Methods and compositions for the diagnosis of sepsis using gamma peptide nucleic acids
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Publication Number
US-11091814-B2
Publication Date
2021-08-17
Expiration Date
Abstract
The present disclosure provides for compositions of γPNA probes. Additionally, the present disclosure provide for methods and kits using γPNA probes for the diagnosis of sepsis.
Core Innovation
The invention provides a method for detecting bacterial and/or fungal genomic material in a sample by using γPNA capture probes. The method contacts the sample with a plurality of γPNA capture probes that include at least one sequence selected from multiple SEQ ID NOS capture sets, and their complementary sequences and functional equivalents. The approach includes a heating step to facilitate interaction between the γPNA capture probes and the sample.
After heating, the γPNA capture probes invade targeted bacterial and/or fungal genomic material. The detecting step is performed by detecting the presence of one or more bacterial and/or fungal genomic material, based on the invaded genomic material. The method is framed as enabling detection without relying on culturing, by targeting bacterial and/or fungal genomic material in the sample using γPNA probe invasion.
In further embodiments, detecting includes the use of γPNA reporter probes, where the γPNA reporter probes comprise at least one sequence selected from defined SEQ ID NOS reporter ranges, including complementary sequences and functional equivalents. The embodiments describe that the γPNA reporter probes are invaded into targeted genomic material as part of the detection workflow. The document also discloses optional preprocessing of genomic material, including enzymatic amplification, and supports probe immobilization on substrates using avidin-family and related chemistries with functional moieties.
Claims Coverage
The document includes one independent claim and dependent claims that refine the detection workflow, define additional probe sequence ranges for detection, add optional preprocessing, and constrain probe and support characteristics. The independent claim covers the core sequence-based γPNA capture invasion and detection steps; the dependent claims further specify reporter probe sequences, amplification, and immobilization/support details.
Sequence-defined γPNA capture invasion for genomic detection
A method for detecting bacterial and/or fungal genomic material in a sample by contacting the sample with a plurality of γPNA capture probes comprising at least one sequence selected from the group consisting of SEQ ID NOS: 1-18, 19-22, 23-28, 29-34, 35-38, 39-57, 58-72, 73-91, 92-94, 95-97, 98-110, 111-113, 114-117, 118-119, 120-121, 122-153, 154-166, 167-190, 191-193, 194-196, 197-211, 212-215, 216-230, complementary sequence thereof, and functional equivalents thereof; heating the γPNA capture probes and the sample; invading a plurality of bacterial and/or fungal genomic material by the γPNA capture probes; and detecting a presence of one or more bacterial and/or fungal genomic material.
γPNA reporter invasion using defined reporter sequence ranges
Detecting comprises adding γPNA reporter probes that comprise at least one sequence selected from the group consisting of SEQ ID NOS: 231-248 and 249-309, including complementary sequences thereof and functional equivalents thereof; heating the γPNA capture probes, the γPNA reporter probes, and the sample; and invading the γPNA reporter probes into targeted genomic material to detect it.
Optional enzymatic amplification preprocessing of genomic material
Enzymatic amplification of genomic material in the sample is performed before the contacting step (a).
Probe functional composition with biotin or hapten
The method includes a γPNA probe that comprises biotin or a hapten.
Immobilized support substrate formats
A support substrate is chosen from defined options including magnetic bead, well, plate, test tube, stick, plastic slide, glass slide, or biochip.
Avidin-family coating on support substrate
The support substrate is coated with Avidin, Neutravidin, or Streptavidin.
Claim coverage centers on sequence-selected γPNA capture probes that invade bacterial and/or fungal genomic material after heating, followed by detecting the presence of invaded genomic material. Dependent claims extend this by defining reporter probe sequence ranges and reporter invasion, optionally adding enzymatic amplification preprocessing, and specifying probe functional composition and support substrate/coating constraints.
Stated Advantages
Enables detection of bacterial and/or fungal genomic material in a sample using γPNA capture probes and probe invasion followed by detecting presence.
Provides an additional detection workflow using γPNA reporter probes with defined reporter sequence ranges.
Supports detection configurations that include optional enzymatic amplification preprocessing.
Enables probe immobilization using specified support substrate formats and avidin-family coatings.
Documented Applications
Sepsis diagnosis by detecting sepsis-related bacterial and/or fungal genomic material using γPNA probes without culturing.
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