A method for amplifying a target nucleic acid from a biological sample in a container, the container comprising: a flexible material defining a plurality of fluidly connected reaction zones fluidly connected by channels, the fluidly connected reaction zones including at least a first-stage PCR reaction zone; the container comprising an amplification mixture for first-stage PCR in the first-stage PCR reaction zone, wherein the amplification mixture includes a thermostable polymerase, primers configured for amplification of a target nucleic acid, and a molecular crowder for increasing the effective concentration of primers and polymerase, the method comprising the steps of: introducing the biological sample into the first-stage PCR reaction zone of the container; mixing the thermostable polymerase, the primers configured for amplification of the target nucleic acid, and the molecular crowder with the biological sample to create the amplification mixture, wherein the molecular crowder is provided in an amount that is at least 3% w/v of the amplification mixture; and amplifying the target nucleic acid by polymerase chain reaction by thermally cycling the amplification mixture through a plurality of at least two-step amplification cycles comprising at least a denaturation temperature and an annealing temperature, wherein each cycle is completed in a cycle time less than 40 seconds per cycle; wherein the molecular crowder is a Ficoll or a mixture of Ficolls. a flexible material defining a plurality of fluidly connected reaction zones fluidly connected by channels, the fluidly connected reaction zones including at least a first-stage PCR reaction zone; the container comprising an amplification mixture for first-stage PCR in the first-stage PCR reaction zone, wherein the amplification mixture includes a thermostable polymerase, primers configured for amplification of a target nucleic acid, and a molecular crowder for increasing the effective concentration of primers and polymerase, the method comprising the steps of: introducing the biological sample into the first-stage PCR reaction zone of the container; mixing the thermostable polymerase, the primers configured for amplification of the target nucleic acid, and the molecular crowder with the biological sample to create the amplification mixture, wherein the molecular crowder is provided in an amount that is at least 3% w/v of the amplification mixture; and amplifying the target nucleic acid by polymerase chain reaction by thermally cycling the amplification mixture through a plurality of at least two-step amplification cycles comprising at least a denaturation temperature and an annealing temperature, wherein each cycle is completed in a cycle time less than 40 seconds per cycle; wherein the molecular crowder is a Ficoll or a mixture of Ficolls. introducing the biological sample into the first-stage PCR reaction zone of the container; mixing the thermostable polymerase, the primers configured for amplification of the target nucleic acid, and the molecular crowder with the biological sample to create the amplification mixture, wherein the molecular crowder is provided in an amount that is at least 3% w/v of the amplification mixture; and amplifying the target nucleic acid by polymerase chain reaction by thermally cycling the amplification mixture through a plurality of at least two-step amplification cycles comprising at least a denaturation temperature and an annealing temperature, wherein each cycle is completed in a cycle time less than 40 seconds per cycle; wherein the molecular crowder is a Ficoll or a mixture of Ficolls. <EOS>