title: Comparison of a conventional and nested PCR for diagnostic confirmation and genotyping of Orientia tsutsugamushi.
abstract: A nested polymerase chain reaction (PCR) targeting the 56-kDa antigen gene is currently the most commonly used molecular technique for confirmation of scrub typhus and genotyping of Orientia tsutsugamushi. In this study, we have compared the commonly used nested PCR (N-PCR) with a single-step conventional PCR (C-PCR) for amplification and genotyping. Eschar samples collected from 24 patients with scrub typhus confirmed by IgM enzyme-linked immunosorbent assay were used for DNA extraction following which amplifications were carried out using nested and C-PCR methods. The amplicons were sequenced and compared to other sequences in the database using BLAST. Conventional PCR showed a high positivity rate of 95.8% compared to the 75% observed using N-PCR. On sequence analysis, the N-PCR amplified region showed more variation among strains than the C-PCR amplified region. The C-PCR, which is more economical, provided faster and better results compared to N-PCR.
authors.affiliation: Medicine Unit I and Infectious Diseases, Christian Medical College, Vellore, Tamil Nadu 632004, India.
authors.forename: Jeshina
authors.initials: J
authors.lastname: Janardhanan
authors.affiliation: Department of Clinical Microbiology, Christian Medical College, Vellore, Tamil Nadu 632004, India.
authors.forename: John Antony Jude
authors.initials: JA
authors.lastname: Prakash
authors.affiliation: Medicine Unit I and Infectious Diseases, Christian Medical College, Vellore, Tamil Nadu 632004, India.
authors.forename: Ooriapadickal C
authors.initials: OC
authors.lastname: Abraham
authors.affiliation: Medicine Unit I and Infectious Diseases, Christian Medical College, Vellore, Tamil Nadu 632004, India. Electronic address: georgemvarghese@hotmail.com.
authors.forename: George M
authors.initials: GM
authors.lastname: Varghese
mesh_terms: D006801:Humans; D058889:Molecular Typing / Q000379:methods*; D012285:Orientia tsutsugamushi / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012612:Scrub Typhus / Q000175:diagnosis / Q000382:microbiology*
keywords: 56 kDa TSA; Conventional PCR; Genotyping; Nested PCR; Scrub typhus