US 12,410,461 B2
Method of preparing clinical samples for nucleic acid amplification
Chiahao Tsui, Somerville, MA (US)
Assigned to Day Zero Diagnostics, Inc., Watertown, MA (US)
Appl. No. 17/434,293
Filed by Day Zero Diagnostics, Inc., Boston, MA (US)
PCT Filed Feb. 28, 2020, PCT No. PCT/US2020/020275
§ 371(c)(1), (2) Date Aug. 26, 2021,
PCT Pub. No. WO2020/176822, PCT Pub. Date Sep. 3, 2020.
Application 17/434,293 is a continuation of application No. 16/289,324, filed on Feb. 28, 2019, granted, now 10,544,446.
Claims priority of provisional application 62/812,085, filed on Feb. 28, 2019.
Prior Publication US 2022/0127660 A1, Apr. 28, 2022
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/6806 (2018.01); C12Q 1/6848 (2018.01)
CPC C12Q 1/6806 (2013.01) [C12Q 1/6848 (2013.01)] 19 Claims
 
1. A method for preparing a clinical sample from a subject comprising microbes, cells of the subject, and at least one polyanionic polymer nucleic acid amplification inhibitor that binds at least one protein in the sample, comprising the steps of:
(a) obtaining the clinical sample from the subject comprising the microbes, the at least one protein, and the at least one polyanionic polymer nucleic acid amplification inhibitor;
(b) separating the clinical sample into:
(i) a first fraction comprising the cells of the subject, and
(ii) a second fraction comprising the microbes and the at least one polyanionic polymer nucleic acid amplification inhibitor bound to the at least one protein;
(c) adding to the second fraction a protease which degrades the at least one protein; and
(d) separating the second fraction into:
(i) a third fraction comprising the microbes and any remaining cells from the subject, and
(ii) a fourth fraction comprising the at least one polyanionic polymer nucleic acid amplification inhibitor;
wherein the microbe is a pathogenic microbe, wherein the pathogenic microbe is Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epidermidis), Streptococcus agalactiae (S. agalactiae), Enterococcus faecalis (E. faecalis), Enterococcus faecium (E. faecium), Escherichia coli (E. coli), or Klebsiella pneumoniae (K. pneumoniae).