| CPC C12Q 1/6848 (2013.01) [C12P 19/34 (2013.01); C12Q 1/6806 (2013.01); C12Q 1/6825 (2013.01); G16B 30/00 (2019.02); G16B 30/10 (2019.02)] | 14 Claims |
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1. A method for amplifying a target nucleic acid characteristic of a pathogen in a whole blood sample obtained from a subject, wherein the whole blood sample comprises subject-derived cells or cell debris, the method comprising:
(a) lysing the cells in the whole blood sample to form a lysate;
(b) adding to the lysate a buffer solution comprising a buffering agent to form a reaction mixture, wherein the buffer solution has a moderately alkaline pH of 8.5 to 9.5 at ambient temperature;
(c) following step (b), heating the reaction mixture to form a denatured reaction mixture and centrifuging the denatured reaction mixture prior to step (d);
(d) adding a thermostable nucleic acid polymerase to the denatured reaction mixture, wherein the final concentration of the thermostable nucleic acid polymerase is at least 0.02 units per microliter of the denatured reaction mixture, or at least 2.4×10−5 micrograms of a thermostable nucleic acid polymerase per microliter of denatured reaction mixture; and
(e) amplifying the target nucleic acid characteristic of a pathogen to form an amplified solution comprising an amplicon, wherein the amplifying is performed by polymerase chain reaction (PCR),
wherein the whole blood sample is about 0.2 mL to about 5 mL.
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