RNA target loss during solid phase hybridization of body fluids--a quantitative study.
Recent applications of nucleic acid hybridization to the diagnosis of viral infections are limited by the sensitivity of the assay system and, hence, by the amount of target viral nucleic acid in the specimen. In the case of single-stranded RNA viruses, RNase activity in body fluids poses a potential obstacle to optimizing sensitivity. We added radioactively labelled, purified, single-stranded enteroviral RNA to various body fluids and noted significant loss of this 'target' RNA during the course of routine hybridization procedures. We confirmed that RNase activity was responsible for much of the target loss. Further characterization of the RNase activity found it to be rapidly acting (less than 15 s), concentrated (equally active at 10-fold dilution of body fluid) and potent--resulting in RNA breakdown products too small to be retained by any of six commercially available membrane filters. The RNase was active only in solution and was effectively inhibited by treatment of the body fluid with inhibitors prior to contact of the fluid with RNA. In contrast, when RNA within virions was added to body fluids, it was largely retained by membrane filters during the hybridization procedure. We conclude that detection of single-stranded RNA viruses in body fluids will depend upon the quantity of intact virions in the specimen. RNase inhibitors should be immediately added to body fluid specimens after collection to minimize the loss of RNA which will occur if virions are disrupted in transport and handling of the specimen.
Rotbart
H A
HA
Department of Pediatrics, University of Colorado School of Medicine, Denver 80262.
Levin
M J
MJ
Murphy
N L
NL
Abzug
M J
MJ
D000653:Amniotic Fluid / Q000201:enzymology; D000818:Animals; D001826:Body Fluids / Q000032:analysis* / Q000201:enzymology; D029822:Enterovirus B, Human / Q000235:genetics; D009693:Nucleic Acid Hybridization; D012367:RNA, Viral / Q000032:analysis* / Q000378:metabolism; D012260:Ribonucleases / Q000097:blood / Q000134:cerebrospinal fluid / Q000378:metabolism / Q000652:urine
D012367:RNA, Viral; D012260:Ribonucleases