AIMS: To compare the nested polymerase chain reaction (PCR) assay for the detection of cytomegalovirus (CMV) DNA in peripheral blood leucocytes and plasma obtained from heparinised blood; to determine the efficiency of various DNA extraction methods to minimise inhibition of plasma PCR and their effect on the sensitivity of plasma PCR; to determine the inhibitory effect of heparin, dextran, and EDTA on the CMV PCR assay. METHODS: 217 heparinised blood specimens from 58 bone marrow transplant patients were processed and the sensitivities and specificities of the PCR assays using peripheral blood leucocytes and plasma (with simple, Instagene, and Geneclean extraction methods) were compared to those of conventional CMV culture. In a separate experiment, dilutions of heparin, dextran, and EDTA were included in PCR assays. RESULTS: The detection of CMV DNA using peripheral blood leucocytes for PCR assay was significantly more sensitive (100%) than when using plasma (60%). Instagene and Geneclean extraction removed inherent inhibition but did not improve the sensitivity of the plasma PCR reaction. Heparin had an inhibitory effect on PCR. CONCLUSIONS: PCR assay using peripheral blood leucocytes is better than plasma for guiding the prescription of ganciclovir to bone marrow transplant patients. Heparin is inhibitory to the plasma PCR reaction.
AIMS: To compare the nested polymerase chain reaction (PCR) assay for the detection of cytomegalovirus (CMV) DNA in peripheral blood leucocytes and plasma obtained from heparinised blood; to determine the efficiency of various DNA extraction methods to minimise inhibition of plasma PCR and their effect on the sensitivity of plasma PCR; to determine the inhibitory effect of heparin, dextran, and EDTA on the CMV PCR assay. METHODS: 217 heparinised blood specimens from 58 bone marrow transplant patients were processed and the sensitivities and specificities of the PCR assays using peripheral blood leucocytes and plasma (with simple, Instagene, and Geneclean extraction methods) were compared to those of conventional CMV culture. In a separate experiment, dilutions of heparin, dextran, and EDTA were included in PCR assays. RESULTS: The detection of CMV DNA using peripheral blood leucocytes for PCR assay was significantly more sensitive (100%) than when using plasma (60%). Instagene and Geneclean extraction removed inherent inhibition but did not improve the sensitivity of the plasma PCR reaction. Heparin had an inhibitory effect on PCR. CONCLUSIONS: PCR assay using peripheral blood leucocytes is better than plasma for guiding the prescription of ganciclovir to bone marrow transplant patients. Heparin is inhibitory to the plasma PCR reaction.
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