title: Comparison of multiplex RT-PCR and real-time HybProbe assay for serotyping of dengue virus using reference strains and clinical samples from India.
abstract: BACKGROUND
  Dengue virus serotyping is crucial from clinical management and epidemiological point of view.
  
  
  OBJECTIVE
  To compare efficacy of two molecular detection and typing methods, namely, multiplex reverse transcription polymerase chain reaction (RT-PCR) and real-time Hybprobe assay using a panel of known dilution of four reference Dengue virus strains and a panel of sera collected from clinically suspected dengue patients.
  
  
  METHODS
  This study was conducted at a tertiary-care teaching hospital in Delhi, India.
  
  
  METHODS
  Dengue serotype specific virus strains were used as prototypes for serotyping assays. Viral load was quantified by quantitative real time reverse transcription polymerase chain reaction (qRT-PCR). Acute phase serum samples were collected from 79 patients with clinically suspected Dengue fever on their first day of presentation during September-October 2012. Viral RNA from serum and cell culture supernatant was extracted. Reverse transcription was carried out. Quantitative detection of DENV RNA from reference strain culture supernatants and each of the 79 patient samples by real-time PCR was performed using light cycler Taqman master mix kit. Serotyping was done by multiplex RT-PCR assay and Hybprobe assay.
  
  
  RESULTS
  The multiplex RT-PCR assay, though found to be 100% specific, couldn't serotype either patient or reference strains with viral load less than 1000 RNA copies/ml. The Hybprobe assay was found to have 100% specificity and had a lower limit of serotype detection of merely 3.54 RNA copies/ml.
  
  
  CONCLUSIONS
  HybProbe assay has an important role especially in situations where serotyping is to be performed in clinical samples with low viral load.
authors: Department of Microbiology, Maulana Azad Medical College, New Delhi, India.
authors: Anita
authors: A
authors: Chakravarti
authors: Department of Microbiology, Maulana Azad Medical College, New Delhi, India.
authors: Mayank Singh
authors: MS
authors: Chauhan
authors: Department of Microbiology, ESI-PGIMSR and ESIC Medical College, Kolkata, West Bengal, India.
authors: Sayantan
authors: S
authors: Banerjee
authors: Department of Microbiology, University College of Medical Sciences and GTB Hospital, New Delhi, India.
authors: Priyamvada
authors: P
authors: Roy
mesh_terms: D003715:Dengue / Q000821:virology; D003716:Dengue Virus / Q000145:classification* / Q000235:genetics*; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D007194:India; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000097:blood / Q000235:genetics / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012703:Serotyping / Q000379:methods*; D062606:Tertiary Care Centers; D019562:Viral Load
chemical_list: D012367:RNA, Viral