High-performance liquid chromatography to assess the effect of serum storage conditions on the detection of hepatitis C virus by the polymerase chain reaction.
The effect of various serum storage conditions on the detection of hepatitis C virus (HCV) by the polymerase chain reaction (PCR) was assessed. 50 microliters aliquots of serum from four HCV PCR positive patients were subjected, in triplicate, to: (a) storage at -20 degrees C for 1, 5, 10 days; (b) storage at room temperature (RT) for 1, 2, 7 days; (c) 1, 3, 5, and 10 successive freeze-thaw cycles; (d) incubation at 37 degrees C, and 56 degrees C for 1, 3, 24 h; and (e) storage in guanidium-thiocyanate extraction buffer at RT, and 4 degrees C for 1, 5, 10 days. PCR products were detected by agarose gel electrophoresis (AGE) and quantitatively by high-performance liquid chromatography (HPLC). Only storage in extraction buffer at RT for 5-10 days and incubation at 56 degrees C for 24 h appeared to result in a loss of > or = 50% of detectable HCV PCR product. Up to 10 successive freeze-thaw cycles, storage at -20 degrees C for up to 10 days or at RT for up to 7 days, storage in extraction buffer at RT for 1 day or at 4 degrees C for up to 10 days, and incubation at 37 degrees C for up to 24 h resulted in minimal PCR signal loss. HPLC was a reproducible method of detecting and quantitating HCV PCR products, and was more sensitive than AGE.
Quan
C M
CM
Department of Microbiology, Toronto Hospital, Ontario, Canada.
Krajden
M
M
Zhao
J
J
Chan
A W
AW
D001793:Blood Preservation; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004587:Electrophoresis, Agar Gel; D016174:Hepacivirus / Q000302:isolation & purification*; D006526:Hepatitis C / Q000097:blood / Q000382:microbiology; D006801:Humans; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000032:analysis; D013048:Specimen Handling
D012367:RNA, Viral