Methods using four-layer filter for PCR sample preparation
In accordance with the present invention, there is provided a device, method and kit for detecting any genetic material containing moieties. The present invention enables identification of known or unknown virions or bacteria contained in a fluid therein, such as virions in a blood sample. It also provides for an amenable and highly automatable device for massed screenings and the discovery of sequence variants of known virions, previously undetected virions, and other genetic material containing moieties. In one aspect, the present invention provides a method for storage and analysis of a nucleic acid containing moiety in a biological sample, the method comprising providing a device comprising a plurality of layers, wherein the plurality of layers comprises a collection layer comprising a neutral porous matrix and a dry anticoagulant or liquefying agent, a filter layer comprising a dry solid medium comprising a solid matrix having a particle size limit that precludes eukaryotic cells, but not prokaryotic cells or virions, a separator layer, and an isolation layer comprising a dry solid medium comprising a neutral solid matrix having sorbed thereto a composition comprising a detergent; applying a biological sample to the collection layer; filtering remaining components of the biological sample through the collection layer to the filter layer; filtering remaining components of the biological sample through the filter layer to the separator layer; filtering remaining components of the biological sample through the separator layer to the isolation layer; retaining nucleic acid components in the isolation layer while removing a plurality of non-nucleic acid components; drying the isolation layer; providing at least one primer; and analyzing the nucleic acid components using PCR techniques, wherein at least two PCR primers are used.
1. A method for storage and analysis of a nucleic acid containing moiety in a biological sample, the method comprising:
a. providing a device comprising a plurality of layers, wherein the plurality of layers comprises:
i. a collection layer comprising a neutral porous matrix and a dry anticoagulant or liquefying agent;
ii. a filter layer comprising a dry solid medium comprising a solid matrix having a particle size limit that precludes eukaryotic cells, but not prokaryotic cells or virions;
iii. a reagent free separator layer; and
iv. an isolation layer comprising a dry solid medium comprising a neutral solid matrix having sorbed thereto a composition comprising a detergent;
b. applying a biological sample to the collection layer;
c. filtering remaining components of the biological sample through the collection layer to the filter layer;
d. filtering remaining components of the biological sample through the filter layer to the separator layer;
e. filtering remaining components of the biological sample through the separator layer to the isolation layer;
f. retaining nucleic acid components in the isolation layer while removing a plurality of non-nucleic acid components;
g. drying the isolation layer;
h. providing at least one primer; and
i. analyzing the nucleic acid components using PCR techniques, wherein at least two PCR primers are used.
2. The method of claim 1 , wherein the detergent of the isolation layer of step a comprises SDS.
3. The method of claim 1 , wherein the composition of the isolation layer of step a further comprises:
a. a weak base; and
b. a chelating agent.
4. The method of claim 3 , wherein the weak base comprises Tris.
5. The method of claim 3 , wherein the chelating agent comprises EDTA.
6. The method of claim 1 , wherein the isolation layer of step a further comprises a primer.
7. The method of claim 1 , wherein the filter layer of step a further comprises an anticoagulant or a liquefying agent.
8. The method of claim 7 , wherein the anticoagulant comprises EDTA or Tris-EDTA.
9. The method of claim 7 , wherein the liquefying agent comprises thiol.
10. The method of claim 1 , further comprising a protecting step between step g and step h, wherein the protecting step comprises placing a cap on at least a portion of the device, wherein the cap comprises:
a. a capping layer comprising a general denaturing agent; and
b. an adhesive edge.
11. The method of claim 10 , wherein the general denaturing agent comprises SDS.
12. The method of claim 10 , wherein the general denaturing agent comprises:
a. a weak base;
b. a chelating agent; and
c. an anionic surfactant or detergent.
13. The method of claim 12 , wherein the weak base comprises Tris.
14. The method of claim 12 , wherein the chelating agent comprises EDTA.
15. The method of claim 12 , wherein the detergent comprises SDS.
16. The method of claim 3 , wherein the composition of the isolation layer of step a further comprises:
iii. uric acid or a urate salt.
17. The method of claim 12 , wherein the general denaturing agent further comprises:
iii. uric acid or a urate salt.