title: Hepatitis C Virus RNA Real-Time Quantitative RT-PCR Method Based on a New Primer Design Strategy.
abstract: Viral nucleic acids are unstable when improperly collected, handled, and stored, resulting in decreased sensitivity of currently available commercial quantitative nucleic acid testing kits. Using known unstable hepatitis C virus RNA, we developed a quantitative RT-PCR method based on a new primer design strategy to reduce the impact of nucleic acid instability on nucleic acid testing. The performance of the method was evaluated for linearity, limit of detection, precision, specificity, and agreement with commercial hepatitis C virus assays. Its clinical application was compared to that of two commercial kits--Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) and Kehua. The quantitative RT-PCR method delivered a good performance, with a linearity of R(2) = 0.99, a total limit of detection (genotypes 1 to 6) of 42.6 IU/mL (95% CI, 32.84 to 67.76 IU/mL), a CV of 1.06% to 3.34%, a specificity of 100%, and a high concordance with the CAP/CTM assay (R(2) = 0.97), with a means ± SD value of -0.06 ± 1.96 log IU/mL (range, -0.38 to 0.25 log IU/mL). The method was superior to commercial assays in detecting unstable hepatitis C virus RNA (P < 0.05). This quantitative RT-PCR method can effectively eliminate the influence of RNA instability on nucleic acid testing. The principle of primer design strategy may be applied to the detection of other RNA or DNA viruses.
authors.affiliation: National Center for Clinical Laboratories, Beijing Hospital, Beijing, People's Republic of China; Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, People's Republic of China.
authors.forename: Lida
authors.initials: L
authors.lastname: Chen
authors.affiliation: Department of Rheumatology, China-Japan Friendship Hospital, Beijing, People's Republic of China.
authors.forename: Wenli
authors.initials: W
authors.lastname: Li
authors.affiliation: Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, People's Republic of China.
authors.forename: Kuo
authors.initials: K
authors.lastname: Zhang
authors.affiliation: Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, People's Republic of China.
authors.forename: Rui
authors.initials: R
authors.lastname: Zhang
authors.affiliation: National Center for Clinical Laboratories, Beijing Hospital, Beijing, People's Republic of China; Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, People's Republic of China.
authors.forename: Tian
authors.initials: T
authors.lastname: Lu
authors.affiliation: National Center for Clinical Laboratories, Beijing Hospital, Beijing, People's Republic of China; Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, People's Republic of China.
authors.forename: Mingju
authors.initials: M
authors.lastname: Hao
authors.affiliation: National Center for Clinical Laboratories, Beijing Hospital, Beijing, People's Republic of China; Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, People's Republic of China.
authors.forename: Tingting
authors.initials: T
authors.lastname: Jia
authors.affiliation: Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, People's Republic of China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Sun
authors.affiliation: Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, People's Republic of China.
authors.forename: Guigao
authors.initials: G
authors.lastname: Lin
authors.affiliation: Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, People's Republic of China.
authors.forename: Lunan
authors.initials: L
authors.lastname: Wang
authors.affiliation: National Center for Clinical Laboratories, Beijing Hospital, Beijing, People's Republic of China; Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, People's Republic of China. Electronic address: jmli@nccl.org.cn.
authors.forename: Jinming
authors.initials: J
authors.lastname: Li
mesh_terms: D005838:Genotype; D016174:Hepacivirus / Q000235:genetics*; D006526:Hepatitis C / Q000175:diagnosis* / Q000821:virology; D006801:Humans; D057230:Limit of Detection; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D012313:RNA / Q000235:genetics*; D020871:RNA Stability / Q000235:genetics*; D012367:RNA, Viral / Q000097:blood* / Q000235:genetics / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D019562:Viral Load
chemical_list: C034477:RNA primers; D012367:RNA, Viral; D012313:RNA