Detection and genotyping of human group A rotaviruses by oligonucleotide microarray hybridization

Abstract

A rapid and reliable method for the identification of five clinically relevant G genotypes (G1 to G4 and G9) of human rotaviruses based on oligonucleotide microarray hybridization has been developed. The genotype-specific oligonucleotides immobilized on the surface of glass slides were selected to bind to the multiple target regions within the VP7 gene that are highly conserved among individual rotavirus genotypes. Rotavirus cDNA was amplified in a PCR with primers common to all group A rotaviruses. A second round of nested PCR amplification was performed in the presence of indodicarbocyanine-dCTP and another pair of degenerate primers also broadly specific for all genotypes. The use of one primer containing 5'-biotin allowed us to prepare fluorescently labeled single-stranded hybridization probe by binding of another strand to magnetic beads. The identification of rotavirus genotype was based on hybridization with several individual genotype-specific oligonucleotides. This approach combines the high sensitivity of PCR with the selectivity of DNA-DNA hybridization. The specificity of oligonucleotide microchip hybridization was evaluated by testing 20 coded rotavirus isolates from different geographic areas for which genotypes were previously determined by conventional methods. Analysis of the coded specimens showed that this microarray-based method is capable of unambiguous identification of all rotavirus strains. Because of the presence of random mutations, each individual virus isolate produced a unique hybridization pattern capable of distinguishing different isolates of the same genotype and, therefore, subgenotype differentiation. This strain information indicates one of several advantages that microarray technology has over conventional PCR techniques.

FIG. 1.

Diversity profile of rotavirus VP7 gene based on analysis of nucleotide sequences of 150 individual strains. The diversity score is calculated by counting the number of mismatches between all combinations of the sequences at each position in the array of aligned sequences. Matching nucleotides are those that can bind the same complementary base. Arrows show the positions of optimized primers for synthesis of fluorescent samples for this microarray assay.

FIG. 2.

PCR amplification of 20 coded rotavirus templates with different pairs of designed primers: LID1 and G-373 (A), LID1 and G-529 (B), and LID1 and G-922 (C). Lane M, 100-bp molecular size markers.

FIG. 3.

Microarray hybridization patterns of 20 rotavirus samples under code. Microarray images (Cy5 fluorescence signal) are sorted into separate rows based on their G genotype. The sample code is shown in the left bottom corner of each image. The typical Cy3 image (QC) of the array is shown at the bottom of the figure, showing the layout of oligonucleotides in the microarray. Random 21-base oligonucleotides are spotted into the first two positions of row C, whereas QCprb oligonucleotide is spotted into positions 9 and 10.

FIG. 4.

Quantitative fluorescence (Cy5) profiles of rotavirus microarrays. Normalized fluorescent signals from each oligonucleotide are shown on the y axis. Numbered locations of probes are shown on the x axis. All G1 genotype-specific oligonucleotides have numbers from 1 to 10, G2 genotype from 11 to 19, G3 genotype from 21 to 29, G4 genotype from 31 to 39, and G4 genotype from 41 to 50. All fluorescence data were obtained from two or three independent hybridizations on different microchips.