US 12,325,882 B2
Fast and portable microfluidic detection system as an alternative to Salmonella's classical culture method
Hatice Esra Aǧel, Kocaeli (TR); Şaban Yilmaz, Kocaeli (TR); and Hasan Saǧcan, Istanbul (TR)
Assigned to TUBITAK, Ankara (TR)
Appl. No. 17/418,082
Filed by TUBITAK, Ankara (TR)
PCT Filed Dec. 25, 2019, PCT No. PCT/IB2019/061357
§ 371(c)(1), (2) Date Jun. 24, 2021,
PCT Pub. No. WO2020/136595, PCT Pub. Date Jul. 2, 2020.
Claims priority of application No. 2018/20388 (TR), filed on Dec. 25, 2018.
Prior Publication US 2022/0098645 A1, Mar. 31, 2022
Int. Cl. C12Q 1/689 (2018.01); B01L 3/00 (2006.01); B01L 7/00 (2006.01); C12Q 1/6853 (2018.01); C12R 1/42 (2006.01); B82Y 5/00 (2011.01)
CPC C12Q 1/689 (2013.01) [B01L 3/502715 (2013.01); B01L 7/52 (2013.01); C12Q 1/6853 (2013.01); B01L 2200/04 (2013.01); B01L 2200/16 (2013.01); B01L 2300/0819 (2013.01); B01L 2300/0825 (2013.01); B01L 2300/0829 (2013.01); B82Y 5/00 (2013.01); C12R 2001/42 (2021.05)] 2 Claims
 
1. A Salmonella pathogen detection method, characterized in that the method comprises following process steps;
a. sample preparation and pre-enrichment, completed in 4 hours,
b. separation of dead and live bacteria, by the application of DNase I 25U, at 37° C. for 30 min; and PMA 5-50 μM, in the dark, at 25° C. for 7 min, in no particular order,
c. DNA isolation where, Proteinase K and solution A comprising 10-100 mM Tris-HCl, 10-100 KCl, 1-10 mM MgCl2, pH 8.2 are applied together,
d. Loop-mediated isothermal DNA amplification with 4 primers which are F3, B3, FIP, BIP primers comprising nucleotide sequence SEQ ID NOs:1, 2, 3, and 4 respectively and the 5′ end of the FIP primer is marked with FAM, specifically designed for InvA gene,
e. hybridization of amplicons from Step (d) with unique probe which is comprising nucleotide SEQ ID NO: 5, is attached in single-use disposable cassettes, which are hybridized in disposable cassettes, and evaluation of results with naked eye,
f. after performing steps a-e, display result on a portable mini-heating device.