buffer, and a chip.

5. The LAMP chip according to claim 4, wherein in the LAMP primer sets, an outer primer pair and an inner primer pair corresponding to any one of the pathogenic bacteria have a molar ratio of 1:8.

6. The LAMP chip according to claim 4, wherein the reaction buffer comprises Bst DNA Polymerase, a 10×Isothermal Amplification Reaction Buffer, BSA-A, dNTP, an MgSO.sub.4 aqueous solution, and a fluorescent dye.

7. The LAMP chip according to claim 4, wherein the chip comprises an isothermal amplification microfluidic chip.

8. The LAMP chip according to claim 4, wherein an amplification reaction cell of the LAMP chip comprises: an H. parasuis reaction cell, an S. choleraesuis reaction cell, a B. bronchiseptica reaction cell, a P. multocida reaction cell, an S. suis reaction cell, an E. rhusiopathiae reaction cell, an Actinobacillus pleuropneumoniae reaction cell, and a negative control reaction cell.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 shows a schematic diagram of a disc-type microfluidic chip and a primer spotting cell provided in examples;

[0024] FIG. 2 shows a specificity result of the disc-type microfluidic chip of A. pleuropneumoniae;

[0025] FIG. 3 shows a specificity result of the disc-type microfluidic chip of H. parasuis;

[0026] FIG. 4 shows a specificity result of the disc-type microfluidic chip of S. choleraesuis;

[0027] FIG. 5 shows a specificity result of the disc-type microfluidic chip of B. bronchiseptica;

[0028] FIG. 6 shows a specificity result of the disc-type microfluidic chip of P. multocida;

[0029] FIG. 7 shows a specificity result of the disc-type microfluidic chip of S. suis;

[0030] FIG. 8 shows a specificity result of the disc-type microfluidic chip of E. rhusiopathiae;

[0031] FIG. 9 shows a sensitivity result of the disc-type microfluidic chip of A. pleuropneumoniae;

[0032] FIG. 10 shows a sensitivity result of the disc-type microfluidic chip of H. parasuis;

[0033] FIG. 11 shows a sensitivity result of the disc-type microfluidic chip of S. choleraesuis;

[0034] FIG. 12 shows a sensitivity result of the disc-type microfluidic chip of B. bronchiseptica;

[0035] FIG. 13 shows a sensitivity result of the disc-type microfluidic chip of P. multocida;

[0036] FIG. 14 shows a sensitivity result of the disc-type microfluidic chip of S. suis; and

[0037] FIG. 15 shows a sensitivity result of the disc-type microfluidic chip of E. rhusiopathiae.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0038] The present disclosure provides LAMP primer sets for detecting porcine susceptibility-related pathogenic bacteria, where the porcine susceptibility-related pathogenic bacteria include: A. pleuropneumoniae, H. parasuis, S. choleraesuis, B. bronchiseptica, P. multocida, S. suis, and E. rhusiopathiae; and the LAMP primer sets include an A. pleuropneumoniae primer set, an H. parasuis primer set, an S. choleraesuis primer set, a B. bronchiseptica primer set, a P. multocida primer set, an S. suis primer set, and an E. rhusiopathiae primer set;

[0039] The A. pleuropneumoniae primer set includes a forward outer primer F3 with the nucleotide sequence set forth in SEQ ID NO: 1, a reverse outer primer B3 with the nucleotide sequence set forth in SEQ ID NO: 2, a forward inner primer FIP with the nucleotide sequence set forth in SEQ ID NO: 3, and a reverse inner primer BIP with the nucleotide sequence set forth in SEQ ID NO: 4;

[0040] The H. parasuis primer set includes a forward outer primer F3 with the nucleotide sequence set forth in SEQ ID NO: 5, a reverse outer primer B3 with the nucleotide sequence set forth in SEQ ID NO: 6, a forward inner primer FIP with the nucleotide sequence set forth in SEQ ID NO: 7, and a reverse inner primer BIP with the nucleotide sequence set forth in SEQ ID NO: 8;

[0041] The S. choleraesuis primer set includes a forward outer primer F3 with the nucleotide sequence set forth in SEQ ID NO: 9, a reverse outer primer B3 with the nucleotide sequence set forth in SEQ ID NO: 10, a forward inner primer FIP with the nucleotide sequence set forth in SEQ ID NO: 11, and a reverse inner primer BIP with the nucleotide sequence set forth in SEQ

[0042] The B. bronchiseptica primer set includes a forward outer primer F3 with the nucleotide sequence set forth in SEQ ID NO: 13, a reverse outer primer B3 with the nucleotide sequence set forth in SEQ ID NO: 14, a forward inner primer FIP with the nucleotide sequence set forth in SEQ ID NO: 15, and a reverse inner primer BIP with the nucleotide sequence set forth in SEQ ID NO: 16;

[0043] The P. multocida primer set includes a forward outer primer F3 with the nucleotide sequence set forth in SEQ ID NO: 17, a reverse outer primer B3 with the nucleotide sequence set forth in SEQ ID NO: 18, a forward inner primer FIP with the nucleotide sequence set forth in SEQ ID NO: 19, and a reverse inner primer BIP with the nucleotide sequence set forth in SEQ ID NO: 20;

[0044] The S. suis primer set includes a forward outer primer F3 with the nucleotide sequence set forth in SEQ ID NO: 21, a reverse outer primer B3 with the nucleotide sequence set forth in SEQ ID NO: 22, a forward inner primer FIP with the nucleotide sequence set forth in SEQ ID NO: 23, and a reverse inner primer BIP with the nucleotide sequence set forth in SEQ ID NO: 24; and

[0045] The E. rhusiopathiae primer set includes a forward outer primer F3 with the nucleotide sequence set forth in SEQ ID NO: 25, a reverse outer primer B3 with the nucleotide sequence set forth in SEQ ID NO: 26, a forward inner primer FIP with the nucleotide sequence set forth in SEQ ID NO: 27, and a reverse inner primer BIP with the nucleotide sequence set forth in SEQ ID NO: 28. The LAMP primer sets have a high specificity and desirable sensitivity.

[0046] The present disclosure further provides a kit of porcine susceptibility-related pathogenic bacteria, including the LAMP primer sets and a reaction buffer. In some embodiments, the reaction buffer includes a 10×Isothermal Amplification Reaction Buffer, BSA-A, dNTP, an MgSO.sub.4 aqueous solution, a fluorescent dye, and Bst DNA Polymerase; and the fluorescent dye is preferably SYTO™9.

[0047] The present disclosure further provides a LAMP chip for detecting porcine susceptibility-related pathogenic bacteria, including the LAMP primer sets, a reaction buffer, and a chip. In some embodiments, the chip includes an isothermal amplification microfluidic chip, preferably a 32-well reaction cell disc-type microfluidic chip, and more preferably a microfluidic chip shown in FIG. 1. In some embodiments, the microfluidic chip used in the experiment of the present disclosure is a 4×8 microfluidic chip produced by Shanghai Igenetec Diagnostics Co., Ltd.; the microfluidic chip preferably includes 4 test zones; each of the test zone preferably includes a sample hole and a reaction cell; the test zones preferably include 8 reaction cells; and the reaction cells preferably include: an H. parasuis reaction cell, an S. choleraesuis reaction cell, a B. bronchiseptica reaction cell, a P. multocida reaction cell, an S. suis reaction cell, an E. rhusiopathiae reaction cell, an A. pleuropneumoniae reaction cell, and a negative control reaction cell. The present disclosure provides a real-time detection technology combining the microfluidic chip with a LAMP technology, which may establish an efficient, rapid, sensitive and specific real-time detection technology, and provides more significance in the detection of porcine susceptibility-related pathogenic bacteria and a basis for boosting the rapid detection of pathogenic bacteria.

[0048] In some embodiments of the present disclosure, in the LAMP primer sets, an outer primer pair and an inner primer pair corresponding to any one of the pathogenic bacteria have a molar ratio of preferably 1:8; the reaction buffer preferably includes an isothermal amplification buffer and an isothermal amplification enzyme solution; the isothermal amplification buffer preferably includes a 10×Isothermal Amplification Reaction Buffer, BSA-A, dNTP, a MgSO.sub.4 aqueous solution, and a fluorescent dye, and the isothermal amplification enzyme solution preferably includes Bst DNA Polymerase; and the fluorescent dye is more preferably SYTO™9. The LAMP chip may effectively improve the detection efficiency of the LAMP chip by selecting a specific reaction buffer.

[0049] In some embodiments of the present disclosure, after the LAMP primer sets of different pathogenic bacteria is obtained, the LAMP primer sets of the corresponding pathogenic bacteria are coated into the reaction cells corresponding to the microfluidic chip, to obtain a microfluidic chip coated with the LAMP primer sets. The outer primer pair and the inner primer pair corresponding to the pathogenic bacteria are preferably mixed in a molar ratio of 1:8, and then added to the reaction cells of the microfluidic chip; in some embodiments, a LAMP primer set corresponding to a pathogenic bacterium is added in each reaction cell; after the LAMP primer sets of all pathogenic bacteria are added to the reaction cells, vacuum heating and drying, compressing, film sealing and molding treatment are preferably conducted, such that the LAMP primer set is coated into the reaction cells.

[0050] After the microfluidic chip coated with the LAMP primer set is obtained, an unknown sample is preferably coated onto an injection zone of the microfluidic chip for amplification. The unknown sample, the isothermal amplification buffer (the 10×Isothermal Amplification Reaction Buffer, the BSA-A, the dNTP, the MgSO.sub.4 aqueous solution, and the fluorescent dye), and the isothermal amplification enzyme solution (Bst DNA Polymerase) are preferably mixed to obtain a reaction mixture before the coating of the unknown sample; the reaction mixture is coated to the injection zone of the microfluidic chip coated with the LAMP primer set, followed by sealing with a sealing film, and the microfluidic chip is placed in a detection device for real-time amplification detection.

[0051] After the amplification is completed, in some embodiments of the present disclosure, a fluorescence intensity-time curve is plotted based on an amplification trend in each reaction cell through changes of fluorescence values, to determine whether a sample in each amplification reaction well is negative or positive. Namely, if the unknown sample shows an S-shaped curve, and a blank control shows no amplification curve, it is determined that the unknown sample is positive for the corresponding pathogenic bacteria.

[0052] In order to further illustrate the present disclosure, the LAMP primer sets for detecting porcine susceptibility-related pathogenic bacteria, and the kit, the LAMP chip and the use based on the same provided by the present disclosure are described in detail below with reference to the accompanying drawings and examples, but the accompanying drawings and the examples should not be construed as limiting the protection scope of the present disclosure.

Example 1

[0053] Design and preparation of LAMP primer sets for detecting porcine susceptibility-related pathogenic bacteria

[0054] 1. Sequence Acquisition:

[0055] (1) Obtaining an A. pleuropneumoniae APX IV gene sequence: a nucleic acid sequence of the APX IV gene was downloaded from the GenBank public database

TABLE-US-00001 (as set forth in SEQ ID NO: 29: CCGGCAACGACAGTAAGATTGAAGGCACTAAAATCA CCCGTAGGATTGCGGGTAAAGAAGTTACGCTTGATA TTGCCAATCAGAAAATTGAAAAAGGCGTGTCAGAGA AATTGGGGCTGTCTGTTAGTGGTTCGGATATCATTA AATTGTTGTTTGGAGCATTGACTCCAACTTTAAATA GAATGTTGCTATCACAACTCATCCAGTCTTTTTCCG ATAGCTTGGCTAAACTTGATAATCCCTTAGCCCCTT ACACTAAAAATGGCGTGGTTTATGTCACCGGCAAAG GGAATGATGTGCTTAAAGGAACTGAACATGAGGATT TGTTTCTCGGTGGTGAGGGGAATGATACTTATTATG CGAGAGTAGGCGATACAATTGAAGACGCCGACGGCA AAGGTAAAGTCTATTTTGTGAGAGAAAAAGGGGTAC CTAAGGCGGATCCTAAGCGGGTAGAGTTTAGCGAGT ACATAACGAAAGAAGAAATAAAAGAGGTTGAAAAGG GGTTATTAACCTACGCAGTTTTAGAAAATTATAATT GGGAAGAGAAAACGGCGACTTTCGCTCATGCGACTA TGCTTAATGAGCTTTTTACTGATTATACTAATTATC GTTATGAAGTTAAAGGACTAAAATTGCCCGCCGTTA AAAA);

[0056] (2) Obtaining an H. parasuis OMP P2 gene sequence:

[0057] a nucleic acid sequence of the OMP P2 gene

TABLE-US-00002 (as set forth in SEQ ID NO: 30: TCTTGCGCCAGTTCTTACGAAGTCAATTTTCTCTTT AACATTACCTGTTTTTTCAACACCATGACCACCATC AACAGCTACAGTAAATGGAGCATTGACATATTTAAG ACCAAAGTATACACCATCTTTGTCTTTCTTATTAAC AGATCCAGATTTATAGTCATCATGAGTATAACCTGC TGCCACAGTTACAGATTGACTTTCCGCAATCTTAGC TGTGTATTTAGCACCTAAACCAAAGCCAGATTTAGC AGAACCTACTTTTACGCCTCCCTTATCATCACGCTC ATTTGCAACATTATAGTTAGCACCTAACGTCAAACC TTCAATGCCTGTATAGGTATAGTTAATTGCTGAATC AGAATCTGAAGTAAGGATATCAAAACCTTTTTTGTT TGTGTTGTTTGCTGAATATTTAATTCCACCAGTACC AACACCGTATACTTTATCAAAACCAGCTTGACCAAT GCTATCACCGATTACAGCTTGTTTACCAAAAGAAAT TTCATGACCATAGCCACCTAAACCGACGTAAGCATA TTTTGTTTTAACATCGCCCCATCCTGCAGCATTTTT AGAATTACTGTCAAGGCGAGTCTCATAAC);

[0058] (3) Obtaining an S. choleraesuis invA gene sequence:

[0059] a nucleic acid sequence of the invA gene

TABLE-US-00003 (as set forth in SEQ ID NO: 31: ATGCAACATTTGGATATCGCTGAATTAGTTCGTTCC GCACTGGAAGTAAGTGGTTGCGATCCTTCACTCATC GGAGGAATAGATAGCCATTCAACAATTGTTCTGGAT TTATTTGCATTGCCAAGTATCTGTATCAGCGTCAAG GACGATGATGTATGGATCTGGGCGCAATTGGGTGCT GACAGCATGGTGGTATTACAACAGCGGGCTTATGAA ATCTTAATGACCATAATGGAAGGATGCCATTTTGCC CGCGGCGGGCAATTACTACTGGGGGAGCAGAATGGG GAGCTAACGCTTAAAGCCTTAGTGCATCCGGATTTT TTATCTGACGGTGAAAAGTTCTCTACTGCCTTGAAT GGGTTTTACAACTATCTGGAAGTTTTTAGTCGGTCG CTAATGAGATG);

[0060] (4) Obtaining a B. bronchiseptica DNT gene sequence:

[0061] a nucleic acid sequence of the DNT gene

TABLE-US-00004 (as set forth in SEQ ID NO: 32: ATCGCGGGCGTGCTCTGCGATCTCGAGAGCGCGCAG CGCACGTTGCCCGTCGTATTGGCCAGGTTTCGGCCC CTTGGCGTGCTTGCGCGATTCAGAAGGCTGGAGCAG GAAACCGCGGGCATGCTGCTTGGCGACCAGGAGCCG GAGCCTCGGGGCTTCATCAGTTTTACCGATTTTCGC GATAGCGACGCGTTCGCCAGCTACGCGGAGTATGCG GCCCAGTTCAACGACTATATCGATCAATACAGCATA CTCGAGGCGCAGCGGCTGGCGCGGATTCTGGCCCTG GGCTCGCGGATGACGGTCGATCAATGGTGCCTTCCC CTGCAGAAAGTACGGCACTACAAGGTGCTGACATCG CAGCCAGGGCTGATCGCGCGTGGAATCGAAAATCAC AACAGGGGCATTGAATATTGCCTGGGGCGGCCGCCG CTGACCGATCTGCCGGGTCTTTTCACCATGTTCCAG CTCCATGATTCCAGCTGGCTGTTGGTATCGAACATC AACGGTGAGCTTTGGTCTGATGTCCTTGCGAACGCT GAGGTGA);

[0062] (5) Obtaining a P. multocida knit1 gene sequence:

[0063] a nucleic acid sequence of the knit1 gene

TABLE-US-00005 (as set forth in SEQ ID NO: 33: GCTGTAAACGAACTCGCCACTTTTTGTTTCATTTGG ACTGACACGATCAAACCGTTGAACACGAAGAAAAAG ACCAAAATAGGTAACCAATACACGATAAATAAATTA AACCGCTCTGCCGTTAATGGCTTCAATAATGGCCAT AAGAAACGTAACTCAACATGGAAATATTGATAAATC AGACTGACAAGGAAATATAAACCGGCAAATAACAAT AAGCTGAGTAATAAATAACGTCCAATCAGTTGCGCC GTTGTCAAGGAAGCAGATTGGCTCAACACACCAAAC TCCGCCCAACAAAACTGTGCTTTTCTTTGCCACACG CCAAATAAAAGACTACCGACAAGCCCACTCACAACG AGCCATAAAATAATGCCATTTCCCATTTCAAGTGGC ATAAAACTCAATTTCGCGGCAATCGGTTCATTCGCA CCGCCCCACTGGGTAAATAGCGGAT);

[0064] (6) Obtaining an S. suis gdh gene sequence:

[0065] a nucleic acid sequence of the gdh gene

TABLE-US-00006 (as set forth in SEQ ID NO: 34: GCAGCGTATTCTGTCAAACGAGCGCGGCGTTTTTCT TTGATGTCCACCAAGAGGTCGAAGTCGATACCAGTT TCGTCAATGATGTAACCATTTGAGTCTGAAACAGAA ATAACTTTTGCACCAAGTTCAGTCGCTTTTTGAACA GCATATTGGGCAACGTTACCAGAACCTGAGATAAGG ACAGTTTGGTCTTTGAAGGATTTACCGTTTGCTGCC AACATGTTATCAGTGAAGTAAACCAAACCGTAACCA GTTGCTTCTGGGCGGATCAATGAACCACCGAAGCCA AGAGGTTTACCAGTCAAGACACCTGCATCAAACTGG CGGAGGCGTTTGTATTGACCGTACATGTAACCGATC TCACGACCACCGACACCGATGTCACCAGCAGGGACG TCAAGTGAAGGTCCGATGTGTTTTTGCAATTCAGTC ATGAAGCTTTGGCAGAAGCGCATGATTTCAGCATCA GTTTTTCCTTTAGGATCAAAGTCTGAACCACCTTTA CCACCGCCGATTGGAAGACCAGTCAAGACGTTTTTG AAGATTTGCTCAAAACCGAGGAACTTCAAGATGGAT TGGTTTACAGTTGGGTGGAAGCGAAGACCGCCTTTA TAAGGACCTACAGCTGAGTTGAACTGAACACGGTAG CCACGGTTGACTTGAACATTTCCATCTTTATCTGTC CATGG);
and

[0066] (7) Obtaining an E. rhusiopathiae spaA gene sequence:

[0067] a nucleic acid sequence of the spaA gene

TABLE-US-00007 (as set forth in SEQ ID NO: 35: ATGAAAAAGAAAAAACACCTATTTCCGAAAGTAAGT CTTATGTCGTGCTTACTTTTAACAGCAATGCCACTA CAAACAGCTTTTGCTGATTCGACAGATATTTCTGTG ATTCCACTAATCGGTGAACAAGTTGGATTGCTCCCA GTTTTACCTGGGACAGGGGTACATGCTCAGGAATAC AACAAAATGACTGATGCTTATATTGAAAAATTGGTA TCTCTAATTAATCAAAAAGTGAAGCCGTTTCTTATA AATGAACCAAAGGGGTACCAAAGTTTCGAAGCAGTG AATGAAGAGATTAACTCGATTGTAAGTGAACTTAAA AATGAAGGAATGAGTCTTCAAAACATTCACCATATG TTTAAACAAAGCATCCAAAACCTAGCAACTAGAATC GGCTACAGAAGTTTTATGCAGGATGCTATGTATCTT GAAAATTTTGAAAGATTAACGATTCCTGAACTTGAT GAAGCATACGTTGATTTACTCGTGAATTACGAGGTG AAACACCGTATTTTAGTAAAATATGAAGGTAAAGTT AAAGGTAGAGCTCCCTTAGAAGCATTTATAGTTCCT CTAAGAGATAGAATTCGTAGTATGAATGAAATTGCT GCAGAAGTAAATTATTTACCTGAAGCGCATGAGGAT TTCTTAGTTTCAGATTCAAGCGAGTATAATGACAAA CTAAATAATATCAACTTTGCTTTGGGTCTAGGGGTC AGCGAGTTTATTGACTATAACCGGCTCGAAAATATG ATGGAAAAAGAACTTCATCCACTGTATCTTGAACTT TATGCTATGCGGAGAAATCGCCAAATTCAAGTTGTA AGAGATGTATATCCAAACTTGGAACGTGCGAACGCG GTTGTTGAATCCTTAAAGACAATTAAAGATATAAAA CAAAGAGGGAAGAAACTACAGGAACTTCTTGAAATT TATATCCAAAGAAGTGGAGATGTTCGAAAACCAGAT GTACTCCAACGATTTATTGGAAAATATCAATCAGTA GTTGATGAAGAAAAAAATAAACTTCAAGATTATTTA GAATCAGATATTTTTGATTCATATAGTGTGGATGGC GAGAAAATAAGAAATAAAGAAATTACACTCATCAAT AGAGATGCATACTTATCTATGATTTACAGAGCTCAA TCGATTTCGGAAATTAAGACGATTCGTGCAGATTTA GAATCACTTGTCAAATCATTCCAAAATGAAGAAAGT GACTCTAAAGTAGAGCCTGAAAGTCCCGTTAAAGTA GAAAAACCAGTTGATGAAGAAAAACCTAAAGATCAA AAGAAGCTAGTTGATCAATCAAAACCCGAATCGAAT TCAAAAGAAGGGTGGATTAAGAAAGATAATAAGTGG TTCTATATTGAGAAATCAGGTGGAATGGCAACAGGT TGGAAGAAGGTAGCAGACAAATGGTACTACCTCGAT AATACGGGTGCTATAGTTACGGGTTGGAAGAAGGTA GCAAACAAATGGTACTATCTTGAAAAATCAGGTGCG ATGGCAACAGGATGGAAGAAAGTATCAAACAAGTGG TACTACCTTGAAAACTCAGGTGCAATGGCAACAGGA TGGAAGAAAGTATCAAACAAGTGGTACTACCTTGAA AATTCAGGCGCAATGGCTACAGGATGGAAAAAGGTA GCAAACAAATGGTACTACCTTGAAAACTCAGGTGCG ATGGCAACAGGATGGAAGAAAGTATCGAACAAGTGG TACTACCTTGAAAACTCAGGCGCAATGGCTACAGGA TGGAAAAAGGTAGCAAACAAATGGTACTACCTTGAT AAATCAGGAATGATGGTTACAGGTTCAAAATCTATT GATGGTAAAAAGTATGCATTTAAGAACGATGGAAGT TTAAAATAG).

[0068] 2. Primer Design

[0069] (1) Specific gene primers: conserved segments of the above-mentioned specific genes were determined, and LAMP primer sets were designed for the conserved segments.

[0070] (2) Synthesis of primers: the primer sequences in Table 1 were entrusted to synthesize by Tsingke Biotechnology Co., Ltd.

[0071] In this example, the 28 primers designed were shown in Table 1.

TABLE-US-00008 TABLE 1 Primer sequences in primer sets Primer Primer sequence ID (5′-3′) Pathogenic bacteria SEQ ID F3: A. pleuropneumoniae NO. 1 CCCTTAGCCCCTTACACTA (APP) SEQ ID B3: NO. 2 CGCTTAGGATCCGCCTTA SEQ ID FIP: NO. 3 CACCACCGAGAAACAAAT CCTCGGCGTGGTTTATGT CACC SEQ ID BIP: NO. 4 AGGCGATACAATTGAAGA CGCCGGTACCCCTTTTTC TCTCACCAC SEQ ID F3: H. parasuis (HPS) NO. 5 ACCTACTTTTACGCCTCC SEQ ID B3: NO. 6 GCATTGGTCAAGCTGGTT SEQ ID FIP: NO. 7 CAGGCATTGAAGGTTTGA CGTTTATCATCACGCTCA TTTGC SEQ ID BIP: NO. 8 ACCTTTTTTGTTTGTGTT GTTTGCTAAAGTATAGGT GTTGGTACTG SEQ ID F3: S. choleraesuis NO. 9 CATTGCCAAGTATCTGTAT (Sal) CAGC SEQ ID B3: NO. 10 CCGGATGCACTAAGGCTTT A SEQ ID FIP: NO. 11 GGAAGGATGCCATTTTGC CCGGTTAGCTCCCCATTC TGCTC SEQ ID BIP: NO. 12 TGAGTGGGCTTGTCGGTA GTCAACACACCAAACTCT GC SEQ ID F3: B. bronchiseptica NO. 13 TGACGGTCGATCAATGGTG (Bb) SEQ ID B3: NO. 14 AGCCAGCTGGAATCATGGA SEQ ID FIP: NO. 15 TCGATTCCACGCGCGATC AGTCCCCTGCAGAAAGTA CGG SEQ ID BIP: NO. 16 GGCATTGAATATTGCCTG GGGCAACATGGTGAAAAG ACCCGG SEQ ID F3: P. multocida  NO. 17 CGTTGTCAAGGAAGCAGA (Pm) SEQ ID B3: NO. 18 TCCGCTATTTACCCAGTG SEQ ID FIP: NO. 19 CGAGCCATAAAATAATGC CATTTCCGTGCGAATGAA CCGATTG SEQ ID BIP: NO. 20 TGAGTGGGCTTGTCGGTA GTCAACACACCAAACTCT GC SEQ ID F3: S. suis (SS) NO. 21 ACACCGATGTCACCAGCA SEQ ID B3: NO. 22 TCGCTTCCACCCAACTGTA SEQ ID FIP: NO. 23 TGCGCTTCTGCCAAAGCT TCAGACGTCAAGTGAAGG TCCG SEQ ID BIP: NO. 24 CCACCTTTACCACCGCCG ATAGTTCCTCGGTTTTGA GCAA SEQ ID F3: E. rhusiopathiae NO. 25 CGGCTCGAAAATATGATGG (ER) SEQ ID B3: NO. 26 GAACATCTCCACTTCTTTG G SEQ ID FIP: NO. 27 ACGTTCCAAGTTTGGATA TACATCTTCATCCACTGT ATCTTGAACT SEQ ID BIP: NO. 28 GCGAACGCGGTTGTTGAA TCCTGTAGTTTCTTCCCT CTTTGT

Example 2

[0072] Preparation and Use of LAMP Chip for Detecting Porcine Susceptibility-Related Pathogenic Bacteria

[0073] 1. Preparation of a LAMP Chip for Detecting Porcine Susceptibility-Related Pathogenic Bacteria

[0074] In the present disclosure, the microfluidic chip for detecting porcine susceptibility-related pathogenic bacteria included the following components:

[0075] (1) Isothermal Amplification Buffer

[0076] The isothermal amplification buffer included water as a solvent, and solutes and concentrations thereof were as follows: 1.4 mM dNTPs, a 10×Isothermal Amplification Reaction Buffer, a 100 mM MgSO.sub.4 aqueous solution, 10% BSA-A by mass percentage, and SYTO™9. A reaction system of microfluidic LAMP was shown in Table 2.

TABLE-US-00009 TABLE 2 Reaction system of microfluidic LAMP Final Component Volume concentration 10× ThermoPol Buffer 2.5 μL 1× MgSO.sub.4 (100 mM) 1.5 μL 6 mM Bst DNA Polymerase   1 μL 320 U/mL Large Fragment dNTPMix (10 mM) 3.5 μL 1.4 mM 10% BSA-A   3 μL SYTO ™9 0.5 μL Template DNA   2 μL SW Water Making up to 25 μL

[0077] (2) Isothermal Amplification Enzyme Solution

[0078] The isothermal amplification enzyme solution included water as a solvent, and solute and concentration were as follows: Bst DNA Polymerase Large Fragment 320 U/mL.

[0079] (3) 32-Well Reaction Cell Disc-Type Microfluidic Chip Loaded with Primer Pairs

[0080] The 32-well reaction cell disc-type microfluidic chip was a 4×8 microfluidic chip, produced by Shanghai Igenetec Diagnostics Co., Ltd. A schematic diagram of the microfluidic chip was shown in FIG. 1,

[0081] In FIG. 1, the reaction cells 7, 15, 23, and 31 in the outer circle were immobilized with the LAMP primers of A. pleuropneumoniae provided in Example 1 (SEQ ID NOs: 1 to 4);

[0082] the reaction cells 1, 9, 17, and 25 in the outer circle were immobilized with the LAMP primers of H. parasuis provided in Example 1 (SEQ ID NOs: 5 to 8);

[0083] the reaction cells 2, 10, 18, and 26 marked in the outer circle were immobilized with the LAMP primers of S. choleraesuis provided in Example 1 (SEQ ID NOs: 9 to 12);

[0084] the reaction cells 3, 11, 19, and 27 in the outer circle were immobilized with the LAMP primers of B. bronchiseptica provided in Example 1 (SEQ ID NOs: 13 to 16);

[0085] the reaction cells 5, 13, 21, and 29 in the outer circle were immobilized with the LAMP primers of P. multocida provided in Example 1 (SEQ ID NOs: 17 to 20);

[0086] the reaction cells 6, 14, 22, and 30 in the outer circle were immobilized with the LAMP primers of S. suis provided in Example 1 (SEQ ID NOs: 21 to 24);

[0087] the reaction cells 7, 15, 23, and 31 in the outer circle were immobilized with the LAMP primers of E. rhusiopathiae provided in Example 1 (SEQ ID NOs: 25 to 28); and

[0088] the reaction cells 8, 16, 24, and 32 were blank controls, with no amplification primers coated.

[0089] A method for coating the primers into the disc-type microfluidic chip was as follows: the outer primers and the inner primers corresponding to the pathogenic bacteria were mixed according to a molar ratio of 1:8, and then mixed with trehalose to prepare corresponding mixed solutions. In each mixed solution, the inner primer had a final concentration of 1.6 μM, the outer primer had a final concentration of 0.2 μM, and the trehalose had a mass percentage of 0.5%.

[0090] Each mixed solution was added to reaction cells corresponding to the chip, and the LAMP primers were coated into the reaction cells after vacuum heating, compressing, film sealing and stamping; all the LAMP primers of the pathogenic bacteria were coated into the reaction cells of the chip to obtain the LAMP chip for later use.

[0091] 2. Use of LAMP Chip for Detecting Various Porcine Susceptibility-Related Pathogenic Bacteria

[0092] (1) Extraction of a Genome

[0093] A bacterial genome and a non-target bacterial genome were extracted using an extraction kit provided by TIANGEN, to obtain a target bacterial genome and a non-target bacterial genome of the porcine pathogenic bacteria.

[0094] The genomic DNA was extracted from clinical samples by the boiling method.

[0095] (2) Preparation of a Reaction System

[0096] 75 μL of the isothermal amplification mixed solution including 3 μL of the isothermal amplification enzyme solution and 6 μL of the target bacterial genome and the non-target bacterial genome of the porcine pathogenic bacteria was mixed evenly by vortex shaking, and injected into the sample hole of the LAMP chip, followed by attaching a parafilm.

[0097] (3) Isothermal Amplification and Detection

[0098] The LAMP chip was placed in a microfluidic chip detection instrument, centrifuged quickly at 1,500 rpm/min for 15 sec and then 3,500 rpm/min for 30 sec, and reacted at 63° C. for 1 h.

[0099] (4) Determination of Results

[0100] The results are shown in FIG. 2 to FIG. 8, where corresponding “S” shape in the reaction cell means positive; and flat curve corresponding to the reaction cell means negative.

[0101] As can be seen from FIG. 2, the detection results of the reaction cells 7, 15, 23, and 31 of the primer set immobilized with A. pleuropneumoniae are “S” shape, showing positive, indicating that the unknown sample is a clinical sample positive for the A. pleuropneumoniae.

[0102] As can be seen from FIG. 3, the detection results of the reaction cells 1, 9, 17, and 25 of the primer set immobilized with H. parasuis are “S” shape, showing positive, indicating that the unknown sample is a clinical sample positive for the H. parasuis.

[0103] As can be seen from FIG. 4, the detection results of the reaction cells 2, 10, 18, and 26 of the primer set immobilized with S. choleraesuis are “S” shape, showing positive, indicating that the unknown sample is a clinical sample positive for the S. choleraesuis.

[0104] As can be seen from FIG. 5, the detection results of the reaction cells 3, 11, 19, and 27 of the primer set immobilized with B. bronchiseptica are “S” shape, showing positive, indicating that the unknown sample is a clinical sample positive for the B. bronchiseptica.

[0105] As can be seen from FIG. 6, the detection results of the reaction cells 5, 13, 21, and 29 of the primer set immobilized with P. multocida are “S” shape, showing positive, indicating that the unknown sample is a clinical sample positive for the P. multocida.

[0106] As can be seen from FIG. 7, the detection results of the reaction cells 6, 14, 22, and 30 of the primer set immobilized with S. suis are “S” shape, showing positive, indicating that the unknown sample is a clinical sample positive for the S. suis.

[0107] As can be seen from FIG. 8, the detection results of the reaction cells 7, 15, 23, and 31 of the primer set immobilized with E. rhusiopathiae are “S” shape, showing positive, indicating that the unknown sample is a clinical sample positive for the E. rhusiopathiae.

[0108] The results show that the LAMP primer sets for detecting porcine susceptibility-related pathogenic bacteria and the LAMP chip based on the same provided by the present disclosure may accurately detect the A. pleuropneumoniae, the H. parasuis, the S. choleraesuis, the B. bronchiseptica, the P. multocida, the S. suis, and the E. rhusiopathiae.

[0109] The results compared with conventional PCR detection are shown in Table 2.

TABLE-US-00010 TABLE 2 Detection results of clinical samples LAMP chip Detection PCR Detection Type Positive Negative rate (%) Positive Negative rate (%) A. pleuropneumoniae (APP) 4 116 3.3 4 116 3.3 H. parasuis (HPS) 1 119 0.8 1 119 0.8 S. choleraesuis (Sal) 2 118 1.6 2 118 1.6 B. bronchiseptica (Bb) 1 119 0.8 1 119 0.8 P. multocida (PW) 4 116 3.3 4 116 3.3 S. suis (SS) 7 113 5.8 7 113 5.8 E. rhusiopathiae (ER) 2 118 1.6 2 118 1.6 Total 21 17.5 21 17.5

[0110] As can be seen from Table 2, the detection results of the LAMP chip are the same as those of the conventional PCR detection, indicating that the detection method provided by the present disclosure has a detection rate reaching 100%. In addition, the detection method may specifically detect the A. pleuropneumoniae, the H. parasuis, the S. choleraesuis, the B. bronchiseptica, the P. multocida, the S. suis, and the E. rhusiopathiae, which has a high specificity and no cross reaction with other pathogens.

Example 3

[0111] Sensitivity of LAMP Chip

[0112] Sensitivity detection was conducted using the LAMP chip prepared in Example 2.

[0113] The nucleic acids of the A. pleuropneumoniae, the H. parasuis, the S. choleraesuis, the B. bronchiseptica, the P. multocida, the S. suis, and the E. rhusiopathiae extracted in Example 2 were diluted separately, and then mixed to obtain a mixed nucleic acid.

[0114] The A. pleuropneumoniae sample had concentration gradients as follows: 1.17×10.sup.2 ng/μL, 1.17×10.sup.1 ng/μL, 1.17×10.sup.0 ng/μL, 1.17×10.sup.−1 ng/μL, 1.17×10.sup.−2 ng/μL, 1.17×10.sup.−3 ng/μL, 1.17×10.sup.−4 ng/μL, and 1.17×10.sup.−5 ng/μL.

[0115] The H. parasuis sample had concentration gradients as follows: 1.27×10.sup.2 ng/μL, 1.27×10.sup.1 ng/μL, 1.27×10.sup.0 ng/μL, 1.27×10.sup.−1 ng/μL, 1.27×10.sup.−2 ng/μL, 1.27×10.sup.−3 ng/μL, 1.27×10.sup.−4 ng/μL, and 1.27×10.sup.−5 ng/μL.

[0116] The S. choleraesuis sample had concentration gradients as follows: 1.57×10.sup.2 ng/μL, 1.57×10.sup.1 ng/μL, 1.57×10.sup.0 ng/μL, 1.57×10.sup.−1 ng/μL, 1.57×10.sup.−2 ng/μL, 1.57×10.sup.−3 ng/μL, 1.57×10.sup.−4 ng/μL, and 1.57×10.sup.−5 ng/μL.

[0117] The B. bronchiseptica sample had concentration gradients as follows: 1.20×10.sup.2 ng/μL, 1.20×10.sup.1 ng/μL, 1.20×10.sup.0 ng/μL, 1.20×10.sup.−1 ng/μL, 1.20×10.sup.−2 ng/μL, 1.20×10.sup.−3 ng/μL, 1.20×10.sup.−4 ng/μL, and 1.20×10.sup.−5 ng/μL.

[0118] The P. multocida sample had concentration gradients as follows: 2.05×10.sup.1 ng/μL, 2.05×10.sup.0 ng/μL, 2.05×10.sup.−1 ng/μL, 2.05×10.sup.−2 ng/μL, 2.05×10.sup.−3 ng/μL, 2.05×10.sup.−4 ng/μL, 2.05×10.sup.−5 ng/μL, and 2.05×10.sup.−6 ng/μL.

[0119] The S. suis sample had concentration gradients as follows: 1.13×10.sup.1 ng/μL, 1.13×10.sup.0 ng/μL, 1.13×10.sup.−1 ng/μL, 1.13×10.sup.−2 ng/μL, 1.13×10.sup.−3 ng/μL, 1.13×10.sup.−4 ng/μL, 1.13×10.sup.−5 ng/μL, and 1.13×10.sup.−6 ng/μL.

[0120] The E. rhusiopathiae sample had concentration gradients as follows: 8.3×10.sup.1 ng/μL, 8.3×10.sup.0 ng/μL, 8.3×10.sup.−1 ng/μL, 8.3×10.sup.−2 ng/μL, 8.3×10.sup.−3 ng/μL, 8.3×10.sup.−4 ng/μL, 8.3×10.sup.−5 ng/μL, and 8.3×10.sup.−6 ng/μL.

[0121] The mixed nucleic acid was mixed with an isothermal amplification system (namely the isothermal amplification buffer and the isothermal amplification enzyme solution provided in Example 2), and a resulting sample was injected into the isothermal amplification microfluidic chip (namely the LAMP chip prepared in Example 2), and isothermal amplification was conducted at 63° C. for 1 h.

[0122] The reaction results are shown in FIG. 9 to FIG. 15, where the A. pleuropneumoniae has a minimum limit of detection (LOD) of 1.17 pg/μL; the H. parasuis has a minimum LOD of 12.7 pg/μL; the S. choleraesuis has a minimum LOD of 15.7 pg/μL; the B. bronchiseptica has a minimum LOD of 12.0 pg/μL; the P. multocida has a minimum LOD of 2.05 pg/μL; the S. suis has a minimum LOD of 1.13 pg/μL; and the E. rhusiopathiae has a minimum LOD of 8.3 pg/μL. It showed that the detection method provided by the present disclosure had an extremely high sensitivity.

[0123] In the present disclosure, the LAMP chip may accurately detect the A. pleuropneumoniae, the H. parasuis, the S. choleraesuis, the B. bronchiseptica, the P. multocida, the S. suis, and the E. rhusiopathiae, thereby making up for the time-consuming and labor-intensive defects of the above pathogen detection technology. The chip may also expand a detection range of pathogens, improve a detection sensitivity and specificity, reduce labor intensity, and shorten a detection cycle. The detection method may also visually determine the detection results with naked eyes without expensive PCR detection instruments, making this method fast, simple, and easy to popularize, safe and reliable in scientific research and production practice, and suitable for field operation. For clinical purposes, the detection of porcine susceptibility-related pathogenic bacteria can be conducted within 1 h, and the detection results are not only faster than the commonly-used PCR methods, but also of great significance for rapid auxiliary guidance of treatment and medication. In addition, the multi-indicator typing detection may also be used for epidemiological investigation and epidemic monitoring.

[0124] The present disclosure has been disclosed with preferred examples as above, which shall not be construed as a limitation to the present disclosure. Any person skilled in the art can make changes and variations without departing from the spirit and scope of the present disclosure. The protection scope of the present disclosure shall be defined by the claims.