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US 2020/0270708 A1Published application
Methods of screening for causative agents of onychodystrophy
AssigneeBakotic Pathology Associates, LLC
InventorsIdowu Akinsanmi, Lori Bennett, Liquan Yang, Marianna Agassandian, Rama Murthy Sakamuri, Erik Gustafson, Lilly Kong, Kiran Madanahally Divakar
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Abstract
Provided herein is a real-time PCR-based method of detecting, in a sample, an agent causing onychodystrophy, wherein the agent causing onychodystrophy belongs to a secondary clade member including one or more primary clade members. Also provided are compositions and kits that finds use in implementing the present method.
Claims20
A method of detecting, in a sample, an agent causing onychodystrophy, wherein the agent causing onychodystrophy belongs to a secondary clade member comprising one or more primary clade members, the method comprising:
- i) screening a sample using at least a first and second set of secondary clade-specific primers to determine whether a secondary clade member among a plurality of secondary clade members is present or absent in the sample, wherein the plurality of secondary clade members comprises a dermatophyte, a yeast, and a saprophyte, wherein the screening comprises:
- performing a first real time polymerase chain reaction (PCR) in a first reaction mixture using the first set of secondary clade-specific primers and a first hydrolysis probe specific for a DNA region amplified by the first set of secondary clade-specific primers, the first hydrolysis probe comprising a fluorescent reporter dye and a quencher;
- and
- performing a second real time PCR in a second reaction mixture using the second set of secondary clade-specific primers and a second hydrolysis probe specific for a DNA region amplified by the second set of secondary clade-specific primers, the second hydrolysis probe comprising a fluorescent reporter dye and a quencher;
- and
- ii) if the secondary clade member is determined to be present in the sample, performing a second screen of the sample to determine whether an an agent causing onychodystrophy is present or absent in the sample using primary clade-specific primers that are specific to a primary clade member that belongs to the secondary clade member, wherein the second screen comprises performing at least a third real time PCR in a third reaction mixture using the primary clade-specific primers and a third hydrolysis probe specific for a DNA region amplified by the primary clade-specific primers, the third hydrolysis probe comprising a fluorescent reporter dye and a quencher.
3 more elements
A method of detecting a yeast and/or a dermatophyte in a sample, the method comprising:
- i) screening a sample using at least a first set of yeast-specific primers and at least first set of dermatophyte-specific primers to determine whether a yeast and/or dermatophyte is present or absent in the sample, wherein the screening comprises:
- performing a first real time polymerase chain reaction (PCR) in a first reaction mixture using the first set of yeast-specific primers and a first hydrolysis probe specific for a DNA region amplified by the first set of yeast-specific primers, the first hydrolysis probe comprising a fluorescent reporter dye and a quencher;
- and
- performing a second real time PCR in a second reaction mixture using the first set of dermatophyte-specific primers and a second hydrolysis probe specific for a DNA region amplified by the first set of dermatophyte-specific primers, the second hydrolysis probe comprising a fluorescent reporter dye and a quencher;
- and
- ii) if the yeast and/or dermatophyte is determined to be present in the sample, performing a second screen of the sample to determine whether a genus and/or species of the yeast and/or dermatophyte is present or absent in the sample using yeast and/or dermatophyte genus and/or species-specific primers, wherein the second screen comprises performing at least a third real time PCR in a third reaction mixture using the yeast and/or dermatophyte genus and/or species-specific primers and a third hydrolysis probe specific for a DNA region amplified by the yeast and/or dermatophyte genus and/or species-specific primers, the third hydrolysis probe comprising a fluorescent reporter dye and a quencher.
3 more elements
A method of detecting a saprophyte and/or Pseudomonas aeruginosa in a sample, the method comprising:
- i) screening a sample using at least a first set of saprophyte-specific primers and at least first set of Pseudomonas aeruginosa-specific primers to determine whether a saprophyte and/or Pseudomonas aeruginosa is present or absent in the sample, wherein the screening comprises:
- performing a first real time polymerase chain reaction (PCR) in a first reaction mixture using the first set of saprophyte-specific primers and a first hydrolysis probe specific for a DNA region amplified by the first set of saprophyte-specific primers, the first hydrolysis probe comprising a fluorescent reporter dye and a quencher;
- and
- performing a second real time PCR in a second reaction mixture using the first set of Pseudomonas aeruginosa-specific primers and a second hydrolysis probe specific for a DNA region amplified by the first set of Pseudomonas aeruginosa-specific primers, the second hydrolysis probe comprising a fluorescent reporter dye and a quencher;
- and
- ii) if the saprophyte is determined to be present in the sample, performing a second screen of the sample to determine whether a genus and/or species of the saprophyte is present or absent in the sample using saprophyte genus and/or species-specific primers, wherein the second screen comprises performing at least a third real time PCR in a third reaction mixture using the saprophyte genus and/or species-specific primers and a third hydrolysis probe specific for a DNA region amplified by the saprophyte genus and/or species-specific primers, the third hydrolysis probe comprising a fluorescent reporter dye and a quencher.
3 more elements
Dependencies are inferred from the claim text.