# US 2020/0399682 A1 · Oligonucleotide, Set of Oligonucleotides, Method for Simultaneous Detection of Neisseria Meningitidis, Streptococcus Pneumoniae and Haemophilus Influenzae, and Kit > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2020/0399682 A1 - Document ID: US20200399682A1 - Status: Published application - Kind: A1 - Application number: 16/971,632 - Filed: 2019-02-20 - Published: 2020-12-24 - Earliest claimed: 2018-02-20 - Assignees: FundaçãO Oswaldo Cruz - Applicants: FundaçãO Oswaldo Cruz - Inventors: IVANO RAFAELLE VICTORIO DE FILIPPIS CAPASSO; ANTONIO EUGENIO CASTRO CARDOSO DE ALMEIDA - Web page: https://patentgrep.com/patent/US20200399682A1 ## CPC - C12Q 1/686: Polymerase chain reaction [PCR] - C12Q 1/689: for bacteria - C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) - C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes - C12N 1/205: Bacterial isolates - C12Q 2600/16: Primer sets for multiplex assays - C12R 2001/21: Haemophilus - C12R 2001/36: Neisseria - C12R 2001/46: Streptococcus {; Enterococcus; Lactococcus} - 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 - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS ## Abstract The present invention provides a real-time PCR method that allows in a single step the simultaneous detection of etiological agents of bacterial meningitis, more specifically, Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae. For this, primers were used to amplify particular regions of the genomes of said bacteria. The presence of bacteria in a sample is indicated by the presence of amplicon, which is detected by means of detection methods appropriate to the PCR methodology employed. ## Claims ### Claim 1 Oligonucleotide, characterized by the fact that it is able to bind to a region of the Neisseria meningitidis nspA gene, and is suitable as a primer, said oligonucleotide comprising at least 10 to 15 consecutive nucleotides of the selected sequences of SEQ ID NOs: 1 and 2. ### Claim 2 Oligonucleotide, characterized by the fact that it is able to bind to a region of the Haemophilus influenzae P6 gene, and is suitable as a primer, said oligonucleotide comprising at least 10 to 15 consecutive nucleotides from the selected sequences of SEQ ID Nos: 3 and 4. ### Claim 3 Oligonucleotide, characterized by the fact that it is able to bind to a region of the Streptococcus pneumoniae ply gene, and is suitable as a primer, said oligonucleotide comprising at least 10 to 15 consecutive nucleotides from the selected sequences of SEQ ID Nos: 5 and 6. ### Claim 4 Oligonucleotide, characterized by the fact that it is able to bind to the nspA gene segment of Neisseria meningitidis, and is suitable as a probe, said oligonucleotide probe comprising at least 8 to 15 consecutive nucleotides of the selected sequence of SEQ ID NO: 7. ### Claim 5 Oligonucleotide, characterized by the fact that it is able to bind to the P6 gene segment of Haemophilus influenzae, and is suitable as a probe, said oligonucleotide probe comprising at least 8 to 15 consecutive nucleotides of the selected sequence of SEQ ID NO: 8. ### Claim 6 Oligonucleotide, characterized by the fact that it is able to bind to the ply gene segment of the Streptococcus pneumoniae, and is suitable as a probe, said probe oligonucleotide comprising at least 8 to 15 consecutive nucleotides of the selected sequence of SEQ ID NO: 9. ### Claim 7 (depends on 4, 5, 6) Oligonucleotide according to claim 4, 5 or 6, characterized by the fact that it is marked with a detectable mark, preferably a fluorescent cluster. ### Claim 8 (depends on 7) Oligonucleotide according to claim 7, characterized in that the fluorescent cluster comprises a donor fluorophore pair and a quencher. ### Claim 9 Oligonucleotide set, characterized by the fact that it comprises at least two oligonucleotides selected from sequences comprising SEQ ID NOs: 1-6. ### Claim 10 (depends on 9) An oligonucleotide set according to claim 9, characterized in that it additionally comprises at least one oligonucleotide selected from sequences comprising SEQ ID NOs: 7-9. ### Claim 11 (depends on 1, 2, 3) Method for simultaneous detection of Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae, characterized by the fact that it comprises the steps of: a) producing at least one amplicon using at least two oligonucleotides, such oligonucleotides as defined in claims 1 to 3, and b) detecting the presence of amplicons. ### Claim 12 (depends on 11) Method according to claim 11, characterized in that said step of producing at least one amplicon comprises at least one of amplification by multiplex PCR, or in real time. ### Claim 13 (depends on 11) Method according to claim 11, characterized in that said step of detecting an amplicon comprises detecting the melting temperatures (TM) of the amplicons. ### Claim 14 (depends on 13) Method according to claim 13, characterized in that the amplicon of N. meningitidis has a TM of 85.8° C.; the amplicon of H. influenzae has a TM of 80° C.; and S. pneumoniae amplicon has a TM of 77° C. ### Claim 15 (depends on 11) Method according to claim 11, characterized in that said step of detecting an amplicon comprises detecting at least one probe oligonucleotide. ### Claim 16 (depends on 15) Method according to claim 15, characterized in that the probe nucleotide comprises at least one oligonucleotide selected from sequences comprising SEQ ID NOs: 7-9. ### Claim 17 (depends on 15) Method according to claim 15, characterized in that the probe oligonucleotide is attached to a fluorophore and a quencher. ### Claim 18 (depends on 11, 12, 13, 14) Method according to any one of claims 11 to 14, characterized in that it allows discrimination between infections by Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae. ### Claim 19 (depends on 1, 2, 3) Kit for diagnosis and discrimination of infection by Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae, characterized by the fact that it comprises at least one oligonucleotide as defined in any one of claims 1 to 3, and b) optionally, instructions for use. ### Claim 20 (depends on 4, 5, 6, 19) Kit according to claim 19, characterized in that it additionally comprises at least one set of oligonucleotides as defined in any one of claims 4 to 6. ### Claim 21 (depends on 20) Kit according to claim 20, characterized in that the oligonucleotides are probe oligonucleotides, and are attached to at least one fluorophore and a quencher. ### Claim 22 (depends on 19, 20) Kit according to any one of claims 19 to 20, characterized in that it also includes a negative control and/or a positive reaction control.