Dot hybridization assay for distinction of rotavirus serotypes

Abstract

We have developed a hybridization assay that permits distinction of rotavirus serotypes 1, 2, 3, and 4. The serotype of rotaviruses from stool samples or tissue culture was recognized by hybridization of specific probes to (i) blots of viral double-stranded RNAs electrophoresed in agarose gels (Northern blots) or (ii) heatdenatured double-stranded RNAs directly dotted on nylon membranes. The probes consisted of 32P-labeled cDNA synthesized by reverse transcription of in vitro derived rotavirus mRNA from rotavirus serotypes 1 to 4. To prepare these probes, mRNAs were primed with a 17-mer nucleotide common to all four serotypes whose sequence is complementary to bases 375 to 391 of the rotavirus gene encoding the VP7 glycoprotein (gene 8 or 9 depending on the rotavirus strain). The resulting downstream transcripts encompassed areas of major sequence divergence among the four serotypes. Hybridization at high stringency (50°C, 50% formamide, 4x SSC [lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate]) was performed for 16 to 48 h. Autoradiograms of the washed membranes allowed recognition of the rotavirus serotype present in the blotted or dotted specimens since each of them hybridized preferentially to one of the four probes. Twenty-four laboratory specimens and 103 clinical specimens from Washington, D.C., Venezuela, and Chile were "serotyped" with this assay. The results were similar to those obtained with a monoclonal antibody serotyping assay.

Further characterisation of human rotaviruses isolated from asymptomatically infected neonates in South Africa

“…These ss transcripts served as the template for producing radiolabelled probes to the VP7 (serotype-specific) and the VP4 (type-specific for the virulence phenotypes of the virus) genes. Avian myeloblastosis virus (AMV) reverse transcriptase was used to produce the copy DNA probes with synthetic oligonucleotide primers directed at nucleotides 375 to 391 of the VP7 gene mRNA (5' GATCCTGTTGGCCATCC 3') and nucleotides 551 to 530 of the VP4 mRNA (5' GGTGTTTCAC-CATGAAACGTCC 3') as developed by Flores et al [1989] and kindly donated to this laboratory. Approximately 100 ng of the VP4 or VP7 primers were mixed with 1-4 pg of total ssRNA.…”

“…Several identical membranes were prepared from each set of RNAs and each membrane contained controls of known VP7 and VP4 specificity. Hybridisation was performed as described by Flores et al [1989]. Briefly, the membranes were pre-hybridised for 1-2 h r a t 51°C in a mixture containing 50% formamide, 4 x SSC, 40 mM sodium buffer (pH 6,5), 0,2%) SDS, and 2 x Denhardt's solution.…”

Development of specific nucleic acid probes for the differentiation of porcine rotavirus serotypes

“…Dot blot hybridization assays have been utilized by a number of groups both for the detection of human rotavirus in clinical samples (Flores et al, 1983;Pedley and Mc-Crae, 1984) and for the detection and characterization of enteric coronavirus infection of baby pigs (Shockley et al, 1987 ). Additional studies have been reported in which improved cDNA probes of gene-specific segments of the rotavirus genome were used to characterize human rotavirus by subgroup and serotype (Dimitrov et al, 1985;Lin et al, 1987;Flores et al, 1989).…”

“…Practical application of the dot blot hybridization assay may require some modification. These include densitometry to minimize subjective comparisons of the reactions, the use of probes representing gene 4 that encodes VP4, and the construction of cDNA probes from selected regions in which highly conserved bases at the 5' end have been removed (Flores et al, 1989). Specific regions of DNA can now be amplified using synthetic oligonucleotides by polymerase chain reaction (PCR) and used as probes.…”

Rapid and accurate viral diagnosis

“…In another study spot hybridization was found to be more sensitive than culture for detection of CMV in buffy coats (Spector et al, 1984). When applied to viruses not routinely isolated, such as rotavirus (Flores et al, 1983), enteric adenoviruses (Stalhandske et al, 1983(Stalhandske et al, , 1985Takiff et al, 1985), parvoviruses (Clewley, 1985;Anderson et al, 1985); papovaviruses (Gibson et al, 1985;Wickenden et al, 1985) and Epstein-Barr virus (Andiman et al, 1983), spot hybridization could prove useful. Detection of HBV-DNA in serum by spot hybridization correlates with active virus replication (Carloni et al, 1987).…”