Development and evaluation of a sensitive enzyme-linked oligonucleotide-sorbent assay for detection of polymerase chain reaction-amplified hepatitis C virus of genotypes 1-6.
A high-throughput polymerase chain reaction (PCR)-based enzyme-linked oligonucleotide-sorbent assay (ELOSA) was developed for use in the diagnostic testing of serum from patients who may be infected with different hepatitis C virus (HCV) genotypes. Twelve genotype-specific 5'-aminated DNA-coated probes were designed based on the variable 5'-untranslated region sequences of the HCV genotypes 1-6. Using 100 clinical serum samples, the performance of the PCR-ELOSA method was compared with Roche's COBAS Amplicor HCV Monitor V2.0 assay and the VERSANT HCV genotype assay (LiPA), and the overall agreement was 99% at the level of HCV genotypes with a detection range of 2.0 x 10(2) to 1.0 x 10(7)IU/ml for PCR-ELOSA. The PCR-ELOSA was more comprehensive as demonstrated by the fact that approximately 20% of the samples with different subtypes could be discriminated by this method but not by LiPA. In addition, the PCR-ELOSA system showed high accuracy (CV<or=6.36%) and even higher reproducibility (CV<or=5.55%). Thus, this novel PCR-ELOSA system provides a sensitive and versatile alternative to current HCV detection assays.
Institute of Molecular and Cellular Biology & Department of Life Science, National Tsing Hua University, No. 101, Section 2, Kuang-Fu Road, Hsinchu 30013, Taiwan; General Biologicals Corporation, No. 6, Innovation First Road, Hsinchu Science Park, Hsinchu 30076, Taiwan.
Rong-Yuan
RY
Huang
Graduate Institute of Molecular Systems Biomedicine, China Medical University, No. 91, Hsueh-Shih Road, Taichung 40402, Taiwan.
Hao-Teng
HT
Chang
Institute of Molecular and Cellular Biology & Department of Life Science, National Tsing Hua University, No. 101, Section 2, Kuang-Fu Road, Hsinchu 30013, Taiwan.
Chung-Yu
CY
Lan
Department of Computer Science and Engineering & Center for Marine Bioscience and Biotechnology, National Taiwan Ocean University, No. 2, Peining Road, Keelung 20224, Taiwan.
Tun-Wen
TW
Pai
General Biologicals Corporation, No. 6, Innovation First Road, Hsinchu Science Park, Hsinchu 30076, Taiwan.
Chao-Nan
CN
Wu
General Biologicals Corporation, No. 6, Innovation First Road, Hsinchu Science Park, Hsinchu 30076, Taiwan.
Chung-Mei
CM
Ling
Institute of Molecular and Cellular Biology & Department of Life Science, National Tsing Hua University, No. 101, Section 2, Kuang-Fu Road, Hsinchu 30013, Taiwan.
Margaret Dah-Tsyr
MD
Chang
D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005784:Gene Amplification; D005838:Genotype; D014943:Global Health; D016174:Hepacivirus / Q000235:genetics*; D006526:Hepatitis C / Q000453:epidemiology; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D015995:Prevalence; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity
D017931:DNA Primers; D012367:RNA, Viral