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 spin 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 removing an assay inhibitor from a crude sample preparation comprising a nucleic acid, the method comprising:
a) adding insoluble polyvinylpyrrolidone particles having a diameter of 100 micrometers to 130 micrometers to said crude sample preparation prior to isolating the nucleic acid under conditions wherein said assay inhibitor binds to said polyvinylpyrrolidone to produce a complex, wherein said crude sample preparation is a supernatant prepared from a stool sample having a mass of at least one gram;
b) separating said complex from said crude sample preparation to produce a clarified sample preparation comprising said nucleic acid, wherein said separating comprises:
i) placing said crude sample preparation comprising said complex into a spin filter comprising a hollow body, a bottom end, and an open top end opposite the bottom end, wherein said hollow body and said bottom end are made from porous filtering material having a nominal pore size of 20 micrometers; and
ii) centrifuging said spin filter;
wherein during said centrifuging, said complex comprising said inhibitor is retained by said spin filter and a fraction of said crude sample preparation comprising said nucleic acid passes through said porous filtering material to produce said clarified sample preparation from which said assay inhibitor has been removed.
2. The method of claim 1 wherein the stool sample has a mass of at least 4 grams.
3. The method of claim 1 wherein the stool sample has a mass of at least 8 grams.
4. The method of claim 1 wherein said polyvinylpyrrolidone is a polyvinylpolypyrrolidone.
5. A system for removing an assay inhibitor from a crude stool sample preparation comprising a nucleic acid, the system comprising:
a) insoluble polyvinylpyrrolidone particles having a diameter of 100 micrometers to 130 micrometers for binding the assay inhibitor and producing a complex;
b) a spin filter comprising a hollow body, a bottom end, and an open top end opposite the bottom end, wherein said hollow body and said bottom end are made from porous filtering material having a nominal pore size of 20 micrometers, and
c) a collection vessel adapted to receive said spin filter for retaining a clarified sample preparation comprising the nucleic acid.
6. The system of claim 5 wherein said polyvinylpyrrolidone is a polyvinylpolypyrrolidone.
7. The system of claim 5 wherein said polyvinylpyrrolidone is provided in a premeasured form.
8. The system of claim 5 wherein said polyvinylpyrrolidone is provided as a tablet.
9. A kit for removing an assay inhibitor from a crude stool sample preparation comprising a nucleic acid, the kit comprising:
a) insoluble polyvinylpyrrolidone particles having a diameter of 100 micrometers to 130 micrometers;
b) a spin filter comprising a hollow body, a bottom end, and an open top end opposite the bottom end, wherein said hollow body and said bottom end are made from porous filtering material having a nominal pore size of approximately 20 micrometers, and
c) a collection vessel adapted to receive said spin filter.
10. The kit of claim 9 , wherein said polyvinylpyrrolidone is a polyvinylpolypyrrolidone.