US 12,188,082 B2
Method and apparatus for detecting a plurality of target nucleic acid sequences in sample
Sung Moon Ko, Seoul (KR); Ji Hye Han, Seoul (KR); Young Wook Kim, Seoul (KR); and Young Yong Park, Seoul (KR)
Assigned to SEEGENE, INC., Seoul (KR)
Appl. No. 17/043,198
Filed by SEEGENE, INC., Seoul (KR)
PCT Filed Apr. 19, 2019, PCT No. PCT/KR2019/004780
§ 371(c)(1), (2) Date Sep. 29, 2020,
PCT Pub. No. WO2019/203623, PCT Pub. Date Oct. 24, 2019.
Claims priority of application No. 10-2018-0046375 (KR), filed on Apr. 20, 2018.
Prior Publication US 2021/0040542 A1, Feb. 11, 2021
Int. Cl. C12Q 1/6816 (2018.01); G16B 40/10 (2019.01)
CPC C12Q 1/6816 (2013.01) [G16B 40/10 (2019.02); C12Q 2527/107 (2013.01); C12Q 2537/143 (2013.01)] 34 Claims
 
1. A method for detecting a plurality of target nucleic acid sequences in a sample, comprising:
contacting in a single reaction vessel the sample to signal-generating compositions for detecting the plurality of target nucleic acid sequences; wherein the signal-generating compositions form a plurality of duplexes for the plurality of target nucleic acid sequences, each of the plurality of duplexes provides a signal indicating the presence or absence of each corresponding target nucleic acid sequence and the plurality of duplexes have Tm values different from each other;
performing a real-time amplification reaction for at least two cycles for (i) the formation of the plurality of duplexes and (ii) a hybridization and/or dissociation of the formed duplexes;
obtaining at all or partial cycles of the reaction, signal values at at least two detection temperatures in each of a plurality of detection temperature ranges assigned to the plurality of target nucleic acid sequences; wherein each of the plurality of detection temperature ranges comprises a temperature range in which the amount of a duplex for each corresponding target nucleic acid sequence is changed;
obtaining a signal-change value of signal values between two reference temperatures selected from the at least two detection temperatures in each of the plurality of detection temperature ranges such that a data set of cycle/signal-change value for each of the plurality of target nucleic acid sequences is obtained; and
determining the presence or absence of the plurality of target nucleic acid sequences in the sample by the data set of cycle/signal-change value for each of the plurality of target nucleic acid sequences.