Evaluation of different genomic regions of Rotavirus A for development of real time PCR

J Virol Methods. 2019 Apr:266:65-71. doi: 10.1016/j.jviromet.2019.01.017. Epub 2019 Jan 30.

Abstract

The nucleotide alignment of all 11 genes of human Rotavirus A (RVA) strains revealed suitability of NSP2, NSP3 and VP6 genes for the development of real time PCR (qRT-PCR). Evaluation of qRT-PCR assays using known rotavirus ELISA positive and negative fecal specimens showed non-overlapping ranges of Mean ±3SD cycle threshold (Ct) values for NSP3 and VP6 based assays. Using serial dilutions of purified RVA, high sensitivity of VP6 qRT-PCR assay (1.95 × 10-5 pg/μL of RNA) was recorded as compared to NSP2 and NSP3 qRT-PCR assays (1.95 × 10-4 pg/μL of RNA). Further, evaluation of the VP6 qRT-PCR assay involving 266 fecal specimens and frequency polygon analysis of the data indicated cut-off value of 35 for Ct with high sensitivity (126/131, 96%) and specificity (12/12, 100%). This VP6 qRT-PCR assay will be a useful diagnostic tool to evaluate clinical presentations in rotaviral gastroenteritis under different conditions such as breast feeding and administration of rotavirus vaccines.

Keywords: Acute gastroenteritis; Real time polymerase chain reaction; Rotavirus A; Sensitivity; VP6 gene.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigens, Viral / genetics
  • Capsid Proteins / genetics
  • Child, Preschool
  • Feces / virology
  • Gastroenteritis / virology
  • Genome, Viral*
  • Genotype
  • Humans
  • Phylogeny
  • RNA, Viral / genetics
  • Real-Time Polymerase Chain Reaction*
  • Rotavirus / genetics*
  • Rotavirus Infections / virology
  • Sensitivity and Specificity
  • Viral Nonstructural Proteins / genetics

Substances

  • Antigens, Viral
  • Capsid Proteins
  • NSP3 protein, Rotavirus
  • RNA, Viral
  • VP6 protein, Rotavirus
  • Viral Nonstructural Proteins