3. Add 1 U Taq DNA polymerase to a final volume of 50 mL.
4. Overlay all reaction mixtures with 100 mL of sterile mineral oil.
5. As thermal cycling conditions for amplification, use the following parameters: 97°C for 1 min, 46°C for 2 min, 72°C for 1 min (15 cycles), final extension at 72°C for 7 min (1 cycle).
6. Identification of the P-type isolates requires adding sufficient VP4 template to 5 ^L 10X PCR buffer (100 mM Tris-HCl, pH 9.0, 500 mM KCl), 8 ^L of stock dNTP mix, 1.5 mM MgCl2, 2.5 U Taq DNA polymerase, 20 pmol of each P-typing primer, and 20 pmol of the VP4 common primer, Con2 (Tables 1 and 3).
7. The cycling parameters for the P-typing reaction are as follows; 97°C for 1 min, 50°C for 2 min, 72°C for 1 min (20 cycles), with a final extension at 72°C for 5 min (1 cycle).
8. Visualize all PCR products by agarose gel (1.5%) electrophoresis as previously described in Subheading 3.3.1.
1. The RNA isolation procedure described was used to isolate RNA directly from fecal matter. The method can also be applied to tissue culture samples, but the quality and quantity of RNA may not be as good. Alternative methods are also available.
2. Contamination with exogenous ribonucleases can result in the degradation of RNA; therefore proper pretreatment of reagents and experimental apparatus is a prerequisite. To reduce ribonuclease contamination of laboratory equipment, all surfaces and apparatus can be thoroughly wiped with RNase AWAY (Molecular Bioproducts, San Diego, CA).
Glassware and nonsterile plasticware can be immersed in a solution of 0.2% (v/v) DEPC and then autoclaved at 121°C for 30 min. Gloves should be worn at all times during procedures.
3. Caution: care should be taken when handling phenol/chloroform. As phenol is an acid, it can cause skin burns. This reagent should only be handled in a safety cabinet with extraction ventilation.
4. The presence of DNA in RNA samples could potentially interfere with subsequent analysis of the rotaviral genome and therefore it should be removed. DNA is degraded by treatment with a specific endonuclease enzyme, DNase 1. This enzyme digests DNA but does not affect the integrity of RNA.
5. Prepare sufficient gel volumes according to the PAGE apparatus used.
6. Isobutanol is added to prevent oxygen from diffusing into the gel, as this would inhibit polymerization.
7. The loading buffer described in Subheading 2.2.1. can be substituted for another appropriate loading buffer if preferred. The volume of sample that is loaded into the gel will depend on the size of the gel wells and thus can be adjusted accordingly.
8. Using excess RNA template can be detrimental to the RT-PCR assay, as it appears to inhibit the reaction. In general, small volumes of dsRNA template (1-5 ^L) often produce better results.
9. Stronger binding forces exist between RNA-RNA compared with DNA-DNA and DNA-RNA; thus more stringent denaturing conditions are required to prevent strand reannealing. Prolonged denaturation (5 min) and the addition of nucleic acid destabilizers, such as DMSO, in the RT step are often recommended (34).
10. It is not essential but recommended to include RNasin in the assay to inactivate any contaminating ribonucleases that may be present in the reaction.
11. The thermocycler used in this study was a Pharmacia ATAQ controller (Pharmacia, Uppsala, Sweden).
12. The amount of cDNA template used in the serotyping PCR assays can be identified by performing initial template titrations. In general, 1-2 ^L of cDNA template is sufficient for amplification. However, if the RT-PCR products are very weak, use more template.
1. Kapikian, A. Z. and Chanock, R. M. (1996) Rotaviruses. In: Fields Virology (Fields, B. N., et al, eds.). Philadelphia, pp. 1657-1708.
2 Cook, S. M., Glass, R. I., LeBaron, C. W., and Ho, M. S. (1990) Global seasonality of rotavirus infections. Bull. WHO 68, 171-177.
3. Institute of Medicine (1986) Prospects for immunizing against rotavirus. In: New Vaccine Development: Establishing Priorities. Diseases of Importance in Developing Countries, vol. II. National Academy Press Washington, DC, pp. 308-318.
4 Ryan, M. J., Ramsay, M., and Brown, D. (1996) Hospital admissions attributable to rotavirus infection in England and Wales. J. Infect. Dis. 174(suppl 1), S12-S18.
5 Woods, P. A., Gentsch, J. R., Gouvea, V., et al. (1982) Distribution of serotypes of human rotavirus in different populations. J. Clin. Microbiol. 30, 781-785.
6 Gonzalez, S. A. L., Tomasini, L., Tortorici, M. A., and Affranchino, J. L. (1995) VP6 from porcine rotavirus strain CN86: amino acid sequence divergence with conservation of subgroup 11 specificity. J. Gen Virol. 76, 221-224.
7 Fielding, P. A., Lambden, P. R., Caul, W. E. O., and Clarke I. N. (1994) Molecular characterisation of the outer capsid protein (VP4) gene from human group C rotavirus. Virology 204, 442-446.
8 Arista, S., Vizzi, E., Ferraro, D., Cascio, A., and Di Stefano, R. (1997) Distribution of VP7 serotypes and VP4 genotypes among rotavirus strains recovered from Italian children with diarrhea. Arch. Virol. 142, 2065-2071.
9 Gouvea, V., Santos, N., and do Carmo Timenetsky, M. (1994) Identification of porcine rotavirus G types by PCR. J. Clin. Microbiol. 32, 1338-1340.
10 Gusmao, R. H., Mascarenhas, J. D. P., Gabbay, Y. B., et al. (1999) Rotavirus subgroups, G serotypes and electropherotypes in cases of nosocomial infantile diarrhoea in Belem, Brazil. J. Trop. Pediatr. 45, 81-86.
11 Steele, A. D., Parker, S. P., Peenze, I., Pager, C.T., Taylor, M. B., and Cubitt, W.D. (1999) Comparative studies of human rotavirus serotype G8 strains recovered in South Africa and the United Kingdom. J. Gen. Virol. 80, 3029-3034.
12 Gentsch, J. R., Woods, P. A., Ramachandran, M., et al. (1996) Review of G and P typing results from a global collection of rotavirus strains: Implications for vaccine development. J. Infect. Dis. 174(suppl 1), S30-S36.
13. Isegawa, Y., Nakagomi, O., Nakagomi, T., and Ueda, S. (1992) A VP4 sequence conserved in human rotavirus strain AU-1 and feline rotavirus strain FRV-1. J. Gen Virol. 73, 1936-1946.
14 Gault, E., Chikhi-Brachet, R., Delon, S., et al. (1999) Distribution of human rotavirus G types circulating in Paris, France, during the 1997-1998 epidemic: high prevalence of type G4. J. Clin. Microbiol. 37, 2373-2375.
15 Ramachandran, M., Gentsch, J. R., Parashar, U. D., et al., and the National Rotavirus Strain Surveillance System Collaborating Laboratories. (1998) Detection and characterisation of novel rotavirus strains on the United States. J. Clin. Microbiol. 36, 3223-3229.
16. Estes, M. (1996) Rotaviruses and their replication. In: Fields Virology, vol II (Fields, B. N., Knipe, D. M., and Howley, P. M., eds.). Lippincott-Raven, Philadelphia, PA, pp. 1625-1655.
17. O' Halloran, F., Lynch, M., Cryan, B., O' Shea, H., and Fanning, S. (2000) Molecular characterization of rotavirus in Ireland: detection of novel strains circulating in the population. J. Clin. Microbiol. 38, 3370-3374.
18 Unicomb, L. E., Podder, G., Gentsch, J. R., et al. (1999) Evidence of high-frequency genomic reassortment of group A rotavirus strains in Bangladesh: emergence of type G9 in 1995. J. Clin. Microbiol. 37, 1885-1891.
19 Santos, N., Lima, R. C. C., Pereira, C. F. A., and Gouvea, V. (1998) Detection of rotavirus types G8 and G10 among Brazilian children with diarrhea. J. Clin. Microbiol. 36, 2727-2729.
20 Leite, J. P. G., Alfieri, A. A., Woods, P. A., Glass, R. I., and Gentsch, J. R. (1996) Rotavirus G and P types circulating in Brazil: characterisation by RT-PCR, probe hybridisation and sequence analysis. Arch. Virol. 141, 2365-2374.
21 Burke, B., and Desselberger, U. (1996) Rotavirus pathogenicity. Virology 218, 299-305.
22. Okada, J., Urasawa, T., Kobayashi, N., et al. (2000) New P serotype of group A human rotavirus closely related to that of a porcine rotavirus. J. Med. Virol. 60, 63-69.
23 Sereno, M. M. and Gorziglia, M. I. (1994) The outer capsid protein VP4 of murine rotavirus strain Eb represents a tentative new P type. Virology 216, 500-504.
24 Ramachandran, M.,. Das, B. K, Vij, A., et al. (1996) Unusual diversity of human rotavirus G and P genotypes in India. J. Clin. Microbiol. 34, 436-439.
25 Bishop, R. F., Unicomb, L. E., and Barnes, G. L. (1991) Epidemiology of rotavirus serotypes in Melbourne, Australia, from 1973 to 1989. J. Clin. Microbiol. 29, 862-868.
26 Sethi, . K., Olive, D. M., Strannegard, O. O., and Al-Nakib, W. (1988) Molecular epidemiology of human rotavirus infections based on genome segment variations in viral strains. J. Med. Virol. 26, 249-259.
27. Lourenco, M. H., Nicolas, J. C., J. Cohen, J., Scherrer, R., and Bricout, F. (1981) Study of human rotavirus genome by electrophoresis: attempt of classification among strains isolated in France. Ann. Virol. 132, 161-173.
28. Fukai, K., Sakai, T., and Kamata, H. (1998) Distribution of the G serotypes and P genotypes of bovine group A rotavirus isolated in Japan. Aust. Vet. J. 76, 418-422.
29 Rodger, S. M. and Holmes, I. H. (1979) Comparison of the genomes of simian, bovine and human rotaviruses by gel electreophoresis and detection of genomic variation among bovine isolates. J. Virol. 30, 839-846.
30 Palombo, E. A., Bugg, H. C., Masendycz, P. J., Coulson, B. S., Barnes, G. L., and Bishop, R. F. (1996) Multiple gene reassortants responsible for an outbreak of gastroenteritis in central and northern Australia. J. Gen Virol. 77, 1223-1227.
31. Ramig, R. F. and Ward, R. L. (1999) Genomic segment reassortment in rotaviruses and other reoviridae. Adv. Virus Res. 39, 163-207.
32 Patton, J. T. (1995) Structure and function of the rotavirus RNA-binding proteins. J. Gen. Virol. 76, 2633-2644.
33 Gentsch, J. R., Glass, R. I., Woods, P. A., et al. (1992) Identification of group A rotavirus gene 4 types by polymerase chain reaction. J. Clin. Microbiol. 30, 1365-1373.
34 Gouvea, V., Glass, R. I., Woods, P., et al. (1990) Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens. J. Clin. Microbiol. 28, 276-282.
35 Ushijima, H., Koike, H., Mukoyama, A., Hasegawa, A., Nishimura, S., and Gentsch, J. (1992) Detection and serotyping of rotavirus in stool specimens by using reverse transcription and polymerase chain reaction. J. Med. Virol. 38, 292-297.
36 Gouvea, V., Santos, N., and Timenetsky, M. D. C. (1994) Identification of bovine and porcine rotavirus G types by PCR. J. Clin. Microbiol. 32, 1338-1340.
37 Alfieri, A. A., Leite, J. P. G., Nakagomi, O., et al. (1996) Characterisation of human rotavirus genotype PG5 from Brazil by probe hybridisation and sequence. Arch. Virol. 141,2353-2364.
38 Palombo, E. A., Clark, R., and Bishop, R. F. (2000) Characterisation of a "European-like" serotype G8 human rotavirus isolated in Australia. J. Med. Virol. 60, 56-62.
39 Palombo, E. A., Masendycz, P. J., Bugg, H. C., Bogdanovic-Sakran, N., Branes, G. L., and Bishop, R. F. (2000) Emergence of G9 in Australia. J. Clin. Microbiol. 38, 1305-1306.
40. Das B. K., Gentsch, J. R., Cicirello, H. G., et al. (1994) Characterization of rotavirus strains from newborns in New Delhi, India. J. Clin. Microbiol. 32, 1820-1822.
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