# US 2013/0157265 A1 · Composition, method and kit for detecting bacteria by means of sequencing > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2013/0157265 A1 - Document ID: US20130157265A1 - Status: Published application - Kind: A1 - Application number: 13/502,395 - Filed: 2009-10-22 - Published: 2013-06-20 - Earliest claimed: 2009-10-22 - Inventors: Jesus Mingorance Cruz; Pablo Castan Garcia; Pedro Manuel Franco De Sarabia Rosado - Web page: https://patentgrep.com/patent/US20130157265A1 ## CPC - C12Q 1/689: for bacteria - C12Q 1/686: Polymerase chain reaction [PCR] - C12Q 2600/16: Primer sets for multiplex assays - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## Abstract The present invention describes a method for detecting the presence and type of a microorganism present in a sample by means of stabilization and sequencing techniques and subsequent analysis of microsequences in genes encoding the ribosomal RNA most conserved, and on specific areas of the 16-S region with taxonomic value. ## Claims ### Claim 1 A method for taxon-specific detection, differentiation and identification of eubacteria in a biological sample by means of sequencing analysis techniques, specifically pyrosequencing of three regions of the 16S bacterial ribosomal RNA gene, characterized in that it comprises the following steps: a. Stabilizing the reaction mixture by gelling by means of adding a stabilizing aqueous solution to the medium and subsequently drying to a degree of humidity between 10% and 30%. b. PCR amplification reaction of the total DNA extracted from the sample using the primer pairs specified in Table 2 (SEQ. ID. No. 1 to 6). c. Sequencing by synthesis, specifically pyrosequencing the PCR products obtained in step a. using the same non-biotinylated primers indicated in Table 2 and used for amplifying the total DNA extracted from the sample (SEQ. ID. No. 2, 4 and 6). ### Claim 2 (depends on 1) The method according to claim 1, characterized in that the amplification reaction is carried out by means of a nucleic acid (PCR) amplification reaction consisting of an initial denaturation at 95° C. for 5 minutes and 30 denaturation cycles at 95° C. for 1 minute, annealing at 54° C. for 1 minute, extension at 74° C. for 30 seconds, and a final extension at 72° C. for 10 minutes. ### Claim 3 (depends on 1) The method according to claim 1, characterized in that the sequences obtained from PCR amplification are subjected to pyrosequencing and are subsequently identified by means of comparison with the sequences deposited and registered in public or private databases. ### Claim 4 (depends on 1, 3) The method according to claim 1, characterized in that each of the sequences obtained by means of sequencing the fragments amplified by means of the amplification reaction described in claim 3 provides an additional level of information, such that if the sequence obtained by means of the reaction that was amplified using amplification primers SEQ. ID. No. 5 and 6 did not provide a sufficient level of information to assure the precise identification of the eubacteria species present in the sample, the overlap of this sequence with the sequence obtained by means of the reaction which was amplified using amplification primers SEQ. ID. No. 3 and 4 can provide precise information at the eubacterial species and genus level, and if the overlapped sequence still did not provide a sufficient level of coincidence to assure its identification, its posterior overlap with the sequence obtained by means of the reaction which was amplified using amplification primers SEQ. ID. No. 1 and 2 gives in all cases a sequence precisely informing about the family, genus and/or species of the eubacteria present in the sample. ### Claim 5 (depends on 1) The method according claim 1, characterized in that an improvement in the pyrosequencing resolution of between 75% and 90% is obtained in the pyrosequencing reaction and as a result a sequencing of longer fragments is also obtained by using the gelling mixture formed by trehalose, melezitose, glycogen or raffinose and lysine or betaine. ### Claim 6 (depends on 5) The method according to claim 5, characterized in that the gelling mixture is made up of trehalose, melezitose, glycogen and lysine. ### Claim 7 (depends on 1) A kit consisting of three reaction tubes or containers, or a series of three reaction tubes, each of these tubes or containers containing all the elements necessary for performing the PCR reaction (ultrapure DNA polymerase, deoxynucleotides dATP, dCTP, dGTP, dTTP, reaction buffer and reaction primers) described in claim 1, such that the first tube contains primers SEQ. ID. No. 1 and 2, the second tube SEQ. ID. No. 3 and 4, and the third tube primers SEQ. ID. No. 5 and 6. ### Claim 8 (depends on 7) The kit according to claim 7, characterized in that it consists of three tubes or containers containing all the reagents necessary for carrying out the pyrosequencing reaction: high-fidelity ultrapure DNA polymerase, ATP-sulfurylase, luciferase, apyrase, sequencing primer, luciferin, adenosine-5′-phosphosulfate, deoxynucleotides dATP, dCTP, dGTP, dTTP, reaction buffer and pyrosequencing primer, such that pyrosequencing primer SEQ. ID. No. 2 is added in the first tube for the purpose of sequencing the amplicon obtained by means of the amplification performed with amplification primers SEQ. ID. No. 1 and 2, pyrosequencing primer SEQ. ID. No. 4 is added in the second tube for the purpose of sequencing the amplicon obtained by means of the amplification performed with amplification primers SEQ. ID. No. 3 and 4, and pyrosequencing primer SEQ. ID. No. 6 is added in the third tube for the purpose of sequencing the amplicon obtained by means of the amplification performed with amplification primers SEQ. ID. No. 5 and 6. ### Claim 9 (depends on 8) The kit according to claim 8, characterized in that each of the tubes indicated in the preceding claim are stabilized by means of adding a stabilization mixture containing trehalose, melezitose, lysine and glycogen, subsequently being dried by means of applying a vacuum at a temperature of 30° C. ### Claim 10 (depends on 7) The kit according to claim 7, characterized in that it contains all the tubes with the ingredients necessary for carrying out a method for taxon-specific detection, differentiation and identification of eubacteria in a biological sample by means of sequencing analysis techniques, specifically pyrosequencing of three regions of the 16S bacterial ribosomal RNA gene, characterized in that it comprises the following steps: a. Stabilizing the reaction mixture by gelling by means of adding a stabilizing aqueous solution to the medium and subsequently drying to a degree of humidity between 10% and 30%. b. PCR amplification reaction of the total DNA extracted from the sample using the primer pairs specified in Table 2 (SEQ. ID. No. 1 to 6). c. Sequencing by synthesis, specifically pyrosequencing the PCR products obtained in step a. using the same non-biotinylated primers indicated in Table 2 and used for amplifying the total DNA extracted from the sample (SEQ. ID. No. 2, 4 and 6).