US 11,939,623 B2
Methods and reagents for nucleic acids detection
Matías Ricardo Gutiérrez Mostafá, Ñuñoa Santiago (CL); Chantal Loretto Márquez Badilla, Ñuñoa Santiago (CL); and Ana Cecilia Morán, Ñuñoa Santiago (CL)
Assigned to GenoSUR, LLC, Miami, FL (US)
Filed by GenoSUR, LLC, Miami, FL (US)
Filed on Sep. 14, 2020, as Appl. No. 17/020,804.
Claims priority of provisional application 63/026,728, filed on May 19, 2020.
Prior Publication US 2021/0363567 A1, Nov. 25, 2021
Int. Cl. C12Q 1/6806 (2018.01); C12Q 1/6844 (2018.01); C12Q 1/6851 (2018.01); C12Q 1/686 (2018.01); C12Q 1/70 (2006.01)
CPC C12Q 1/6806 (2013.01) [C12Q 1/6844 (2013.01); C12Q 1/6851 (2013.01); C12Q 1/686 (2013.01); C12Q 1/70 (2013.01)] 14 Claims
 
1. A method for detecting genetic material, the method comprising:
(a) obtaining a stabilized biological sample, the stabilized biological sample comprising a biological sample and a transport solution, wherein the transport solution comprises a chaotropic agent and a chelating agent, and wherein the transport solution stabilizes nucleic acid molecules in the stabilized biological sample at room temperature, wherein the nucleic acid molecules comprise genetic material from a pathogen, a host, or a combination of the pathogen and the host;
(b) without prior extraction of the nucleic acid molecule present in the stabilized biological sample, mixing the stabilized biological sample with an activating solution to form a stabilized and activated biological sample, the activating solution comprising a buffer having pH from 6.8 to 8.2;
(c) combining the stabilized and activated biological sample to an amplification reaction mixture, wherein the amplification reaction mixture antagonizes the effect of inhibitors of enzymatic reactions, wherein the enzymatic reaction comprises DNA polymerization and reverse transcription of RNA, wherein the amplification reaction mixture comprises an amplification solution, DNA polymerase enzymes, a DNA polymerase buffer, and primer sets and probes specific for the nucleic acid molecules, wherein the amplification solution comprises a non-ionic surfactant at a concentration of 0.5 to 5 percent (vol./vol.);
(d) subjecting the amplification reaction mixture to a polymerase chain reaction (PCR), wherein the PCR amplifies the nucleic acid molecule;
(e) detecting the pathogen in the stabilized biological sample when a signal generated in the polymerase chain reaction surpasses a threshold signal for the polymerase chain reaction.