(WO2019222222) Methods for microbial identification in clinical specimens by differential ribosomal rna probe hybridization 机翻标题: 暂无翻译,请尝试点击翻译按钮。

源语言标题
(WO2019222222) Methods for microbial identification in clinical specimens by differential ribosomal rna probe hybridization
公开号/公开日
WO2019/222222 WO2019/222222 / 2019-12-26 2019-11-21
申请号/申请日
WOUS2019/032227 / 2019-05-14
发明人
CHURCHILL BERNARDCHURCHMAN SCOTT ADAMHAAKE DAVID ARNOLDHALFORD COLIN WYNNKNAUF ROGERMONTI GABRIEL;
申请人
MICROBIOTIX UNIVERSITY OF CALIFORNIA;
主分类号
IPC分类号
C07H-021/04 C12Q-001/00 C12Q-001/68
摘要
(WO2019/222222) A method of identifying a target microbe in a specimen and including the steps of a) obtaining a specimen, b) lysing the specimen to release a plurality of rRNA molecules from one or more first target microbes in the specimen; c) contacting the specimen with a plurality of first oligonucleotide probe sets configured to selectively bind to rRNA molecules released from the target microbe thereby forming a plurality of first hybridized complexes, each first hybridized complex including one first capture probe and one first detector probe and one of the plurality of rRNA molecules, and d) analyzing the first hybridized complexes to identify a first target microbe in the specimen.
机翻摘要
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地址
代理人
(WO2019222222) TOMSA, Michael S. ([US])
代理机构
;
优先权号
2018US-62671389
主权利要求
(WO2019/222222) CLAIMS 1. A method of identifying a first target microbe in a specimen, the method comprising:   (a) lysing the specimen to release rRNA molecules from the first target microbe thereby producing a lysate;   (b) contacting the rRNA molecules with a first oligonucleotide probe set configured to selectively bind to the rRNA molecules; each first oligonucleotide probe set comprising a first capture probe and a first detector probe, thereby forming a first hybridized complex, the first hybridized complex comprising rRNA molecules bound to the first oligonucleotide probe set; and   (c) analyzing the first hybridized complex to identify the first target microbe. 2. The method of claim 1 , wherein the first capture probe comprises an oligonucleotide adapted to hybridize with a first target sequence of the rRNA molecules released in step  (a); and wherein the first detector probe comprises a detectably labeled oligonucleotide adapted to hybridize with a second sequence of the rRNA molecules. 3. The method of claim 1 or 2, wherein the method includes the further step of neutralizing the lysate of the specimen produced in step (a). 4. The method of any of claims 1 to 3, the method comprising the further step of washing away unhybridized first detector probes and/or unhybridized first capture probes from step  (b). 5. The method of any of claims 1 to 4, wherein the oligonucleotide probe sets further comprise a helper probe, wherein the helper probe includes sequences adapted to hybridize with a helper sequence on the capture probe. 6. The method of any of claims 1 to 5, wherein step (b) comprises contacting the rRNA molecules with a panel comprising a plurality of oligonucleotide probe sets, each probe set adapted to selectively bind to rRNA molecules released from a pre-determined microorganism. 7. The method of any one of claims 1 to 6, wherein the microorganism is selected from the group consisting of: Escherichia coli (EC), Klebsiella pneumoniae (KP), Proteus mirabilis (PM), Pseudomonas aeruginosa (PA), Staphylococcus saprophyticus (SS), Staphylococcus aureus (SA), Streptococcus agalactiae (GB), Enterococcus faecium (EM), Candida albicans (CA), Enterobacteriaceae (EB), Enterococci (EF), Candida species (CN), Citrobacter freundii (CF), Serratia marcescens (SM), Enterobacter cloacae (EL), Enterobacter hormaechei (EH), and Klebsiella oxytoca (KX), Klebsiella aerogenes (KA), Morganella morganii (MM), Acinetobacter baumanii (AB), Streptococcus pyogenes (SY), Streptococcus pneumoniae (SP), Streptococcus viridans (SV), Stenotrophomonas maltophilia (XM) and Staphylococcus epidermidis (SE). 8. The method of any one of claims 1 to 7, wherein the first oligonucleotide probe set is configured to selectively bind to 16S rRNA sequences. 9. The method of any one of claims 1 to 7, wherein the first oligonucleotide probe set is configured to selectively bind to 23S rRNA sequences. 10. The method of any one of claims 1 to 9, the method further comprises identifying a second target microbe in the specimen by:   (d) contacting the rRNA molecules with a second oligonucleotide probe set configured to selectively bind to rRNA molecules released from the second target microbe; the second oligonucleotide probe set comprising a second capture probe and second detector probe, thereby forming a second hybridized complex, the hybridized complex comprising rRNA molecules bound to the second oligonucleotide probe set; and   (e) analyzing the second hybridized complexes to identify the second target microbe.   1 1 . The method of claim 10, wherein the second capture probe comprises an oligonucleotide adapted to hybridize with a first target sequence of the rRNA molecules released in step (a); and wherein the second detector probe comprises a detectably labeled oligonucleotide adapted to hybridize with a second sequence of the rRNA molecules, 12. The method of claim 10 or 1 1 , wherein the steps (d) and (e) are performed simultaneously with steps (b) and (c). 13. The method of any one of claims 10 to 12, wherein the first and second oligonucleotide probe sets are configured to selectively bind to rRNA molecules released from different pre-determined microorganisms. 14. The method of any one of claims 10 to 13, wherein the second oligonucleotide probe set is configured to selectively bind to rRNA molecules released from a microorganism selected from the group consisting of: Escherichia coli (EC), Klebsiella pneumoniae (KP), Proteus mirabilis (PM), Pseudomonas aeruginosa (PA), Staphylococcus saprophyticus (SS), Staphylococcus aureus (S A), Streptococcus agalactiae ( GB), Enterococcus faecium (EM), Candida albicans (CA), Enterobacteriaceae (EB), Enterococci (EF), Candida species (CN), Citrobacter freundii (CF), Serratia marcescens (SM), Enterobacter cloacae (EL), Enterobacter hormaechei (EH), and Klebsiella oxytoca (KX), Klebsiella aerogenes (KA), Morganella morganii (MM), Acinetobacter baumanii (AB), Streptococcus pyogenes (SY), Streptococcus pneumoniae (SP), Streptococcus viridans (SV), Stenotrophomonas maltophilia (XM) and Staphylococcus epidermidis (SE). 15. The method of any one of claims 1 to 14, wherein step (a) comprises at least one of mechanical lysis, chemical lysis and a combination of mechanical and chemical lysis. 16. The method of any one of claims 1 to 15, wherein the first capture probes are secured to a substrate during steps (b) and (c). 17. The method of claim 16, wherein the substrate is substantially planar and the first capture probes are secured within a first detection region. 18. The method of claim 16 or 17 wherein the substrate comprises at least one of plastic, metal, glass, nitrocellulose, organic material and a magnetic-bead based platform. 19. The method of claim 18, wherein the magnetic-bead based platform comprises a Luminex MAG P IX™ system. 20. The method of any one of claims 1 to 19, wherein step (c) is carried out by a quantitative real-time PCR (qPCR) system.   21 . The method of any one of claims 1 to 19, wherein step (c) comprises at least one of excitation and imaging of fluorescent-tagged detector probes; bioluminescence using luciferase-type enzymes; and amperometric current using an electrochemical sensor. 22. The method of any one of claims 1 to 21 , wherein the specimen is suspected of containing pathogens associated with at least one of a urinary tract infection and sepsis. 23. The method of any one of claims 1 to 22, wherein the specimen is a clinical specimen. 24. The method of claim 23, wherein the clinical specimen comprises at least one of urine, blood, serum, plasma, saliva, tears, gastric fluids, digestive fluids, stool, mucus, sputum, sweat, earwax, oil, semen, vaginal fluid, glandular secretion, breast milk, synovial fluid, pleural fluid, lymph fluid, amniotic fluid, feces, cerebrospinal fluid, wounds, burns, tissue homogenates and an inoculum derived therefrom that is generated during conventional laboratory testing procedures. 25. The method of any one of claims 1 to 24, wherein steps (a) to (c) are conducted in sequence on the specimen without an intervening culturing or incubation step. 26. The method of any one claims 1 to 25, wherein the first oligonucleotide probe set further comprises a helper probe having sequences adapted to hybridize with a helper sequence on the first capture probe to help immobilize the first capture probe prior to steps 1 (b) and 1 (c). 27. An oligonucleotide probe set for use in identifying a first target microbe in a specimen, wherein the oligonucleotide probe set comprises a capture probe and a detector probe, the capture probe and detector probe each being adapted to selectively hybridize to a first target sequence of rRNA molecules released from the first target microbe. 28. The oligonucleotide probe set of claim 27, wherein the probe set further comprises a helper probe adapted to hybridize with a helper sequence of the capture probe to help immobilize the first capture probe. 29. The oligonucleotide probe set of claims 27 or 28, wherein the probe set is adapted to selectively bind to rRNA molecules released from a pre-determined microorganism, preferably a microorganism selected from the group consisting of Escherichia coli (EC), Klebsiella pneumoniae (KP), Proteus mirabilis (PM), Pseudomonas aeruginosa (PA), Staphylococcus saprophyticus (SS), Staphylococcus aureus (SA), Streptococcus agalactiae (GB), Enterococcus faecium ( EM), Candida albicans (CA), Enterobacteriaceae (EB), Enterococci (EF), Candida species (CN), Citrobacter freundii (CF), Serratia marcescens (SM), Enterobacter cloacae (EL), Enterobacter hormaechei (EH), and Klebsiella oxytoca (KX), Klebsiella aerogenes (KA), Morganella morganii (MM), Acinetobacter baumanii (AB), Streptococcus pyogenes (SY), Streptococcus pneumoniae (SP), Streptococcus viridans (SV), Stenotrophomonas maltophilia (XM), Staphylococcus epidermidis (SE) and combinations of any two or more of these. 30. The oligonucleotide probe set of claim 27 or 28, wherein the oligonucleotide probe set is adapted to hybridize with rRNA molecules released from substantially all eubacteria (EU) or similar microbes.   31 . The oligonucleotide probe set of claim 27 or 28, wherein the oligonucleotide probe set is adapted to hybridize 16S rRNA sequences. 32. The oligonucleotide probe set of claim 27 or 28, wherein the oligonucleotide probe set is adapted to hybridize 23S rRNA sequences. 33. A probe panel disposed on a substrate, the probe panel comprising a plurality of detection regions, each detection region comprising an oligonucleotide probe set configured to selectively bind to RNA molecules released from a pre-determined microorganism, each detection region comprising a different oligonucleotide probe set. 34. The probe panel of claim 33, wherein each oligonucleotide probe set comprises a first capture probe having an oligonucleotide adapted to hybridize with a first target sequence of the rRNA molecules of the pre-determined microorganism, and a first detector probe having a detectably labeled oligonucleotide adapted to hybridize with a second sequence of the rRNA molecules of the pre-determined microorganism. 35. The probe panel of claim 33 or 34, wherein the pre-determined microorganism is selected from the group consisting of: Escherichia coli (EC), Klebsiella pneumoniae (KP), Proteus mirabilis (PM), Pseudomonas aeruginosa (PA), Staphylococcus saprophyticus (SS), Staphylococcus aureus (SA), Streptococcus agalactiae (GB), Enterococcus faecium (EM), Candida albicans (CA), Enterobacteriaceae (EB), Enterococci (EF), Candida species (CN), Citrobacter freundii (CF), Serratia marcescens (SM), Enterobacter cloacae (EL), Enterobacter hormaechei (EH), and Klebsiella oxytoca (KX), Klebsiella aerogenes (KA), Morganella morganii (MM), Acinetobacter baumanii (AB), Streptococcus pyogenes (SY), Streptococcus pneumoniae (SP), Streptococcus viridans (SV), Stenotrophomonas maltophilia (XM), Staphylococcus epidermidis (SE) and any combination of two or more of these. 36. The probe panel of any of claims 33 to 35, wherein the number of detection regions is in the range of between 3 and 100. 37. The probe panel of any of claims 33 to 35, wherein the number of detection regions is in the range of between 5 and 50. 38. The probe panel of any of claims 33 to 35, wherein the number of detection regions is in the range of between 10 and 25. 39. The probe panel of any one of claims 33 to 38, wherein the substrate comprises at least one of plastic, metal, glass, nitrocellulose, organic material and a magnetic-bead based platform. 40. The system of claim 39, wherein the magnetic-bead based platform comprises a Luminex MAG P IX™ system.   41 . A system for identifying at least two microbes in a specimen, the system comprising:   (a) the probe panel of any one of claims 33 to 40; and   (b) a detection apparatus configured to detect hybridized complexes present in any detection regions of the probe panel. 42. The system of claim 41 , wherein the detection apparatus comprises a plurality of sensors, each sensor associated with a single detection region and configured to detect a signal from a hybridized complex when present in the single detection region. 43. The system of claim 42, wherein the plurality of sensors are operable simultaneously. 44. The system of claim 41 , wherein the detection apparatus comprises a single sensor, moveable to be associated with each detection region and configured to detect a signal from a hybridized complex when present in a detection region proximal to the single sensor. 45. The system of any one of claims 41 to 44, wherein the specimen is selected from the group consisting of urine, blood, serum, plasma, saliva, tears, gastric fluids, digestive fluids, stool, mucus, sputum, sweat, earwax, oil, semen, vaginal fluid, glandular secretion, breast milk, synovial fluid, pleural fluid, lymph fluid, amniotic fluid, feces, cerebrospinal fluid, wounds, burns, tissue homogenates and an inoculum derived therefrom that is generated during conventional laboratory testing procedures.
法律状态
PENDING
专利类型码
A3 A2
国别省市代码
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