Related Experiment Video
Updated: Aug 18, 2026
Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses
Published on: December 3, 2011
Use of non-radioactive probes for VP4 typing of human rotaviruses
1Department of Gastroenterology, Royal Children's Hospital, Parkville, Victoria, Australia. gastro@cryptic.rch.unimelb.edu.au
Abstract:
The rotavirus outer capsid proteins VP4 and VP7 determine the P- and G-serotypes, respectively, of the virus. Three types of VP4 protein are commonly found in human rotaviruses (P4, P6 and P8) which are encoded by distinct VP4 gene alleles. We developed a non-radioactive Northern hybridization method for the P-genotyping of rotavirus field isolates. Double-stranded RNA was isolated from faecal specimens of rotavirus positive patients. Digoxigenin (DIG)-labelled cDNA probes derived from the VP4 gene of the standard strains RV5 (P4), ST3 (P6) and RV4 (P8) were used to discriminate between the different alleles. Although the P4 probe exhibited cross-reactivity with some P8 samples, the P6 and P8 probes were found to be type-specific. In addition, the probes did not react with standard strains representative of other defined human and animal rotavirus P-types. Use of these probes on viral RNA of faecal origin allowed approximately 70% of samples to be assigned a P-type. This method complements PCR- and EIA-based P-typing methods, is relatively inexpensive and is readily applicable to large numbers of samples, thus proving useful for epidemiological studies.
Insights
A new Northern hybridization method effectively P-genotypes rotavirus field isolates using DIG-labeled VP4 gene probes. This cost-effective technique aids in epidemiological studies by identifying rotavirus P-types in fecal samples.
Area of Science:
- Virology
- Molecular Biology
- Epidemiology
Background:
- Rotavirus outer capsid proteins VP4 and VP7 define P- and G-serotypes.
- Human rotaviruses commonly exhibit P4, P6, and P8 VP4 protein types, encoded by distinct VP4 gene alleles.
Purpose of the Study:
- To develop a non-radioactive Northern hybridization method for P-genotyping rotavirus field isolates.
- To assess the specificity and applicability of digoxigenin (DIG)-labeled VP4 gene probes for rotavirus P-typing.
Main Methods:
- Isolation of double-stranded RNA from rotavirus-positive fecal specimens.
- Utilizing DIG-labeled cDNA probes from standard rotavirus strains (RV5 (P4), ST3 (P6), RV4 (P8)) for Northern hybridization.
- Assessing probe cross-reactivity and type-specificity against various rotavirus P-types.
Main Results:
- P6 and P8 probes demonstrated type-specificity, while the P4 probe showed some cross-reactivity with P8 samples.
- Probes did not react with other defined human and animal rotavirus P-types.
- The method successfully P-typed approximately 70% of fecal rotavirus samples.
Conclusions:
- The developed Northern hybridization method is a valuable tool for rotavirus P-genotyping.
- This technique is cost-effective, applicable to large sample numbers, and complements existing PCR- and EIA-based methods for epidemiological surveillance.
