Specific primer for identifying indoleproducing bacteria and application

Abstract

The invention relates to the technical field of biology, in particular to a specific primer for identifying indoleproducing bacteria and application. Three pairs of specific primers (a first primer pair, a second primer pair and / or a third primer pair) for targeted identification of the indole-producing flora are finally screened according to the design of tryptophan enzyme gene conserved regions of different bacteriophage indole-producing flora and through PCR amplification test, agarose gel electrophoresis and clone sequencing. The kit prepared by using the specific primer obtained by screening can amplify a sample to be detected through a PCR instrument and qualitatively identify the indoleproducing bacteria, and the identification method is simple, efficient and accurate, and has practical application value. The invention provides a gene-level molecular marker for screening the indoleproducing bacteria, and provides a convenient and accurate method for observing and researching the diversity change of indoleproducing bacteria species in human and animal bodies.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly relates to a specific primer for identifying indole-producing bacteria and its use. Background Art

[0002] Chronic kidney disease (CKD) is a common chronic disease worldwide. In recent years, it has shown a rapid growth trend and has become a major public health problem globally. How to delay the progression of CKD to the middle and late stages is of great clinical and socio-economic significance for improving the prognosis of CKD, enhancing the quality of life of patients, and reducing the mortality and disability rates.

[0003] Renal fibrosis is the key pathological change in chronic kidney disease. Indoxyl sulfate is one of the main enterogenic uremic toxins mediating fibrosis in chronic kidney disease. The precursor of indoxyl sulfate - indole is produced by intestinal bacteria through the conversion of tryptophan by tryptophanase. In the normal human intestine, indole secreted by indole-producing bacteria can serve as an intercellular signal to regulate the normal physiological activities of microorganisms. However, in patients with chronic kidney disease, there is an "intestinal leakage" phenomenon. After indole enters the blood and circulates to the liver, it is sulfated to produce indoxyl sulfate. Some studies have found that knocking out the bacterial tryptophanase gene can eliminate the production of indole in vitro, and no bacteria have been found to produce indole through enzymes other than tryptophanase. Therefore, many sci literature regard the tryptophanase gene as a marker gene for identifying indole-producing bacteria. The distribution of indole-producing bacteria is very extensive and can be distributed in the intestines of humans and animals, as well as in the soil. Therefore, it is necessary to understand the species diversity of indole-producing bacteria. However, current researchers have not fully studied the species diversity of indole-producing bacteria. There is no accurate and convenient method to identify the species diversity of indole-producing bacteria.

[0004] Currently, the existing technology identifies and determines indole-producing bacteria through the combined analysis of 16S rRNA gene and whole-genome shotgun sequencing. However, these methods are costly, require high bioinformatics technology, and have a long sequencing cycle. Based on this, developing specific primers for identifying the species diversity of indole-producing bacteria to make the method for identifying and determining indole-producing bacteria simpler, more efficient, and more accurate has practical application value. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a specific primer for identifying indole-producing bacteria in view of the deficiencies of the existing technology in identifying and determining indole-producing bacteria through the combined analysis of 16S rRNA gene and whole-genome shotgun sequencing, which are costly, require high bioinformatics technology, and have a long sequencing cycle.

[0006] Another technical problem to be solved by the present invention is to provide the use of the specific primer in the preparation of a kit for identifying indole-producing bacteria.

[0007] Another technical problem to be solved by the present invention is to provide a kit for identifying indole-producing bacteria.

[0008] The last technical problem to be solved by the present invention is to provide a method for identifying indole-producing bacteria.

[0009] To solve the above technical problems, the technical solutions adopted by the present invention are as follows: In the first aspect, the present invention discloses a specific primer for identifying indole-producing bacteria, and the specific primer is any one or a combination of several groups among the first primer pair, the second primer pair or the third primer pair.

[0010] Among them, the forward primer of the first primer pair has a nucleotide sequence as shown in SEQ ID NO.1; the reverse primer of the first primer pair has a nucleotide sequence as shown in SEQ ID NO.2.

[0011] Among them, the forward primer of the second primer pair has a nucleotide sequence as shown in SEQ ID NO.3; the reverse primer of the second primer pair has a nucleotide sequence as shown in SEQ ID NO.4.

[0012] Among them, the forward primer of the third primer pair has a nucleotide sequence as shown in SEQ ID NO.5; the reverse primer of the second primer pair has a nucleotide sequence as shown in SEQ ID NO.6.

[0013] In some embodiments of the present invention, the first primer pair identified 1 phylum Bacteroidota, 1 class Bacteroidia, 1 order Bacteroidales, 3 families Muribaculaceae , Bacteroidaceae , Rikenellaceae , 4 genera Duncaniella , Bacteroides , unclassified Muribaculaceae , Alistipes , 10 species Duncaniella sp., Muribaculaceae bacterium, Bacteroides stercoris , Bacteroides thetaiotaomicron , uncultured Duncaniella sp., uncultured Alistipes sp., Bacteroides sp. HMSC067B03, Bacteroides uniformis , Alistipes sp., Bacteroides ovatus of indole-producing bacteria.

[0014] In some embodiments of the present invention, the second primer pair identified 5 phyla in the bacterial kingdom, namely Bacillota, Verrucomicrobiota, Acidobacteriota, Chloroflexota, Actinomycetota, 7 classes, namely Clostridia, Thermoanaerobaculia, Vicinamibacteria, Verrucomicrobiia, Candidatus Limnocylindria, Chloroflexia, Thermoleophilia, 5 orders, namely Eubacteriales, Vicinamibacterales, Verrucomicrobiales, Chloroflexales, Thermoleophilales, 4 families Oscillospiraceae , Akkermansiaceae , Roseiflexineae , Thermoleophilaceae , 2 genera Akkermansia , Candidatus Ribeiella , 7 species Oscillospiraceae bacterium, Thermoanaerobaculia bacterium, Vicinamibacterales bacterium, uncultured Akkermansia sp., Candidatus Limnocylindria bacterium, Candidatus Ribeiella danica sp., Thermoleophilaceae bacterium that produce indole.

[0015] In some embodiments of the present invention, the third primer pair identified 1 phylum in the bacterial kingdom, namely Pseudomonadota, 1 class, namely Gammaproteobacteria, 1 order, namely Enterobacterales, 1 family Enterobacteriaceae , 1 genus Escherichia , 1 species Escherichia coli that produce indole.

[0016] Among them, the indole-producing bacteria are intestinal bacteria that convert tryptophan into indole through tryptophanase.

[0017] In the normal human intestine, intestinal bacteria (indole-producing bacteria) convert tryptophan into indole through tryptophanase. Indole can serve as an intercellular signal to regulate the normal physiological activities of microorganisms. However, in patients with chronic kidney disease, there is an "intestinal leakage" phenomenon. After indole enters the bloodstream, it circulates to the liver and is sulfated to produce indoxyl sulfate. Indoxyl sulfate is one of the main gut-derived uremic toxins that mediate fibrosis in chronic kidney disease, and renal fibrosis is the key pathological change in chronic kidney disease.

[0018] Among them, the specific primers for identifying indole-producing bacteria are specifically used to detect the tryptophanase gene in indole-producing bacteria to identify whether it is an indole-producing bacterium.

[0019] In a second aspect, the present invention discloses the use of the specific primers in the preparation of a kit for identifying indole-producing bacteria.

[0020] In a third aspect, the present invention discloses a kit for identifying indole-producing bacteria, which contains any one or several groups of the specific primers.

[0021] Among them, the PCR reaction system used in the kit is a 50 μL system: 0.25 μL of Ex Taq HS, 4 μL of dNTP Mixture, 1 μL of forward primer, 1 μL of reverse primer, 5 μL of 10×Ex Taq Buffer each 1 μL, 36.75 μL of ddH2O, and 2 μL of DNA of the sample to be tested.

[0022] Specifically, the DNA of the sample to be tested is: genomic DNA of rat fecal flora, genomic DNA of human fecal flora, or genomic DNA of soil flora.

[0023] Among them, the PCR reaction conditions used in the kit are: pre-denaturation at 94°C for 10 minutes, 1 cycle; denaturation at 95°C for 30 seconds, annealing at 53°C for 30 seconds, extension at 72°C for 30 seconds for 1 cycle, a total of 30 cycles; final extension at 72°C for 10 minutes for 1 cycle, and the product is stored at 4°C.

[0024] In a fourth aspect, the present invention discloses a method for identifying indole-producing bacteria, which includes the following steps: (1) Extract the DNA of the sample to be tested; (2) Using the DNA of the sample to be tested as a template, perform PCR amplification with the specific primers to obtain a PCR product; (3) Detect the PCR product by 1% agarose gel electrophoresis and judge the result.

[0025] Among them, the specific primers are the first primer pair, the second primer pair, and / or the third primer pair.

[0026] Specifically, since the types of bacteria contained in the sample to be tested are not all the same, the types of bacteria containing the tryptophanase gene among these bacteria are not all the same. Therefore, the tryptophanase gene sequences among different types of bacteria are not completely the same. These incompletely identical tryptophanase gene sequences contain both similar conserved regions and dissimilar non-conserved regions. However, as long as the method described in the present invention is used for PCR amplification with the first primer pair, an amplification band appears at the 440 bp position on the agarose gel; or when PCR amplification is performed with the second primer pair, an amplification band appears at the 425 bp position on the agarose gel; or when PCR amplification is performed with the third primer pair, an amplification band appears at the 406 bp position on the agarose gel, it indicates that the sample to be tested contains tryptophanase and is an indole-producing bacterium.

[0027] Beneficial effects: The present invention is designed based on the conserved regions of the tryptophanase genes of indole-producing bacteria in different phyla, and through PCR amplification experiments, agarose gel electrophoresis, and cloning and sequencing, 3 pairs of specific primers for targeted identification of indole-producing bacteria are finally screened. The kit prepared with the screened specific primers can amplify the sample to be tested through a PCR instrument to directly and qualitatively identify indole-producing bacteria. This identification method is simple, efficient, accurate, the experimental cycle is shortened to about 5 hours, the cost is low, and the requirements for bioinformatics technology are relatively low, having practical application value. The present invention provides a molecular marker at the gene level for microbiology practitioners to screen indole-producing bacteria, and provides a convenient and accurate method for researchers in clinical medicine and basic medicine to observe the changes in the species diversity of indole-producing bacteria in humans and animals. Description of the drawings

[0028] The following further specifically describes the present invention in conjunction with the drawings, and the above and / or other advantages of the present invention will become clearer.

[0029] Figure 1 It is the conserved region of the forward and reverse primer sequences of the first primer pair. Among them, a is the conserved region of the forward primer, b is the conserved region of the reverse primer sequence, and "*" is the conserved base in the forward primer region or the reverse primer region.

[0030] Figure 2 It is the conserved region of the forward and reverse primer sequences of the fourth primer pair. Among them, a is the conserved region of the forward primer, b is the conserved region of the reverse primer sequence, and "*" is the conserved base in the forward primer region or the reverse primer region.

[0031] Figure 3 It is the conserved region of the forward and reverse primer sequences of the second primer pair. Among them, a is the conserved region of the forward primer, b is the conserved region of the reverse primer sequence, and "*" is the conserved base in the forward primer region or the reverse primer region.

[0032] Figure 4 It is the conserved region of the forward and reverse primer sequences of the fifth primer pair. Among them, a is the conserved region of the forward primer, b is the conserved region of the reverse primer sequence, and "*" is the conserved base in the forward primer region or the reverse primer region.

[0033] Figure 5 It is the conserved region of the forward and reverse primer sequences of the third primer pair. Among them, a is the conserved region of the forward primer, b is the conserved region of the reverse primer sequence, and "*" is the conserved base in the forward primer region or the reverse primer region.

[0034] Figure 6 It is the conserved region of the forward and reverse primer sequences of the sixth primer pair. Among them, a is the conserved region of the forward primer, b is the conserved region of the reverse primer sequence, and "*" is the conserved base in the forward primer region or the reverse primer region.

[0035] Figure 7 It is the agarose gel electrophoresis detection result of 6 groups of tryptophanase gene primer pairs. Among them, a is the agarose gel electrophoresis detection result of the first primer pair, the second primer pair, and the third primer pair; b is the agarose gel electrophoresis detection result of the fourth primer pair, the fifth primer pair, and the sixth primer pair.

[0036] Figure 8 It is the motif display of the sequence obtained by amplification with the first primer pair. Among them, a is the binding region of the forward primer of the first primer pair and the DNA template; b is the binding region of the reverse primer of the first primer pair and the DNA template.

[0037] Figure 9 It is the motif display of the sequence obtained by amplification with the second primer pair. Among them, a is the binding region of the forward primer of the second primer pair and the DNA template; b is the binding region of the reverse primer of the second primer pair and the DNA template.

[0038] Figure 10 It is the motif display of the sequence obtained by amplification with the third primer pair. Among them, a is the binding region of the forward primer of the third primer pair and the DNA template; b is the binding region of the reverse primer of the third primer pair and the DNA template.

[0039] Figure 11 It is the binding region of the forward primer of the sixth primer pair and the DNA template.

[0040] Figure 12 It is the phylogenetic tree constructed from the sequences of human, rat fecal flora and soil flora identified by the first, second, and third primer pairs and the reference sequence. The reference sequence is the NCBI Reference Sequence ID or the Genebank plus the name of the strain or family. Detailed implementation method

[0041] The following further specifically describes the present invention in conjunction with specific embodiments, and the above and / or other advantages of the present invention will become clearer.

[0042] In the following examples, the experimental methods are conventional methods unless otherwise specified; the reagents and materials can be obtained from commercial sources unless otherwise specified.

[0043] The present invention has been approved by the Ethics Committee of the Affiliated Hospital of Nanjing University of Chinese Medicine, and the approval number is: 2025DW-030-01.

[0044] Example 1: Primer Design The tryptophanase DNA sequences in the bacterial kingdom in the NCBI database were queried, and a total of 154 tryptophanase gene sequences in 3 phyla (Phylum Bacteroidota, Phylum Actinomycetota, Phylum Pseudomonadota) were collected, and 6 groups of primer pairs were designed according to the 3 phyla respectively.

[0045] 1. Two classes Bacteroidia and Flavobacteriia, two orders Bacteroidales and Flavobacteriales, and seven families Bacteroidaceae , Dysgonomonadaceae , Odoribacteraceae , Porphyromonadaceae, Prevotellaceae , Rikenellaceae, Weeksellaceae , and ten genera Bacteroides , Dysgonomonas , Butyricimonas , Odoribacter , Porphyromonas , Prevotella , Alistipes , Chryseobacterium , Elizabethkingia , Empedobacte r, a total of 40 bacterial species Bacteroides acidifaciens (GeneID89646669), Bacteroides cellulosilyticus (GeneID66307512), Bacteroides clarus (GeneID61678932), Bacteroides congonensis (GeneID82173502), Empedobacter brevis (GeneID84649463), Elizabethkingia anophelis R26(GeneID56685136), Bacteroides eggerthii (GeneID64229610), Bacteroides faecis (GeneID69591221),Bacteroides fluxus (GeneID86048535), Bacteroides intestinalis (GeneID69508995), Bacteroides nordii (GeneID69502888), Bacteroides ovatus (GeneID29451707), Bacteroides salyersiae (GeneID77203696), Bacteroides stercoris (GeneID31797138), Bacteroides thetaiotaomicron (GeneID60927475), Bacteroides uniformis (GeneID66280815), Bacteroides xylanisolvens (GeneID69480792), Elizabethkingia miricola (GeneID66583889), Dysgonomonas mossii (GeneID78083246), Butyricimonas faecihominis (GeneID83158670), Butyricimonas paravirosa (GeneID86890433), Butyricimonas virosa (GeneID77452881), Odoribacter splanchnicus (GeneID69855797), Porphyromonas endodontalis (GeneID84818444), Porphyromonas gingivalis (GeneID29256096), Porphyromonas gulae (GeneID57239471), Prevotella intermedia (GeneID34515074), Prevotella nigrescens (GeneID67366882), Prevotella pallens (GeneID78570559), Prevotella pectinovora (GeneID88847520), Alistipes finegoldii (GeneID79836330), Alistipes onderdonkii subsp. vulgaris (GeneID59808890), Alistipes putredinis (GeneID73803429), Alistipes shahii (GeneID66250428), Chryseobacterium arthrosphaerae (GeneID78303638), Chryseobacterium gleum (GeneID84657101), Chryseobacterium indologenes (GeneID56899753), Elizabethkingia bruuniana (GeneID78268328),Elizabethkingia meningoseptica (GeneID72520441), Empedobacter falsenii (GeneID78399994) tryptophanase gene sequences were aligned using the bioedit software. Then, online primer design was performed using Clustal Omega (https: / / www.ebi.ac.uk / Tools / msa / clustalo / ), and two sets of sequence conserved regions ( Figure 1 、 Figure 2 ) were found, and primer design was performed separately to obtain the first primer pair and the fourth primer pair. The forward primer of the first primer pair is: NGJ_367_F: CAYTTYGAYACNACNAARGG (SEQ ID NO.1); the reverse primer is: NGJ_806_R: GCNANRAANCCDCCNATRTT (SEQ ID NO.2), and the amplified DNA length is 440 bp. The forward primer of the fourth primer pair is NGJ_202_F: GGNGAYGARAGYTAYGCHGG (SEQ ID NO.7); the reverse primer is: NGJ_1040_R: AANANNGCRTGNCCNCC (SEQ ID NO.8), and the amplified DNA length is 839 bp.

[0046] 2. Two classes, Acidimicrobiia and Actinomycetes, three orders, Acidimicrobiales, Kitasatosporales, and Propionibacteriales, under the phylum Actinomycetota, three families Acidimicrobiaceae 、 Streptomycetaceae 、 Propionibacteriaceae , and three genera Ferrimicrobium 、 Streptomyces、Cutibacterium , a total of 12 species Ferrimicrobium acidiphilum (GeneID78373721), Streptomyces antimycoticus (GeneID91525172), Streptomyces caniscabiei (GeneID79936578, GeneID79935556), Streptomyces chartreusis (GeneID91330180), Streptomyces europaeiscabiei (GeneID89653209), Streptomyces hygroscopicus subsp. hygroscopicus (GeneID89483294), Streptomyces mirabilis (GeneID87473902), Streptomyces rochei(GeneID=90940517), Streptomyces scabiei 87.22 (GeneID24307372), Streptomyces stelliscabiei (GeneID86826013), Cutibacterium acnes HL096PA1 (GeneID66621070), Cutibacterium modestum (GeneID73015931)'s tryptophanase gene sequence was aligned using the bioedit software. Then, online primer design was performed using ClustalOmega (https: / / www.ebi.ac.uk / Tools / msa / clustalo / ), and two sets of conserved regions of the sequences were found ( Figure 3 、 Figure 4 ), and primer design was carried out separately to obtain the second primer pair and the fifth primer pair. The forward primer of the second primer pair is: FXJ-1717-F: TTCAARGGNRAYATSGA (SEQ ID NO.3); the reverse primer is: FXJ-2141-R: ADNCCVCCGTAGGT (SEQ ID NO.4), and the amplified DNA length is 425 bp. The forward primer of the fifth primer pair is: FXJ_1394_F1: TGCTSASSGAYTCVGG (SEQ ID NO.9); the reverse primer is: FXJ_1835_R1: GANACNGGYTGNCCVCC (SEQ ID NO.10), and the amplified DNA length is 442 bp.

[0047] 3. Two classes, Betaproteobacteria and Gammaproteobacteria, under the phylum Pseudomonadota, five orders, Neisseriales, Enterobacterales, Pasteurellales, Vibrionales, Aeromonadales, eight families Chromobacteriaceae 、 Aeromonadaceae 、 Enterobacteriaceae 、 Erwiniaceae 、 Morganellaceae 、 Yersiniaceae 、 Pasteurellaceae 、 Vibrionaceae , and 24 genera Chromobacterium 、 Aeromonas 、 Atlantibacter 、 Citrobacter. 、 Cronobacter 、 Enterobacter 、 Escherichia, Klebsiella 、 Kluyvera 、 Leclercia 、 Plesiomonas 、 Raoultella 、Salmonella , Shigella , Pantoea , Morganella , Proteus , Providencia , Yersinia , Haemophilus , Histophilus , Rodentibacter , Grimontia , Vibrio , a total of 83 strains Chromobacterium subtsugae (GeneID89687229), Aeromonas media (NCBI Reference Sequence NZ_JBGWXD010000016.1), Aeromonas rivipollensis (NCBI Reference Sequence NZ_JBGWZY010000036.1, NZ_JBGXAN010000004.1), Atlantibacter hermannii (NCBI Reference Sequence NZ_JAGSQW010000018.1), Citrobacter telavivensis (NCBI Reference Sequence NZ_JBGUBF010000003.1), Citrobacter farmeri (GeneID69618495), Citrobacter koseri (GeneID45136682), Citrobacter sedlakii (GeneID84235355, NCBI Reference Sequence NZ_JAGSQR010000006.1), Cronobacter dublinensis (GeneID45665186), Enterobacter sp. (NCBI ReferenceSequence NZ_DAMCWV010000032.1), Escherichia albertii (NCBI Reference SequenceNZ_JBAFYE010000002.1, GeneID89518607), Escherichia coli(GenBankCAUZHM010000002.1, GenBankCAUZHK010000001.1, GenBankOY754453.1, GenBankOY754349.1, GenBankOY754427.1, GenBankOY754443.1, GenBankCAUZHL010000007.1, GenBankOY757949.1, GenBankOY754452.1, GenBankOY754442.1, GenBankLR963129., GeneID948221), Escherichia fergusonii (GeneID75059599), Escherichia marmotae (GeneID86948642), Escherichia ruysiae (GeneID86861820), Klebsiella grimontii (GeneID64339535), Klebsiella michiganensis (GeneID66561963), Klebsiella oxytoca (GeneID69796358, NCBI Reference Sequence NZ_JBGREC010000025.1), Klebsiella pasteurii (GeneID77228633, NCBI Reference Sequence NZ_JBHJCW010000058.1), Kluyvera sp. 142359(NCBI Reference Sequence NZ_JBIQOI010000005.1), Leclercia adecarboxylata (NCBI Reference Sequence NZ_JBJDND010000018.1), Plesiomonas shigelloides (NCBI Reference Sequence NZ_JBJKNW010000002.1), Plesiomonas shigelloides (GeneID69705757), Raoultella ornithinolytica (GeneID69887175), Salmonella enterica (NCBI Reference Sequence NZ_JBHEYB010000028.1), Shigella flexneri (NCBI Reference Sequence NZ_QWSM01000195.1), Shigella boydii (GeneID75205423), Shigella dysenteriae (GeneID75173927), Shigella sonnei(GeneID66672392), Pantoea ananatis (GeneID57270242), Pantoea stewartii (GeneID61253334), Morganella morganii (NCBI Reference Sequence NZ_JBEEWI010000004.1, GeneID69679949), Proteus columbae (GeneID84585726), Proteus faecis (GeneID83611838), Proteus terrae (GeneID57333731), Proteus vulgaris (GeneID57310150), Providencia alcalifaciens (GeneID57292356), Providencia huaxiensis (GeneID89491481), Providencia rettgeri (GeneID61416928), Providencia rustigianii (GeneID76393052), Providencia stuartii (GeneID86884287), Providencia vermicola (GeneID88352764), Yersinia enterocolitica (GenBank AM286415.1.), Yersinia frederiksenii (GenBankAALE02000001.1) Yersinia intermedia (GenBank AALF02000001.1), Yersinia kristensenii (NCBI Reference Sequence NZ_JBJLTX010000005.1), Yersinia rochesterensis (GenBank CP009997.1), Yersinia alsatica (GeneID5139036), Yersinia enterocolitica (GeneID77329538), Yersinia frederiksenii (GeneID57904469), Yersinia intermedia (GeneID58048876), Yersinia kristensenii (GeneID61905266), Yersinia massiliensis (GeneID61815729), Yersinia proxima (GeneID69698888), Yersinia rochesterensis (GeneID82549885), Haemophilus haemolyticus(GeneID56957079), Haemophilus somnus (GenBankCP000947.1, GeneID31487572), Rodentibacter caecimuris (GeneID85658124), Rodentibacter pneumotropicus (GeneID61266447), Grimontia hollisae (GeneID58894733), Vibrio natriegens (GeneID70914283), Vibrio anguillarum (GeneID83860782), Vibrio chagasii (GeneID=77341656), Vibrio coralliirubri (GeneID89591910), Vibrio crassostreae (GeneID61503409), Vibrio cyclitrophicus (GeneID50229818), Vibrio diazotrophicus (GeneID77140188), Vibrio kanaloae (GeneID61411458), Vibrio lentus (GeneID69648652), Vibrio metoecu s(GeneID58290080), Vibrio metschnikovii (GeneID79888076), Vibrio mimicus (GeneID57836343), Vibrio navarrensis (GeneID59227612), Vibrio nigripulchritudo (GeneID25061998), Vibrio paracholerae (GeneID89512186), Vibrio parahaemolyticus (GeneID1190879), Vibrio splendidus (GeneID72399092), Vibrio tarriae (GeneID88784263), Vibrio tasmaniensis (GeneID77316069), Vibrio vulnificus (GeneID66967793), Vibrio cholerae (GenBankCP010812.1), Vibrio kanaloae (NCBI Reference Sequence NZ_JBFRRD010000038.1), VibrioThe tryptophanase gene sequence of sp. 10N.247.310.24 (NCBI Reference Sequence NZ_JBFSUD010000040.1) was aligned using the bioedit software. Then, online primer design was performed using ClustalOmega (https: / / www.ebi.ac.uk / Tools / msa / clustalo / ), and two sets of sequence conserved regions ( Figure 5 , Figure 6 ) were found, and primer design was carried out separately to obtain the third primer pair and the sixth primer pair. The forward primer of the third primer pair is: Jjdbj_836_F: CNATGTCNGCNAARAARGA (SEQ ID NO.5); the reverse primer is: Jjdbj_1241_R: ARNGANCCDATYTCNAC (SEQ ID NO.6), and the amplified DNA length is 406 bp. The forward primer of the sixth primer pair is: Jjdbj-838-F: ATGTCNGCNAARAARGA (SEQ ID NO.11); the reverse primer is: Jjdbj-1241-R1: ARNGANCCDATYTC (SEQ ID NO.12), and the amplified DNA length is 404 bp.

[0048] After the above 6 sets of tryptophanase gene primer pairs were designed, they were synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd.

[0049] Example 2: Amplification of tryptophanase gene The method of designing primers by analyzing the conservation of the tryptophanase gene in the bacterial community in Example 1 can only obtain theoretically feasible primers for the tryptophanase gene in the bacterial community to the greatest extent. Whether the primers can successfully achieve the purpose of amplifying the tryptophanase gene still requires PCR amplification experiments, agarose gel electrophoresis, and cloning and sequencing of the primers and templates.

[0050] 1. The genomic DNA of rat and human fecal flora and soil flora was extracted using the Tiangen Fecal Genomic DNA Extraction Kit (DP328) and the Tiangen Soil Genomic DNA Extraction Kit (DP336) according to the operation instructions.

[0051] 2. Amplification of tryptophanase gene Using the 6 sets of tryptophanase gene primers obtained in Example 1, the tryptophanase gene was PCR amplified using the genomic DNA of rats, human fecal flora, and soil flora as templates respectively. The PCR amplification system is as follows: Use TaKaRa Ex Taq HotStart Version (RR006A), add 0.25 μL of TaKaRa Ex Taq HS, 4 μL of dNTP Mixture, 5 μL of 10×Ex Taq Buffer, 1 μL of each of the forward and reverse primers, 2 μL of rat, human fecal flora or soil flora DNA, and 36.75 μL of sterile distilled water, with a total volume of 50 μL. Amplification was carried out using an EasyCycler 96 PCR instrument. The PCR amplification program was: pre-denaturation at 94 °C for 10 minutes, 1 cycle; denaturation at 95 °C for 30 seconds, annealing at 53 °C for 30 seconds, extension at 72 °C for 30 seconds for 1 cycle, a total of 30 cycles; final extension at 72 °C for 10 minutes for 1 cycle, and the product was stored at 4 °C.

[0052] The PCR products were detected by 1% agarose gel electrophoresis, and the results are as Figure 4 shown. As can be seen from Figure 4 a in, after the PCR amplification experiment, bands appeared at the corresponding positions of 440 bp, 425 bp, and 406 bp for the first primer pair, the second primer pair, and the third primer pair; as can be seen from Figure 4 b in, bands did not appear at the corresponding positions of 839 bp and 442 bp for the fourth primer pair and the fifth primer pair, and a band appeared at 404 bp for the sixth primer pair at the corresponding position.

[0053] 3. Gel cutting recovery and sequencing verification Under the ultraviolet lamp, the target bands that appeared at the corresponding length positions of the agarose gel in Step 2 were cut off, and the target fragments of the tryptophanase gene were recovered using the TIANgelMidi Purification Kit (DP209-2), and purified using the UnlversalDNA Purification Kit (DP214). Then the purified target fragment was ligated to the pMD19-T vector by T4 ligase, and then transformed into Escherichia coli DH5α competent cells. Positive clones were screened and sent to Shanghai Sangon Biological Engineering Technology and Service Co., Ltd. for sequencing.

[0054] The MEME 5.5.7 (https: / / meme-suite.org / meme / tools / meme) was used to perform a conservation analysis on the sequenced sequences to study the matching of primer pairs with the sequences. Meanwhile, the obtained sequencing results were aligned through blastx on the NCBI website, the reference amino acid sequences were downloaded, and the Mega12 software and the neighbor-joining method were used for phylogenetic tree analysis.

[0055] (1) Results of the matching of primer pairs with sequences The results of the matching of primer pairs with sequences were shown by the motifs of the sequences obtained by primer pair amplification, and the results were as Figures 8 - 11 shown.

[0056] For the sequence amplified by the first primer pair, Figure 8 the binding site of the forward primer of the first primer pair and the template was at positions 1 to 20 of a. At positions 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, and 20, the bases of the template matched the bases of the forward primer that did not use degenerate bases, and unique bases appeared at all these positions; at positions 3, 6, 9, 12, 15, and 18, it was shown that the bases of the template matched the degenerate bases of the forward primer, and no mutant bases other than degenerate bases appeared at these 6 positions, indicating that the forward primer of the first primer pair had a good match with the template. Figure 8 the binding site of the reverse primer of the first primer pair and the template was at positions 31 to 50 of b. At positions 31, 32, 34, 35, 37, 38, 40, 41, 43, 44, 47, 49, and 50, the bases of the template completely matched the bases of the reverse primer of the first primer pair that did not use degenerate bases, and no mutant bases existed; at positions 33, 36, 39, 42, 45, 46, and 48, the bases of the template matched the degenerate bases of the primer, and no mutant bases other than degenerate bases appeared, indicating that the reverse primer of the first primer pair had a good match with the template.

[0057] For the sequence amplified by the second primer pair, Figure 9 the binding site of the forward primer of the second primer pair and the template was at positions 1 to 17 of a. At positions 1, 2, 3, 4, 5, 7, 8, 11, 13, 14, 16, and 17, the bases of the template matched the bases of the forward primer that did not use degenerate bases, and unique bases appeared at all these positions; at positions 6, 9, 10, 12, and 15, it was shown that the bases of the template matched the degenerate bases of the forward primer, and no mutant bases other than degenerate bases appeared at these 5 positions, indicating that the forward primer of the second primer pair had a good match with the template. Figure 9The binding site of the reverse primer of the second primer pair to the template is located at positions 37 - 50 of b. At positions 37, 38, 39, 40, 41, 42, 43, 44, 46, 47, 50, the bases of the template match exactly with the bases of the reverse primer of the second primer pair that do not use degenerate bases, and there are no mutant bases. At positions 45, 48, 49, the degenerate bases of the template match with those of the primer, and no mutant bases other than the degenerate bases appear, indicating that the reverse primer of the second primer pair has good matching with the template.

[0058] The sequence obtained by amplification with the third primer pair Figure 10 The binding site of the forward primer of the third primer pair to the template is located at positions 1 - 19 of a. At positions 1, 3, 4, 5, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, the bases of the template match with the bases of the forward primer that do not use degenerate bases, and all sequences have unique bases at this site. At positions 2, 8, 11, 14, 17, it shows that the bases of the template match with the bases of the forward primer that use degenerate bases, and no mutant bases other than the degenerate bases appear at these 5 positions, indicating that the forward primer of the third primer pair has good matching with the template. Figure 10 The binding site of the reverse primer of the third primer pair to the template is located at positions 34 - 50 of b. At positions 34, 35, 37, 38, 40, 41, 43, 44, 46, 47, 50, the bases of the template match exactly with the bases of the reverse primer of the third primer pair that do not use degenerate bases, and there are no mutant bases. At positions 36, 39, 42, 45, 48, 49, the degenerate bases of the template match with those of the primer, and no mutant bases outside the range of degenerate bases appear, indicating that the reverse primer of the third primer pair has good matching with the template.

[0059] Figure 11 It is a diagram of the binding region of the forward primer of the sixth primer pair to the DNA template. At positions 1 - 17 in the figure, it is the binding site of the forward primer of the sixth primer pair to the template. At positions 1, 2, 3, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, the bases of the template match with the bases of the forward primer that do not use degenerate bases, and all sequences have unique bases at this site. At positions 6, 9, 12, 15, it shows that the bases of the template match with the bases of the primer that use degenerate bases, and no mutant bases other than the degenerate bases appear at these 4 positions, indicating that the forward primer of the sixth primer pair has good matching with the template. However, at the position of the reverse primer, the reverse primer does not match with the template, and the purpose of effectively amplifying the tryptophanase gene is not achieved. Therefore, although a band appears at the corresponding position in the agarose gel electrophoresis, after cloning and sequencing and analysis of the matching between the primer and the template, the design of this primer pair fails.

[0060] Based on the above matching analysis of the template and primer binding sites, it is shown that the sequence design regions of the first, second, and third primer pairs designed in the present invention are very conserved regions within the tryptophanase gene of indole-producing bacteria. Only when the signature gene of indole-producing bacteria, namely the tryptophanase gene, exists in the bacterial flora, will bands appear at the corresponding positions on the gel electrophoresis pattern. Conversely, if the tryptophanase gene is not present in the bacterial flora, no bands will appear at the corresponding positions on the gel electrophoresis pattern, indicating that the three groups of primer pairs screened out in the present invention are specific for the tryptophanase gene of indole-producing bacteria.

[0061] (2)Phylogenetic tree analysis results Construct phylogenetic trees using the sequences of human and rat fecal flora and soil flora identified by the first, second, and third primer pairs and the reference sequences. The results are as Figure 12 shown. It can be seen from the figure that the first primer pair identified 1 phylum Bacteroidota, 1 class Bacteroidia, 1 order Bacteroidales, 3 families Muribaculaceae , Bacteroidaceae , Rikenellaceae , 4 genera Duncaniella , Bacteroides , unclassified Muribaculaceae , Alistipes , 10 species Duncaniella sp., Muribaculaceae bacterium, Bacteroides stercoris , Bacteroides thetaiotaomicron , uncultured Duncaniella sp., uncultured Alistipes sp., Bacteroides sp. HMSC067B03, Bacteroides uniformis , Alistipes sp., Bacteroides ovatus .

[0062] The second primer pair identified 5 phyla of bacteria, namely Bacillota, Verrucomicrobiota, Acidobacteriota, Chloroflexota, and Actinomycetota; 7 classes, namely Clostridia, Thermoanaerobaculia, Vicinamibacteria, Verrucomicrobiia, Candidatus Limnocylindria, Chloroflexia, and Thermoleophilia; 5 orders, namely Eubacteriales, Vicinamibacterales, Verrucomicrobiales, Chloroflexales, and Thermoleophilales; 4 families Oscillospiraceae , Akkermansiaceae , Roseiflexineae , Thermoleophilaceae , and 2 genera Akkermansia , Candidatus Ribeiella , and 7 species Oscillospiraceae bacterium, Thermoanaerobaculia bacterium, Vicinamibacterales bacterium, uncultured Akkermansia sp., Candidatus Limnocylindria bacterium, Candidatus Ribeiella danica sp., Thermoleophilaceae bacterium.

[0063] The third primer pair identified 1 phylum of bacteria, Pseudomonadota; 1 class, Gammaproteobacteria; 1 order, Enterobacterales; 1 family Enterobacteriaceae , 1 genus Escherichia , and 1 species Escherichia coli。

[0064] In summary, in this example, using the three sets of primer pairs designed by the present invention, a total of 7 phyla, 9 classes, 7 orders, 8 families, 7 genera, and 18 species of indole-producing bacteria were qualitatively identified. Among them, the first primer pair can amplify rat and human fecal samples by PCR amplification; the second primer pair can amplify human fecal samples and soil samples by PCR amplification; the third primer pair can amplify human fecal samples by PCR amplification. It shows that the three sets of primer pairs designed by the present invention can identify indole-producing bacteria in different samples with relatively high species diversity.

[0065] The present invention provides an idea and method for specific primers for identifying indole-producing bacteria and their uses. There are many methods and ways to specifically implement this technical solution. The above description is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be implemented by existing technologies.

Claims

1. A specific primer for identifying indole-producing bacteria, characterized in that, The specific primer is any one or a combination of several of the first primer pair, the second primer pair or the third primer pair; Among them, for the forward primer of the first primer pair, its nucleotide sequence is as shown in SEQ ID NO.1; for the reverse primer of the first primer pair, its nucleotide sequence is as shown in SEQ ID NO.2; For the forward primer of the second primer pair, its nucleotide sequence is as shown in SEQ ID NO.3; for the reverse primer of the second primer pair, its nucleotide sequence is as shown in SEQ ID NO.4; For the forward primer of the third primer pair, its nucleotide sequence is as shown in SEQ ID NO.5; for the reverse primer of the second primer pair, its nucleotide sequence is as shown in SEQ ID NO.

6.

2. The specific primer according to claim 1, characterized in that, The indole-producing bacterium is an intestinal bacterium that converts tryptophan into indole through tryptophanase.

3. The specific primer according to claim 1, characterized in that, The specific primer for identifying indole-producing bacteria specifically detects the tryptophanase gene in indole-producing bacteria to identify whether it is an indole-producing bacterium.

4. Use of the specific primer according to any one of claims 1 to 3 in the preparation of a kit for identifying indole-producing bacteria.

5. A kit for identifying indole-producing bacteria, characterized in that, The kit contains the specific primer described in any one or several of claims 1 to 3.

6. The kit according to claim 5, characterized in that, For the kit, the PCR reaction system used is a 50 μL system: 0.25 μL of Ex Taq HS, 4 μL of dNTP Mixture, 1 μL of forward primer, 1 μL of reverse primer, 1 μL each of 5 μL of 10× Ex Taq Buffer, 36.75 μL of ddH2O, and 2 μL of DNA of the sample to be tested.

7. The kit according to claim 6, characterized in that, The DNA of the sample to be tested is: genomic DNA of rat fecal flora, genomic DNA of human fecal flora or genomic DNA of soil flora.

8. The kit according to claim 5, characterized in that, For the kit, the PCR reaction conditions used are: pre-denaturation at 94°C for 10 minutes, 1 cycle; denaturation at 95°C for 30 seconds, annealing at 53°C for 30 seconds, extension at 72°C for 30 seconds for 1 cycle, a total of 30 cycles; final extension at 72°C for 10 minutes for 1 cycle, and the product is stored at 4°C.

9. A method for identifying indole-producing bacteria, characterized in that, It includes the following steps: (1) Extract the DNA of the sample to be tested; (2) Using the DNA of the sample to be tested as a template, perform PCR amplification with specific primers to obtain PCR products; (3) Detect the PCR products by 1% agarose gel electrophoresis and judge the results; Among them, the specific primer is the first primer pair, the second primer pair and / or the third primer pair described in claim 1.

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