US 12,247,243 B2
Direct detection method for tick-borne infections and a cell technology medium for the method
Béla Pál Bózsik, Budapest (HU); Béla Bózsik, Budapest (HU); and András Pál Bózsik, Budakalász (HU)
Assigned to LYME DIAGNOSTICS KFT., Budakalász (HU)
Appl. No. 17/598,785
Filed by LYME DIAGNOSTICS KFT., Budakalász (HU)
PCT Filed Mar. 18, 2020, PCT No. PCT/HU2020/000011
§ 371(c)(1), (2) Date Sep. 27, 2021,
PCT Pub. No. WO2020/194003, PCT Pub. Date Oct. 1, 2020.
Claims priority of application No. P1900099 (HU), filed on Mar. 28, 2019.
Prior Publication US 2022/0162665 A1, May 26, 2022
Int. Cl. C12Q 1/04 (2006.01); G01N 21/65 (2006.01); G01N 33/49 (2006.01)
CPC C12Q 1/04 (2013.01) [C12Q 1/045 (2013.01); G01N 21/65 (2013.01); G01N 33/491 (2013.01); G01N 2333/20 (2013.01); G01N 2333/43556 (2013.01)] 11 Claims
 
1. A method for detecting tick-borne infections in human/animal body fluid of a subject, the human/animal body fluid comprising a blood sample containing plasma and blood cells, the method comprising the steps of:
adding the blood sample directly into a specified amount of cell technology medium establishing a partially hostile environment for any pathogens infecting the plasma and any pathogens infecting the blood cells, due to the cell technology medium's low-level antibiotic or preservative content aiming at bacteriostatic effect and lowered sugar content;
mixing the blood sample with the cell technology medium to provide a first mixed sample containing a mix of plasma and cell technology medium and the blood cells,
centrifuging the first mixed sample at a speed of 2000 g-4000 g for at least 10 minutes, thereby obtaining the mix of plasma and cell technology medium, the blood cells, and a separating layer between the mix of plasma and cell technology medium, and the blood cells, wherein the separating layer comprises a “buffy coat” layer that contains blood cells;
removing the mix of the plasma and cell technology medium from the blood cells and the buffy coat layer;
mixing part of the blood cells from the “buffy coat” layer and the blood cells located a few cell layers beneath the buffy coat layer with the mix of the plasma and cell technology medium, thereby obtaining a second mixed sample;
concentrating the second mixed sample at a speed of 15000-20000g for 10 minutes, resulting in a sedimented sample; and
testing to detect pathogens in the sedimented sample, including the any pathogens infecting the plasma and sedimented therefrom and the any infected blood cells sedimented therefrom, which blood cells contain pathogens infecting the blood cells.