AUTOMATED CLOSED-VESSEL SYSTEM FOR IN-VITRO DIAGNOSTICS BASED ON POLYMERASE CHAIN-REACTION

An automated system for polymerase chain reaction (PCR) amplification and detection combats false-positive results caused by ''PCR product c arryover.'' The system uses a single vessel for both PCR amplification and the subsequent detection of PCR products, eliminating the need to handle PCR products in an open environment and risk product carryover . The sample and PCR reagents are introduced into one compartment with in the vessel, and amplification occurs as they are thermally cycled. Other compartments contain the reagents for detection of PCR products. Pressure from a roller provides for sequential delivery of the conten ts of the compartments to a detection area. The PCR products are bioti nylated at their 5' ends during amplification through the use of bioti nylated primers. After delivery to the detection area, they are specif ically captured by hybridization with immobilized oligonucleotide prob es. Subsequent reaction with streptavidin-horseradish peroxidase conju gate forms a complex that catalyzes dye formation from a dye precursor . Wash steps minimize nonspecific background. This format is amenable to multiplexing, permitting internal controls, speciation of bacteria, typing of viruses, and panel testing. An HIV assay performed with thi s system demonstrated 100% sensitivity and 95% specificity for 64 pati ents' samples relative to a conventional PCR assay based on P-32 solut ion hybridization. Similarly, an automated closed-vessel assay of cyto megalovirus exhibited 97.5% sensitivity and 100% specificity.