Open AccessQuantitation of hepatitis B virus DNA in serum by ammonium sulfate precipitation and molecular hybridization
Author(s) -
Wen Long T.,
Henneberger Michael,
Nguyen Nicole,
McPherson Richard A.
Publication year - 1994
Publication title -
journal of clinical laboratory analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 50
eISSN - 1098-2825
pISSN - 0887-8013
DOI - 10.1002/jcla.1860080109
Subject(s) - ammonium sulfate , ammonium sulfate precipitation , hepatitis b virus , precipitation , virology , dna , chemistry , ammonium , virus , microbiology and biotechnology , biology , biochemistry , chromatography , physics , organic chemistry , enzyme , size exclusion chromatography , meteorology
We developed a method to quantitate hepatitis B virus (HBV) DNA in serum by ammonium sulfate fractionation and DNA hybridization. Serum samples were precipitated with 45% saturated ammonium sulfate, resuspended in buffer, and spotted on a nylon membrane. Following denaturation in alkali, HBV DNA sequences on the membrane were detected by hybridization with a 32 P‐labeled DNA probe of the entire HBV genome. Bound radioactivity was measured with liquid scintillation counting. Ammonium sulfate fractionation of positive samples increased assay sensitivity by 10–30‐fold compared to no treatment. Sensitivity for detection of cloned HBV DNA added to negative serum was 0.2 pg. Recovery of cloned HBV DNA added to negative serum before fractionation was equivalent to direct spotting of DNA onto the membrane in the absence of serum. This method enhanced HBV DNA recovery from serum into small volumes, thereby expanding the potential analytic range of spot hybridization assays. © 1994 Wiley‐Liss, Inc.