Single-tube nested PCR with room-temperature-stable reagents.

Institut f~r Laboratoriumsund Transfusionsmedizin, Herzund Diabeteszentrum Nordrhein-Westfalen, Universit~itsklinik der Ruhr-Universit~it Bochum, Bad Oeynhausen, Germany The application of nested PCR in medical diagnostics implies that the same type of reaction is performed repetitively in the same laboratory over long periods of time, such as in the longitudinal monitoring of immunosuppressed patients for virus infections, (1) or in the monitoring of donated blood for transmissible infectious agents. (~-s) To ensure interassay conformity, it is desirable to use preformulated PCR reagent mixes that may be prepared in advance in batches and may be kept on stock, instead of preparing a fresh reagent mix for every PCR assay. False-positive reactions in PCR may arise from selfcontamination with amplified DNA ("carryover"). This problem is aggravated in nested PCR, because amplified DNA (first-round PCR product) has to be transferred manually into the tubes for the second-round PCR. To avoid this additional risk of carryover, it is desirable to perform first-round and second-round PCR in one reaction tube without opening it during the entire process.(6) We modified nested PCR so that it is performed in a single tube, using preformulated PCR reaction mixes embedded in a trehalose matrix. (7-1°) These preformulated mixes may be stored for I>6 months at ambient temperature. We compared the performance of this single-tube nested PCR with conventional nested PCR. Blood samples from different patients with virus infections were assayed, including infections with human immunodeficiency virus type 1 (HIV 1), hepatitis C virus (HCV), and cytomegalovirus (CMV).