The heteroduplex mobility assay (HMA) as a pre-sequencing screen for Norwalk-like viruses.
Molecular epidemiological studies of Norwalk-like viruses (NLVs), previously known as small round structured viruses (SRSVs), are dependant currently on DNA sequencing of PCR amplicons, which is expensive and time consuming. The Heteroduplex Mobility assay (HMA) was evaluated as a method for identification of PCR amplicons from the commonly circulating NLV strains without DNA sequencing. The procedure was developed for use with two reference strains, a Mexico virus-like strain (MXV-like; Hu¿NLV¿RBH¿1993¿UK) and the Grimsby virus strain (Hu¿NLV¿Gimsby¿1995¿UK), and was optimised with regards to the annealing and electrophoresis conditions and the electrophoresis gel matrix. Using the optimised conditions, amplicons of less than 90% sequence identity formed visible heteroduplexes, allowing the strains to be placed into three categories; Mexico-like, Grimsby-like and non-Mexico virus/non-Grimsby virus strains. Outbreak strains 'genotyped' previously by DNA sequencing as Mexico virus or Grimsby virus were identified correctly by the heteroduplex mobility assay. The procedure was applied prospectively to strains from 130 outbreaks occurring in the UK between 1997 and 1998. Heteroduplex mobility assay was successful on 120 (92%) strains of which 68 (57%) were GRV-like strains, three (2.5%) were Mexico virus-like strains and 49 (41%) were categorised as non- Mexico/non-Grimsby virus strains. Amplicons from 50 of the 120 strains were sequenced and there was perfect correlation between the heteroduplex mobility assay categorisation and phylogenetic analysis. HMA offers a rapid, robust and far cheaper alternative to sequencing for the identification of prevalent Norwalk-like virus genotypes for molecular epidemiological studies.
Mattick
K L
KL
Regional Virology Laboratory, Leeds Public Health Laboratory, UK.
Green
J
J
Punia
P
P
Belda
F J
FJ
Gallimore
C I
CI
Brown
D W
DW
D017250:Caliciviridae Infections / Q000821:virology*; D020180:Heteroduplex Analysis; D006801:Humans; D009663:Norwalk virus / Q000145:classification* / Q000235:genetics; D009692:Nucleic Acid Heteroduplexes / Q000032:analysis; D010802:Phylogeny; D012367:RNA, Viral / Q000235:genetics; D006113:United Kingdom
D009692:Nucleic Acid Heteroduplexes; D012367:RNA, Viral