Multiplexed polymerase chain reaction for genetic sequence analysis

摘要

A PCR method that comprises the steps: (1) extracting nucleic acids from a clinical sample obtained from an organism to produce a target sample containing background DNA of the organism and suspected of containing one or more pathogen nucleic acids from a predefined set of pathogens; (2) adding to the target sample plurality of PCR primers corresponding to genes found in the predefined set of pathogens; (3) performing a polymerase chain reaction on the combined target sample and PCR primers to amplify subset of the nucleic acids that correspond to the genes to produce an amplified sample; The primers include at least one primer pair for each pathogen and comprise a tail sequence that is non-complementary to the DNA of any of the predefined set of pathogens and to the DNA of the species of the organism. The concentration of at least one primer in the polymerase chain reaction is no more that about 100 nM. The tail sequence can be SEQ ID NO: 1 or SEQ ID NO: 2.