A specific primer for identifying indole-producing bacteria and its use

Abstract

The present invention relates to the field of biotechnology, and more specifically to specific primers for identifying indole-producing bacteria and their uses. The present invention is designed based on conserved regions of the tryptophanase gene of indole-producing bacteria from different phyla. Through PCR amplification experiments, agarose gel electrophoresis, and cloning sequencing, three pairs of specific primers (a first primer pair, a second primer pair, and / or a third primer pair) for targeted identification of indole-producing bacteria were ultimately screened and obtained. A kit prepared using the screened specific primers can be used to amplify test samples using a PCR instrument to qualitatively identify indole-producing bacteria. The identification method is simple, efficient, and accurate, and has practical application value. The present invention provides molecular markers at the gene level for the screening of indole-producing bacteria, and offers a convenient and accurate method for observing and studying changes in the species diversity of indole-producing bacteria in humans and animals.

Description

Technical Field

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

[0002] Chronic kidney disease (CKD) is a common chronic disease worldwide, showing a rapid increase in recent years and becoming a major global public health issue. Delaying the progression of CKD to the middle and late stages is of great clinical and socioeconomic significance for improving CKD prognosis, enhancing patients' quality of life, and reducing mortality and disability rates.

[0003] Renal fibrosis is a key pathological change in chronic kidney disease (CKD). Indoxyl sulfate is one of the major enteric uremic toxins mediating CKD fibrosis. Indole, the precursor of indoxyl sulfate, is produced by intestinal bacteria from tryptophan via tryptophanase. In the normal human intestine, indole secreted by indole-producing bacteria serves as an intercellular signal, regulating normal microbial physiological activity. However, in patients with CKD, a "leaky gut" phenomenon occurs, where indole enters the bloodstream and circulates to the liver, where it is sulfated to produce indoxyl sulfate. Studies have shown that knocking out the bacterial tryptophanase gene can eliminate indole production in vitro. However, no bacteria have been found to produce indole through enzymes other than tryptophanase. Therefore, many scientific papers use the tryptophanase gene as a marker gene for identifying indole-producing bacteria. Indole-producing bacteria are widely distributed, found in the human and animal intestines and soil. Therefore, understanding the species diversity of indole-producing bacteria is crucial. However, research on the species diversity of indole-producing bacteria is currently insufficient. Accurate and convenient methods for identifying the species diversity of indole-producing bacteria are still lacking.

[0004] Currently, existing technologies rely on combined analysis of the 16S rRNA gene and whole-genome shotgun sequencing to identify and characterize indole-producing bacteria. However, these methods are expensive, require high bioinformatics expertise, and require long sequencing cycles. Therefore, developing specific primers to identify and characterize the diversity of indole-producing bacteria would make the identification and characterization of indole-producing bacteria simpler, more efficient, and more accurate, with practical application value. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to address the shortcomings of the existing technology of identifying and identifying indole-producing bacteria by combined analysis of 16S rRNA gene and whole genome shotgun sequencing, which are expensive, require high bioinformatics technology, and have a long sequencing cycle. First, a specific primer for identifying indole-producing bacteria is provided.

[0006] The technical problem that the present invention also aims to solve is to provide a use of specific primers in preparing a kit for identifying indole-producing bacteria.

[0007] The technical problem that the present invention also aims to solve is to provide a kit for identifying indole-producing bacteria.

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

[0009] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0010] In a first aspect, the present invention discloses a specific primer for identifying indole-producing bacteria, wherein 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.

[0011] The nucleotide sequence of the front primer of the first primer pair is shown as SEQ ID NO.1; the nucleotide sequence of the back primer of the first primer pair is shown as SEQ ID NO.2.

[0012] The nucleotide sequence of the front primer of the second primer pair is shown as SEQ ID NO.3; the nucleotide sequence of the back primer of the second primer pair is shown as SEQ ID NO.4.

[0013] The nucleotide sequence of the front primer of the third primer pair is shown as SEQ ID NO.5; the nucleotide sequence of the back primer of the second primer pair is shown as SEQ ID NO.6.

[0014] In some embodiments of the present invention, the first primer pair identified one phylum Bacteroidota, one class Bacteroidia, one order Bacteroidales, and three families Muribaculaceae 、 Bacteroidetes 、 Rikenellaceae , 4 bacterial genera Duncaniella 、 Bacteroides 、unclassified Muribaculaceae 、 Common grebe , 10 strains Duncaniella sp.、 Muribaculaceae bacteria, Bacteroides suffocatus 、 Bacteroides thetaiotaomicron 、uncultured Duncaniella sp., uncultured Common grebe sp.、 Bacteroides sp. HMSC067B03, Bacteroides uniformis 、 Common grebe sp.、 Bacteroides ovatus indole-producing bacteria.

[0015] In some embodiments of the present invention, the second primer pair identified 5 bacterial phyla Bacillota, Verrucomicrobiota, Acidobacteriota, Chloroflexota, Actinomycetota, 7 bacterial classes Clostridia, Thermoanaerobaculia, Vicinamibacteria, Verrucomicrobiia, CandidatusLimnocylindria, Chloroflexia, Thermoleophilia, 5 bacterial orders Eubacteriales, Vicinamibacterales, Verrucomicrobiales, Chloroflexales, Thermoleophilales, 4 bacterial families Oscillospiraceae 、 Akkermansiaceae 、 Roseiflexinae 、 Thermoleophilaceae , 2 genera Akkermansia 、 Candidate Ribeiella , 7 strains Oscillospiraceae bacterium, Thermoanaerobaculia bacterium, Vicinamibacterales bacterium, uncultured Akkermansia sp.、 Candidatus Limnocylindria bacteria, Candidate Danish Ribeiella sp.、 Thermoleophilaceae Indole-producing bacteria of bacterium.

[0016] In some embodiments of the present invention, the third primer pair identified a phylum Pseudomonadota in the bacterial kingdom, a class Gammaproteobacteria, an order Enterobacterales, and a family Enterobacteriaceae , 1 genus Escherichia , 1 strain Escherichia coli indole-producing bacteria.

[0017] The indole-producing bacteria are intestinal bacteria that convert tryptophan into indole through tryptophanase.

[0018] In the normal human intestine, indole-producing bacteria (indole-producing bacteria) convert tryptophan into indole through tryptophanase. Indole acts as an intercellular signal, regulating normal microbial physiological activities. However, in patients with chronic kidney disease (CKD), a "leaky gut" phenomenon occurs. Indole enters the bloodstream and circulates to the liver, where it undergoes sulfation to produce indoxyl sulfate. Indoxyl sulfate is one of the main enteric uremic toxins that mediate fibrosis in CKD, a key pathological change in CKD.

[0019] The specific primers for identifying indole-producing bacteria are specifically used to detect the tryptophanase gene in indole-producing bacteria, thereby identifying whether the bacteria are indole-producing bacteria.

[0020] In a second aspect, the present invention discloses the use of the specific primers in preparing a kit for identifying indole-producing bacteria.

[0021] In a third aspect, the present invention discloses a kit for identifying indole-producing bacteria, wherein the kit contains any one or more groups of the specific primers.

[0022] The PCR reaction system used in the kit is a 50 μL system: 0.25 μL Ex Taq HS, 4 μL dNTP Mixture, 1 μL forward primer, 1 μL rear primer, 5 μL 10×Ex Taq Buffer, 1 μL each, 36.75 μL ddH2O, and 2 μL sample DNA to be tested.

[0023] Specifically, the sample DNA to be tested is: rat fecal flora genomic DNA, human fecal flora genomic DNA or soil flora genomic DNA.

[0024] The PCR reaction conditions used in the kit are as follows: pre-denaturation at 94°C for 10 minutes, one cycle; denaturation at 95°C for 30 seconds, annealing at 53°C for 30 seconds, and extension at 72°C for 30 seconds, for a total of 30 cycles; final extension at 72°C for 10 minutes, for one cycle, and the product is stored at 4°C.

[0025] In a fourth aspect, the present invention discloses a method for identifying indole-producing bacteria, comprising the following steps:

[0026] (1) Extract DNA from the sample to be tested;

[0027] (2) Using the DNA of the sample to be tested as a template, PCR amplification is performed using the specific primers to obtain a PCR product;

[0028] (3) Detect the PCR product using 1% agarose gel electrophoresis and determine the results.

[0029] Wherein, the specific primers are a first primer pair, a second primer pair and / or a third primer pair.

[0030] Specifically, because the bacterial species contained in the test sample vary, the species of bacteria containing the tryptophanase gene within these bacterial species also vary. Therefore, the tryptophanase gene sequences of different bacterial species are not identical. These non-identical tryptophanase gene sequences contain both similar conserved regions and dissimilar non-conserved regions. However, if, according to the method described herein, PCR amplification using the first primer pair results in an amplified band at the 440 bp position on an agarose gel; or PCR amplification using the second primer pair results in an amplified band at the 425 bp position on an agarose gel; or PCR amplification using the third primer pair results in an amplified band at the 406 bp position on an agarose gel, then the test sample contains tryptophanase and is an indole-producing bacterium.

[0031] Beneficial effects:

[0032] The present invention is designed based on the conserved regions of the tryptophanase genes of indole-producing bacteria from different phyla, and through PCR amplification tests, agarose gel electrophoresis and cloning sequencing, three pairs of specific primers for targeted identification of indole-producing bacteria were finally screened. A kit prepared using the specific primers obtained by screening can be used to amplify the sample to be tested by a PCR instrument to directly qualitatively identify indole-producing bacteria. This identification method is simple, efficient, and accurate, with an experimental cycle shortened to about 5 hours, low cost, and low requirements for bioinformatics technology, and has practical application value. The present invention provides molecular markers 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 changes in the species diversity of indole-producing bacteria in humans and animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The present invention will be further described below in detail with reference to the accompanying drawings, and the above and / or other advantages of the present invention will become more apparent.

[0034] Figure 1 is the conserved region of the forward primer and the rear primer sequence of the first primer pair. Wherein, a is the conserved region of the forward primer, b is the conserved region of the rear primer sequence, and "*" is the conserved base in the forward primer region or the rear primer region.

[0035] Figure 2 is the conserved region of the forward primer and the rear primer sequence of the fourth primer pair, wherein a is the conserved region of the forward primer, b is the conserved region of the rear primer sequence, and "*" is a conserved base in the forward primer region or the rear primer region.

[0036] Figure 3is the conserved region of the forward primer and the rear primer sequence of the second primer pair. Wherein, a is the conserved region of the forward primer, b is the conserved region of the rear primer sequence, and "*" is a conserved base in the forward primer region or the rear primer region.

[0037] Figure 4 is the conserved region of the forward primer and the rear primer sequence of the fifth primer pair. Wherein, a is the conserved region of the forward primer, b is the conserved region of the rear primer sequence, and "*" is a conserved base in the forward primer region or the rear primer region.

[0038] Figure 5 is the conserved region of the forward primer and the rear primer sequence of the third primer pair. Wherein, a is the conserved region of the forward primer, b is the conserved region of the rear primer sequence, and "*" is a conserved base in the forward primer region or the rear primer region.

[0039] Figure 6 is the conserved region of the forward primer and the rear primer sequence of the sixth primer pair. Wherein, a is the conserved region of the forward primer, b is the conserved region of the rear primer sequence, and "*" is a conserved base in the forward primer region or the rear primer region.

[0040] Figure 7 The following are the agarose gel electrophoresis results for six tryptophanase gene primer pairs. (a) shows the agarose gel electrophoresis results for the first, second, and third primer pairs; (b) shows the agarose gel electrophoresis results for the fourth, fifth, and sixth primer pairs.

[0041] Figure 8 The motif representation of the sequence obtained by amplification with the first primer pair is shown in Figure 1. Where a is the binding region between the first primer pair's front primer and the DNA template; b is the binding region between the first primer pair's back primer and the DNA template.

[0042] Figure 9 The motif representation of the sequence obtained by amplification with the second primer pair is shown in Figure 1. Where a represents the binding region between the front primer of the second primer pair and the DNA template; b represents the binding region between the back primer of the second primer pair and the DNA template.

[0043] Figure 10 The motif of the sequence amplified by the third primer pair is shown in Figure 1. Here, a represents the binding region between the front primer of the third primer pair and the DNA template; b represents the binding region between the back primer of the third primer pair and the DNA template.

[0044] Figure 11 It is the binding region between the front primer of the sixth primer pair and the DNA template.

[0045] Figure 12Phylogenetic trees constructed from human, rat fecal, and soil microbial sequences identified using the first, second, and third primer pairs and reference sequences. Reference sequences are NCBI Reference Sequence IDs or Genebank plus the species or family name. DETAILED DESCRIPTION

[0046] The present invention will be further described below in conjunction with specific embodiments, and the above and / or other advantages of the present invention will become more apparent.

[0047] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0048] This invention has been approved by the Ethics Committee of the Affiliated Hospital of Nanjing University of Chinese Medicine with approval number: 2025DW-030-01.

[0049] Example 1: Primer design

[0050] The tryptophanase DNA sequences in the bacterial kingdom were searched in the NCBI database, and a total of 154 tryptophanase gene sequences from three bacterial phyla (Bacteroidota, Actinomycetota, and Pseudomonadota) were collected. Six sets of primer pairs were designed based on the three bacterial phyla.

[0051] 1. The two classes Bacteroidia and Flavobacteriia, the two orders Bacteroidales and Flavobacteriales, and the seven families Bacteroidetes 、 Dysgonomonadaceae 、 Odoribacteraceae 、 Porphyromonadaceae、Prevotellaceae 、 Rikenellaceae, Weeksellaceae , 10 bacterial genera Bacteroides 、 Dysgonomonas 、 Butyricimonas 、 Odoric bacteria 、 Porphyromonas 、 Prevotella 、 Common grebe 、 Chryseobacterium 、 Elizabethkingia 、 Empedobactes r, a total of 40 strains Bacteroides acidificis (GeneID89646669), Bacteroides cellulolytic (GeneID66307512), Bacteroides clarus (GeneID61678932), Bacteroides congonensis(GeneID82173502)、 Empedobacter brevis (GeneID84649463)、 Elizabethkingia anopheles R26(GeneID56685136)、 Bacteroides eggerthii (GeneID64229610)、 Bacteroides faeces (GeneID69591221)、 Bacteroides flux (GeneID86048535)、 Intestinal Bacteroides (GeneID69508995)、 Bacteroides nordii (GeneID69502888)、 Bacteroides ovatus (GeneID29451707)、 Bacteroides salyersiae (GeneID77203696)、 Bacteroides suffocatus (GeneID31797138)、 Bacteroides thetaiotaomicron (GeneID60927475)、 Bacteroides uniformis (GeneID66280815)、 Bacteroides xylanisolvens (GeneID69480792)、 Elizabethkingia miricola (GeneID66583889)、 Dysgonomonas mossii (GeneID78083246)、 Butyricimonas of a man of dung (GeneID83158670)、 Butyricimonas paravirosa (GeneID86890433)、 Butyricimonas virosa (GeneID77452881)、 Odoribacter splanchnicus (GeneID69855797)、 Porphyromonas endodontalis (GeneID84818444)、 Porphyromonas gum (GeneID29256096)、 Porphyromonas gulae (GeneID57239471)、 Prevotella intermediate (GeneID34515074)、 Prevotella nigrescens (GeneID67366882)、 Prevotella pale (GeneID78570559)、 Prevotella pectinovora (GeneID88847520)、 Common grebe finegolds (GeneID79836330)、 Alistipes onderdonkii subsp. common (GeneID59808890)、 Rotten sedges (GeneID73803429)、 Alistipes shahii(GeneID66250428), Chryseobacterium arthrosphaerae (GeneID78303638), Chryseobacterium gleum (GeneID84657101), Chryseobacterium indologenes (GeneID56899753), Elizabethkingia bruuniana (GeneID78268328), Elizabethkingia meningosepticemia (GeneID72520441), Empedobacter falsenii The tryptophanase gene sequence of (GeneID78399994) was aligned using bioedit software. Then, Clustal Omega (https: / / www.ebi.ac.uk / Tools / msa / clustalo / ) was used for online primer design, and two sets of sequence conserved regions were found ( Figure 1 、 Figure 2 ), and primers were designed separately to obtain the first and fourth primer pairs. The first primer pair consisted of the forward primer NGJ_367_F: CAYTTYGAYACNACNAARGG (SEQ ID NO. 1); the rear primer NGJ_806_R: GCNANRAANCCDCCNATRTT (SEQ ID NO. 2), amplifying a DNA length of 440 bp. The fourth primer pair consisted of the forward primer NGJ_202_F: GGNGAYGARAGYTAYGCHGG (SEQ ID NO. 7); the rear primer NGJ_1040_R: AANANNGCRTGNCCNCC (SEQ ID NO. 8), amplifying a DNA length of 839 bp.

[0052] 2. Under the phylum Actinomycetota, the two classes Acidimicrobiia and Actinomycetes, the three orders Acidimicrobiales, Kitasatosporales, and Propionibacteriales, and the three families Acidimicrobiaceae 、 Streptomycetaceae 、 Propionibacteriaceae , 3 bacterial genera Ferrimicrobium 、 Streptomyces, Cutibacterium , a total of 12 strains Ferrimicrobium acidophilus (GeneID78373721), Streptomyces antimycoticus (GeneID91525172), Streptomyces caniscabiei (GeneID79936578, GeneID79935556), Streptomyces Chartreuse (GeneID91330180), Streptomyces europaeiscabiei(GeneID89653209), Streptomyces hygroscopicus subsp. hygroscopic (GeneID89483294), Streptomyces wonderful (GeneID87473902), Streptomyces rochei (GeneID=90940517), Streptomyces scabies 87.22 (GeneID24307372), Streptomyces stelliscabiei (GeneID86826013), Cutibacterium acnes HL096PA1 (GeneID66621070), Cutibacterium modestum The tryptophanase gene sequence of (GeneID73015931) was aligned using bioedit software. Then, online primer design was performed using ClustalOmega (https: / / www.ebi.ac.uk / Tools / msa / clustalo / ) to find two sets of sequence conserved regions ( Figure 3 、 Figure 4 ), and primers were designed separately to obtain the second and fifth primer pairs. The second primer pair consisted of the forward primer FXJ-1717-F: TTCAARGGNRAYATSGA (SEQ ID NO. 3); the rear primer FXJ-2141-R: ADNCCVCCGTAGGT (SEQ ID NO. 4), amplifying a DNA length of 425 bp. The fifth primer pair consisted of the forward primer FXJ_1394_F1: TGCTSASSGAYTCVGG (SEQ ID NO. 9); the rear primer FXJ_1835_R1: GANACNGGYTGNCCVCC (SEQ ID NO. 10), amplifying a DNA length of 442 bp.

[0053] 3. The two classes Betaproteobacteria and Gammaproteobacteria, five orders Neisseriales, Enterobacterales, Pasteurellales, Vibrionales, Aeromonadales, and eight families under the phylum Pseudomonadota were Chromobacteriaceae 、 Aeromonadaceae 、 Enterobacteriaceae 、 Erwiniaceae 、 Morganellaceae 、 Yersiniaceae 、 Pasteurellaceae 、 Vibrionaceae , 24 bacterial 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、NCBIReference Sequence NZ_JBGREC010000025.1)、 Klebsiella pasteurii (GeneID77228633、NCBI Reference Sequence NZ_JBHJCW010000058.1)、 Kluyvera sp. 142359(NCBIReference Sequence NZ_JBIQOI010000005.1)、 Leclercia adecarboxylata (NCBIReference Sequence NZ_JBJDND010000018.1)、 Plesiomonas shigelloides (NCBIReference Sequence NZ_JBJKNW010000002.1)、 Plesiomonas shigelloides (GeneID69705757)、 Raoultella ornithinolytica (GeneID69887175)、 Salmonella enterica (NCBI Reference Sequence NZ_JBHEYB010000028.1)、 Shigella flexneri (NCBIReference Sequence NZ_QWSM01000195.1)、 Shigella boydii (GeneID75205423)、 Shigella dysenteriae (GeneID75173927)、 Shigella sonnei(GeneID66672392)、 Pantoea ananatis (GeneID57270242)、 Pantoea stewartii (GeneID61253334)、 Morganella Morgan's (NCBI Reference Sequence NZ_JBEEWI010000004.1、GeneID69679949)、 Proteus pigeons (GeneID84585726)、 Proteus faeces (GeneID83611838)、 Proteus of the earth (GeneID57333731)、 Common Proteus (GeneID57310150)、 Alkalizing Providence (GeneID57292356)、 Providence of Huaxi (GeneID89491481)、 Providence Rettgeri (GeneID61416928)、 Providence rusticus (GeneID76393052)、 Providence of Stuart (GeneID86884287)、 Providence vermiculite (GeneID88352764)、 Yersinia enterocolitica (GenBank AM286415.1.)、 Yersinia frederiksenii (GenBankAALE02000001.1)、 Yersinia intermedia (GenBank AALF02000001.1)、 Yersinia kristensenii (NCBI Reference Sequence NZ_JBJLTX010000005.1)、 Yersinia from Rochester (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 somniferum (GenBankCP000947.1、GeneID31487572)、 Rodentia bacterium caecimuris (GeneID85658124)、 Rodentiabacter pneumotropicus (GeneID61266447)、 Grimontia hollisae (GeneID58894733)、 Vibrio natrigens (GeneID70914283)、 Vibrio eel (GeneID83860782)、 Vibrio chagasii (GeneID=77341656)、 Vibrio coralliirubri (GeneID89591910)、 Vibrio crassostrea (GeneID61503409)、 Vibrio cyclitrophicus (GeneID50229818)、 Vibrio diazotrophicus (GeneID77140188)、 Vibrio kanaloae (GeneID61411458)、 Vibrio lentis (GeneID69648652)、 Vibrio metoecu s(GeneID58290080)、 Vibrio metschnikovii (GeneID79888076)、 Vibrio mimeticus (GeneID57836343)、 Vibrio navarreensis (GeneID59227612)、 Vibrio nigripulchritudo (GeneID25061998)、 Vibrio paracholera (GeneID89512186)、 Vibrio parahaemolyticus (GeneID1190879)、 Vibrio splendens (GeneID72399092)、 Vibrio tarriae (GeneID88784263)、 Tasmanian Vibrio (GeneID77316069)、 Vibrio vulnificus (GeneID66967793)、 Cholera vibrio (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 bioedit software. Then, online primer design was performed using ClustalOmega (https: / / www.ebi.ac.uk / Tools / msa / clustalo / ) to find two sets of sequence conserved regions ( Figure 5 、 Figure 6 ), and primers were designed separately to obtain a third and sixth primer pair. The forward primer of the third primer pair was Jjdbj_836_F: CNATGTCNGCNAARAARGA (SEQ ID NO. 5); the rear primer was Jjdbj_1241_R: ARNGANCCDATYTCNAC (SEQ ID NO. 6), and the amplified DNA length was 406 bp. The forward primer of the sixth primer pair was Jjdbj-838-F: ATGTCNGCNAARAARGA (SEQ ID NO. 11); the rear primer was Jjdbj-1241-R1: ARNGANCCDATYTC (SEQ ID NO. 12), and the amplified DNA length was 404 bp.

[0054] After the primer design was completed, the above 6 groups of tryptophanase gene primer pairs were synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd.

[0055] Example 2: Tryptophanase gene amplification

[0056] Example 1: Designing primers by analyzing the conservation of bacterial tryptophanase genes can only yield theoretically feasible primers for bacterial tryptophanase genes. Whether the primers can successfully amplify the tryptophanase gene still requires PCR amplification, agarose gel electrophoresis, and cloning and sequencing of the primers and templates.

[0057] 1. Use the Tiangen Fecal Genomic DNA Extraction Kit (DP328) and the Tiangen Soil Genomic DNA Extraction Kit (DP336) according to the operating instructions to extract the fecal microbiome genomes of rats and humans and the soil microbiome genomes.

[0058] 2. Tryptophanase gene amplification

[0059] The 6 sets of tryptophanase gene primer pairs obtained in Example 1 were used to amplify the tryptophanase gene using rat, human fecal flora and soil flora genomes as templates. The PCR amplification system was as follows: TaKaRa Ex Taq HotStart Version (RR006A) was used, wherein 0.25 μL of TaKaRa Ex Taq HS was added, 4 μL of dNTP Mixture was added, 5 μL of 10×Ex Taq Buffer was added, 1 μL of each of the front and back primers was added, 2 μL of rat, human fecal flora or soil flora DNA was added, 36.75 μL of sterile distilled water, and the total volume was 50 μL. Amplification was performed using an EasyCycler 96 PCR instrument, and the PCR amplification program was as follows: pre-denaturation at 94°C for 10 minutes, 1 cycle; denaturation at 95°C for 30 seconds, annealing at 53°C for 30 seconds, and extension at 72°C for 30 seconds as 1 cycle, for a total of 30 cycles; final extension at 72°C for 10 minutes for 1 cycle, and the product was stored at 4°C.

[0060] The PCR products were detected by 1% agarose gel electrophoresis. Figure 4 As shown. Figure 4 As can be seen from a in the figure, after PCR amplification test, the first primer pair, the second primer pair, and the third primer pair produced bands at the corresponding positions of 440 bp, 425 bp, and 406 bp; Figure 4 As can be seen from b, the fourth and fifth primer pairs did not produce bands at the corresponding positions of 839 bp and 442 bp, respectively, while the sixth primer pair produced a band at the corresponding position of 404 bp.

[0061] 3. Gel excision recovery and sequencing verification

[0062] Under ultraviolet light, cut the target band appearing at the corresponding length on the agarose gel in step 2. Recover the target fragment of the tryptophanase gene using the TIANgelMidi Purification Kit (DP209-2) and purify it using the Universal DNA Purification Kit (DP214). Ligate the purified target fragment to the pMD19-T vector using T4 ligase and transform it into competent E. coli DH5α cells. Screen for positive clones and send them to Shanghai Sangon Biotechnology Service Co., Ltd. for sequencing.

[0063] Sequences obtained were analyzed for conservation using MEME 5.5.7 (https: / / meme-suite.org / meme / tools / meme) to investigate primer pair-sequence compatibility. Sequencing results were then compared using blastx from the NCBI website. Reference amino acid sequences were downloaded and phylogenetic tree analysis was performed using Mega12 software and the neighbor-joining method.

[0064] (1) Matching results between primer pairs and sequences

[0065] The matching results between primer pairs and sequences are displayed by the motif of the sequence obtained by primer pair amplification. Figures 8 - 11 shown.

[0066] The sequence obtained by amplification of the first primer pair, Figure 8 Positions 1 to 20 of a are the binding sites of the first primer pair, the forward primer, and the template. At positions 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, and 20, the bases in the template and the forward primer that do not use degenerate bases match, and all sequences have unique bases at these positions. At positions 3, 6, 9, 12, 15, and 18, the bases in the template and the forward primer that use degenerate bases match, and no mutant bases other than degenerate bases appear at these six positions, indicating that the first primer pair, the forward primer, and the template have good matching. Figure 8 Positions 31 to 50 in b are the binding sites between the second primer of the first primer pair and the template. At positions 31, 32, 34, 35, 37, 38, 40, 41, 43, 44, 47, 49, and 50, the bases of the template and the second primer of the first primer pair that are not degenerate bases are completely matched, and no mutant bases exist. At positions 33, 36, 39, 42, 45, 46, and 48, the degenerate bases of the template and primers match, and no mutant bases other than degenerate bases appear, indicating that the second primer of the first primer pair and the template have a good match.

[0067] The sequence obtained by amplification of the second primer pair, Figure 9 Positions 1 to 17 of a are the binding sites of the second primer to the front primer and the template. At positions 1, 2, 3, 4, 5, 7, 8, 11, 13, 14, 16, and 17, the bases of the template and the front primer that do not use degenerate bases match, and all sequences have unique bases at these positions. At positions 6, 9, 10, 12, and 15, the bases of the template and the front primer that use degenerate bases match, and no mutant bases other than degenerate bases appear at these five positions, indicating that the second primer to the front primer has a good match with the template. Figure 9Positions 37 to 50 in b are the binding sites between the second primer pair and the template. At positions 37, 38, 39, 40, 41, 42, 43, 44, 46, 47, and 50, the bases of the template and the second primer pair that do not use degenerate bases are completely matched, and no mutant bases exist. At positions 45, 48, and 49, the degenerate bases of the template and primers match, and no mutant bases other than degenerate bases appear, indicating that the second primer pair and the template have a good match.

[0068] The sequence obtained by amplification of the third primer pair, Figure 10 Positions 1 to 19 of a are the binding sites of the third primer pair, the front primer, and the template. At positions 1, 3, 4, 5, 6, 7, 9, 10, 12, 13, 15, 16, 18, and 19, the bases in the template and the front primer that do not use degenerate bases match, and all sequences have unique bases at these positions. At positions 2, 8, 11, 14, and 17, the bases in the template and the front primer that use degenerate bases match, and no mutant bases other than degenerate bases appear at these five positions, indicating that the third primer pair, the front primer, and the template have good matching. Figure 10 Positions 34 to 50 in b are the binding sites between the second primer of the third primer pair and the template. At positions 34, 35, 37, 38, 40, 41, 43, 44, 46, 47, and 50, the bases in the template and the second primer of the third primer pair that are not degenerate bases are completely matched, and no mutant bases exist. At positions 36, 39, 42, 45, 48, and 49, the degenerate bases in the template and primers are matched, and no mutant bases outside the degenerate base range appear, indicating that the second primer of the third primer pair has a good match with the template.

[0069] Figure 11 This figure shows the binding region between the forward primer of the sixth primer pair and the DNA template. Positions 1 to 17 in the figure represent the binding sites of the forward primer of the sixth primer pair. At positions 1, 2, 3, 4, 5, 7, 8, 10, 11, 13, 14, 16, and 17, the template and the forward primers (without degenerate bases) match, and all sequences contain unique bases at these positions. At positions 6, 9, 12, and 15, the template and the forward primers (with degenerate bases) match, and no mutations other than degenerate bases occur at these four positions, indicating a good match between the forward primer and the template of the sixth primer pair. However, at the rear primer positions, the rear primer and the template do not match, resulting in ineffective amplification of the tryptophanase gene. Therefore, although bands were observed at the corresponding positions by agarose gel electrophoresis, clone sequencing and primer-template compatibility analysis indicated that this primer pair was designed unsuccessfully.

[0070] Compatibility analysis of the template and primer binding sites demonstrated that the sequences of the first, second, and third primer pairs designed in the present invention target highly conserved regions within the tryptophanase gene of indole-producing bacteria. Bands appear at corresponding positions on the gel electrophoresis plot only when the tryptophanase gene, a marker gene for indole-producing bacteria, is present in the bacterial flora. Conversely, no bands appear at corresponding positions on the gel electrophoresis plot if the tryptophanase gene is absent, demonstrating that the three primer pairs selected in the present invention are specific for the tryptophanase gene of indole-producing bacteria.

[0071] (2) Phylogenetic tree analysis results

[0072] The human and rat fecal flora and soil flora sequences identified by the first, second, and third primer pairs were used to construct a phylogenetic tree with the reference sequence. The results are as follows Figure 12 As shown in the figure, the first primer pair identified 1 phylum Bacteroidota, 1 class Bacteroidia, 1 order Bacteroidales, and 3 families Muribaculaceae 、 Bacteroidaceae 、 Rikenellaceae , 4 bacterial genera Duncaniella 、 Bacteroides 、unclassified Muribaculaceae 、 Alistipes , 10 strains Duncaniella sp.、 Muribaculaceae bacteria, Bacteroides stercoris 、 Bacteroides thetaiotaomicron 、uncultured Duncaniella sp., uncultured Alistipes sp.、 Bacteroides sp. HMSC067B03, Bacteroides uniformis 、 Alistipes sp.、 Bacteroides ovatus .

[0073] The second primer pair identified five bacterial phyla, Bacillota, Verrucomicrobiota, Acidobacteriota, Chloroflexota, and Actinomycetota; seven bacterial classes, Clostridia, Thermoanaerobaculia, Vicinamibacteria, Verrucomicrobiia, Candidatus Limnocylindria, Chloroflexia, and Thermoleophilia; five bacterial orders, Eubacteriales, Vicinamibacterales, Verrucomicrobiales, Chloroflexales, and Thermoleophilales; and four bacterial families, Oscillospiraceae 、 Akkermansiaceae 、 Roseiflexineae 、 Thermoleophilaceae , 2 genera Akkermansia 、 Candidatus Ribeiella , 7 strains Oscillospiraceae bacterium, Thermoanaerobaculia bacterium, Vicinamibacterales bacterium, uncultured Akkermansia sp.、 Candidatus Limnocylindria bacteria, Candidatus Ribeiella danica sp.、 Thermoleophilaceae bacterium.

[0074] The third primer pair identified one phylum Pseudomonadota, one class Gammaproteobacteria, one order Enterobacterales, and one family Enterobacteriaceae , 1 genus Escherichia , 1 strain Escherichia coli。

[0075] In summary, in this example, the three primer pairs designed according to the present invention qualitatively identified indole-producing bacteria from 7 phyla, 9 classes, 7 orders, 8 families, 7 genera, and 18 species. The first primer pair was able to amplify rat and human fecal samples via PCR; the second primer pair was able to amplify human fecal samples and soil samples via PCR; and the third primer pair was able to amplify human fecal samples via PCR. This demonstrates that the three primer pairs designed according to the present invention can identify indole-producing bacteria from diverse samples with a high species diversity.

[0076] The present invention provides a specific primer for identifying indole-producing bacteria and its application concept and method. There are many methods and approaches to implement this technical solution. The above is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also considered to be within the scope of protection of the present invention. Any components not specified in this embodiment may be implemented using existing technologies.

Claims

1. A specific primer for identifying indole-producing bacteria, characterized in that: The specific primers are a first primer pair, a second primer pair and a third primer pair; The nucleotide sequence of the front primer of the first primer pair is shown in SEQ ID NO.1; the nucleotide sequence of the back primer of the first primer pair is shown in SEQ ID NO.2; The nucleotide sequence of the front primer of the second primer pair is shown in SEQ ID NO.3; the nucleotide sequence of the back primer of the second primer pair is shown in SEQ ID NO.4; The nucleotide sequence of the front primer of the third primer pair is shown in SEQ ID NO.5; the nucleotide sequence of the back primer of the second primer pair is shown in SEQ ID NO.6; The indole-producing bacteria are intestinal bacteria that convert tryptophan into indole through tryptophanase.

2. The specific primer according to claim 1, characterized in that The specific primers for identifying indole-producing bacteria specifically utilize the specific primers to detect the tryptophanase gene in the indole-producing bacteria, thereby identifying whether the bacteria are indole-producing bacteria.

3. Use of the specific primer according to any one of claims 1 to 2 in preparing a kit for identifying indole-producing bacteria.

4. A kit for identifying indole-producing bacteria, characterized in that The kit contains the specific primers according to any one of claims 1 to 2.

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

6. The kit according to claim 5, characterized in that The sample DNA to be tested is: rat fecal flora genomic DNA, human fecal flora genomic DNA or soil flora genomic DNA.

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

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