Removal of PCR inhibitors
View Patent ↗Provided herein is technology relating to processing and preparing samples and particularly, but not exclusively, to methods, systems, and kits for removing assay inhibitors, e.g., compounds that inhibit polymerase chain reaction, from samples comprising nucleic acids. In particular, the technology is directed toward treating crude sample preparations, such as supernatants from homogenized stool samples, with insoluble polyvinylpyrrolidone (PVP) to form PVP-assay inhibitor complexes, and filtration to separate the PVP-assay inhibitor complexes from the crude sample preparations to produce clarified samples that exhibit reduced assay inhibition.
1. A method for amplifying a target nucleic acid from a crude human stool sample preparation comprising a nucleic acid, the method comprising:
a) mixing insoluble cross-linked polyvinylpyrrolidone particles in powder, granule, or tablet form with said crude human stool sample preparation in an amount of 50 milligrams of insoluble cross-linked polyvinylpyrrolidone particles per milliliter of crude sample preparation, under conditions wherein said assay inhibitor binds to said polyvinylpyrrolidone particles to produce an insoluble complex in a suspension;
b) separating said insoluble complex from said suspension, wherein said separating comprises filtering said suspension through porous filtering material, wherein during said filtering said insoluble complex is retained by said porous filtering material, and wherein a liquid fraction of said suspension passes through porous filtering material to produce a clarified sample preparation comprising said nucleic acid;
c) capturing target nucleic acid from said clarified sample preparation and eluting captured target nucleic acid to form an eluted nucleic acid solution; and
d) conducting a polymerase chain reaction in a reaction volume, wherein at least ⅕ th of the reaction volume of said polymerase chain reaction is eluted nucleic acid solution of c).
2. The method of claim 1 wherein said crude human stool sample preparation is a supernatant prepared from a human stool sample.
3. The method of claim 1 , wherein said porous filtering material is in a spin filter, and wherein said filtering comprises centrifuging.
4. The method of claim 1 , wherein said insoluble cross-linked polyvinylpyrrolidone particles comprise particles having a diameter between 100 and 130 micrometers.
5. The method of claim 1 , wherein said porous filtering material comprises pores of less than 45 micrometers.
6. The method of claim 3 , wherein said spin filter comprises a porous frit.
7. The method of claim 1 ,
wherein said target nucleic acid is human DNA.
8. The method of claim 7 , wherein said human DNA is indicative of colon cancer and/or adenoma.