title: Multiplex Reverse Transcriptase-PCR for Simultaneous Detection of Hepatitis B, C, and E Virus.
abstract: BACKGROUND
  The hepatitis B virus (HBV), HCV, and HEV may occur as singly or concurrently in patients of different kind of liver disease. The rapid, reliable, and cost-effective screening of these pathogens is required for the large epidemiological studies. Therefore, a study has been planned to develop a multiplex Reverse Transcriptase-PCR assay which can be used for the screening of maximum number of pathogens at a time.
  
  
  METHODS
  To develop multiplex Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) assay for simultaneous detection of HBV, HCV, and HEV; the serum samples of 54 patients who were positive either singly or in co-infection with for HBV, HCV, and HEV serologically were screened by uniplex PCR/RT-PCR followed by multiplex RT-PCR for HBV, HCV, and HEV using specific primers. These primers can detect most genotypes of these viruses. Multiplex RT-PCR was done in one tube for the identification of viral DNA/RNA using a mixture of three pairs of specific primers for hepatitis B, C, and E viruses. Representative positive samples of these viruses by uniplex/multiplex RT-PCR were also confirmed by sequencing followed by alignment with reference strains sequence.
  
  
  RESULTS
  The specificity of multiplex PCR was 100% with high sensitivity 89%, 87%, and 74% for HBV, HCV, and HEV respectively. The sensitivity and specificity of RT-multiplex PCR demonstrated a good correlation with that of uniplex PCR.
  
  
  CONCLUSIONS
  The study suggests that multiplex RT-PCR can serve as a simple and reliable assay for rapid, sensitive, and cost-effective method for simultaneous detection of super-infections with HEV particularly in Asian countries as a cause of decompensation of chronic liver disease.
authors.affiliation: Department of Medicine, Maulana Azad Medical College, University of Delhi, New Delhi, India.
authors.forename: Gunjan
authors.initials: G
authors.lastname: Garg
authors.affiliation: Department of Biotechnology & Molecular Medicine, Pt. B.D. Sharma Post Graduate Institute of Medical Sciences, Rohtak, India.
authors.forename: Deepak
authors.initials: D
authors.lastname: Kumar
authors.affiliation: Department of Medicine, Maulana Azad Medical College, University of Delhi, New Delhi, India.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Asim
authors.affiliation: Department of Biosciences, Jamia Millia Islamia, New Delhi, India.
authors.forename: Syed Akhtar
authors.initials: SA
authors.lastname: Husain
authors.affiliation: Ambedkar Center for Biomedical Research, University of Delhi, New Delhi, India.
authors.forename: Bhudev C
authors.initials: BC
authors.lastname: Das
authors.affiliation: Department of Medicine, Maulana Azad Medical College, University of Delhi, New Delhi, India.
authors.forename: Premashis
authors.initials: P
authors.lastname: Kar
keywords: ELISA, enzyme linked immunosorbent assay; HAV, hepatitis A virus; HBV, hepatitis B virus; HCV, hepatitis C virus; HEV, hepatitis E virus; NATs, nucleic acid amplification technologies; NPV, negative predictive value; PCR, polymerase chain reaction; PPV, positive predictive value; RT-PCR, Reverse Transcriptase-Polymerase Chain Reaction; hepatitis B virus; hepatitis C virus; hepatitis E virus; liver diseases; multiplex PCR