title: Development of a Multiplex Real-Time PCR Assay for the Detection of Treponema pallidum, HCV, HIV-1, and HBV.
abstract: Treponema pallidum, hepatitis C virus (HCV), human immunodeficiency virus (HIV)-1, and hepatitis B virus (HBV) are major causes of sexually transmitted diseases passed through blood contact. The development of a sensitive and efficient method for detection is critical for early diagnosis and for large-scale screening of blood specimens in China. This study aims to establish an assay to detect these pathogens in clinical serum specimens. We established a TaqMan-locked nucleic acid (LNA) real-time polymerase chain reaction (PCR) assay for rapid, sensitive, specific, quantitative, and simultaneous detection and identification. The copy numbers of standards of these 4 pathogens were quantified. Standard curves were generated by determining the mean cycle threshold values versus 10-fold serial dilutions of standards over a range of 10(6) to 10(1) copies/μL, with the lowest detection limit of the assay being 10(1) copies/μL. The assay was applied to 328 clinical specimens and compared with enzyme-linked immunosorbent assay (ELISA) and commercial nucleic acid testing (NAT) methods. The assay identified 39 T. pallidum-, 96 HCV-, 13 HIV-1-, 123 HBV-, 5 HBV/HCV-, 1 T. pallidum/HBV-, 1 HIV-1/HCV-, and 1 HIV-1/T. pallidum-positive specimens. The high sensitivity of the assay confers strong potential for its use as a highly reliable, cost-effective, and useful molecular diagnostic tool for large-scale screening of clinical specimens. This assay will assist in the study of the pathogenesis and epidemiology of sexually transmitted blood diseases.
authors: ABSL-III Laboratory at Center for Animal Experiment, State Key Laboratory of Virology, Wuhan University School of Medicine.
authors: Li
authors: L
authors: Zhou
authors: Rui
authors: R
authors: Gong
authors: Xuan
authors: X
authors: Lu
authors: Yi
authors: Y
authors: Zhang
authors: Jingfeng
authors: J
authors: Tang
mesh_terms: D002681:China; D004269:DNA, Bacterial / Q000097:blood; D004279:DNA, Viral / Q000097:blood; D015658:HIV Infections / Q000175:diagnosis*; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification; D016174:Hepacivirus / Q000235:genetics / Q000302:isolation & purification; D006509:Hepatitis B / Q000175:diagnosis*; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification; D006526:Hepatitis C / Q000175:diagnosis*; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000097:blood; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013587:Syphilis / Q000175:diagnosis*; D014210:Treponema pallidum / Q000235:genetics / Q000302:isolation & purification
chemical_list: D004269:DNA, Bacterial; D004279:DNA, Viral; D012367:RNA, Viral