Methods and systems for amplification and detection in biological samples
AssigneeT2 Biosystems, Inc.
InventorsMohamed Nabuan NAUFER, Daniel Alejandro GAMERO, Jessica Lee SNYDER, Joseph MARTURANO, Mark DALTON, Christopher STEELE, Benjamin GALLO
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Abstract
The invention features methods for amplification and detection of target nucleic acids in complex biological samples, for example, whole blood. The invention also features diagnostic and therapeutic methods based on amplification and detection of target nucleic acids characteristic of pathogens present in complex biological samples containing cells and/or cell debris, for example, whole blood.
Claims113
A method for amplifying and detecting a target pathogen nucleic acid in a biological sample suspected of containing one or more pathogen cells, the method comprising:
- (a) lysing one or more pathogen cells within the biological sample by contacting the biological sample with a lysis agent to form a solution containing both subject cell nucleic acid and pathogen nucleic acid, and optionally:
- (i) centrifuging the product of step (a) to form a supernatant and a pellet;
- (ii) discarding some or all of the supernatant of step (i) and optionally washing the pellet once;
- (iii) centrifuging the product of step (ii) to form a supernatant and a pellet;
- (iv) discarding some or all of the supernatant of step (iii) and mixing the pellet of step (iii) with a buffer;
- (b) amplifying the pathogen nucleic acid in the solution of step (a) by 6 to 20 cycles of PCR to form an amplified solution, wherein the PCR is symmetric PCR;
- (c) adding a pathogen-specific fluorescent probe to the amplified solution;
- (d) measuring a signal from the pathogen-specific fluorescent probe in the amplified solution of step (c) to obtain a baseline measurement;
- (e) further amplifying the pathogen nucleic acid in the amplified solution of step (c) by 30 to 50 cycles of PCR, wherein the PCR is asymmetric PCR;
- (f) measuring the signal from the pathogen-specific fluorescent probe in the amplified solution of step (e);
- and
- (g) obtaining a final signal measurement by subtracting the baseline measurement from step (d) from the signal from step (f), wherein the presence of the target pathogen in the biological sample is detected based on the final signal measurement.
A method for amplifying and detecting a target pathogen nucleic acid in a biological sample, the method comprising:
- (a) contacting a biological sample suspected of containing one or more pathogen cells with a lysis agent, thereby lysing cells within the biological sample;
- (b) centrifuging the product of step (a) to form a supernatant and a pellet;
- (c) discarding some or all of the supernatant of step (b) and washing the pellet once;
- (d) centrifuging the product of step (c) to form a supernatant and a pellet;
- (e) discarding some or all of the supernatant of step (d) and mixing the pellet of (d) with a buffer to form a solution containing both subject cell nucleic acid and pathogen nucleic acid;
- (f) amplifying the pathogen nucleic acid in the solution of step (e) by 6 to 20 cycles of polymerase chain reaction (PCR) to form an amplified solution;
- (g) adding a pathogen-specific fluorescent probe to the amplified solution;
- (h) measuring a signal from the pathogen-specific fluorescent probe in the amplified solution of step (g) to obtain a baseline measurement;
- (i) further amplifying the pathogen nucleic acid in the amplified solution of step (g) by 30 to 50 cycles of PCR;
- (j) measuring the signal from the pathogen-specific fluorescent probe in the amplified solution of step (i);
- and
- (k) obtaining a final signal measurement by subtracting the baseline measurement from step (h) from the signal from step (j), wherein the presence of the target pathogen in the biological sample is detected based on the final signal measurement.
Dependencies are inferred from the claim text.