Standardization and validation of multiplex RT-PCR for the simultaneous detection of avian rotavirus species RVA, RVD, RVF, and RVG
DOI:
https://doi.org/10.34119/bjhrv8n5-274Keywords:
fecal incontinence, molecular diagnostic techniques, poultry diseases, validation studyAbstract
Rotaviruses (RV) are considered significant pathogenic agents responsible for gastroenteritis in humans and various animals, including young birds and mammals. Four RV species – RVA, RVD, RVF, and RVG – exclusively infect avian hosts. In Brazil, RV has been detected in poultry, as well as in domestic and wild birds. This study aimed to standardize protocols for the detection of RVA, RVD, RVF, and RVG in birds using the Multiplex RT-PCR technique. A total of 21 fecal specimens were processed by preparing 10% fecal suspensions in 0.01 M Tris-Ca²⁺ buffer. Viral genomes were subsequently extracted. The Multiplex RT-PCR was performed using specific primers targeting genes of RVA, RVD, RVF, and RVG. Positive RV amplification and validation of the Multiplex RT-PCR were confirmed by 2% agarose gel electrophoresis. Amplicons were then purified and sequenced using the Sanger method. Primer sets were evaluated for specificity and sensitivity, while the Multiplex RT-PCR protocol was assessed for reproducibility, repeatability, and agreement via the Kappa coefficient. All primers were specific, with a detection threshold of 5 × 10⁻7 ng/µL. Reproducibility and repeatability were 91.6% and 93.54%, respectively. The Kappa coefficient was 0.82, indicating an almost perfect level of agreement. Therefore, the developed assays were deemed specific and sensitive tools for the simultaneous detection of RV species infecting avian hosts.
References
ALFIERI, A. F., RESENDE, M., RESENDE, J. S., & ALFIERI, A. A. Atypical rotavirus infections among broiler chickens in Brazil. Arquivo Brasileiro de Medicina Veterinária e Zootecnia 41 (1989): 81-82.
ALFIERI, ALICE FERNANDES, CLAUDIA YURIKA TAMEHIRO, AND AMAURI ALCINDO ALFIERI. Virus entéricos RNA fita dupla, segmentado, em aves: Rotavírus, Reovírus e Picobirnavírus. Semina: Ciências Agrárias 21.1 (2000): 101-113.
AliView, A. L. A fast and lightweight alignment viewer and editor for large datasets., 2014, 30. DOI: https://doi. org/10.1093/bioinformatics/btu531, 3276-3278.
BENNETT, SUSAN, AND RORY N. GUNSON. The development of a multiplex real-time RT-PCR for the detection of adenovirus, astrovirus, rotavirus and sapovirus from stool samples. Journal of Virological Methods 242 (2017): 30-34.
BERGELAND, MARTIN E., John P. McAdaragh, and Ivan Stotz. Rotaviral enteritis in turkey poults. (1977): 129-130.
BESERRA, L. A., BARBOSA, C. M., BERG, M., BRANDÃO, P. E., SOARES, R. M., & GREGORI, F. (2020). Genome constellations of rotavirus a isolated from avian species in Brazil, 2008–2015. Brazilian Journal of Microbiology, 51, 1363-1375.
BESERRA, L. A., BERNARDES, N. T., BRANDÃO, P. E., & GREGORI, F. (2015). Monitoring and molecular characterization of group Drotavirus in Brazilian poultry farms. Pesquisa Veterinária Brasileira, 35, 536-540.
BESERRA, LAILA ANDREIA RODRIGUES, AND FÁBIO Gregori. Description of rotavirus F in broilers from Brazilian poultry farms. Avian Diseases 58.3 (2014): 458-461.
BEZERRA, D. A. M., DA SILVA, R. R., KAIANO, J. H. L., DE SOUZA OLIVEIRA, D., GABBAY, Y. B., LINHARES, A. C., & MASCARENHAS, J. D. A. P. (2014). Detection, epidemiology and characterization of VP6 and VP7 genes of group D rotavirus in broiler chickens. Avian Pathology, 43(3), 238-243.
BEZERRA, D. A. M., DA SILVA, R. R., KAIANO, J. H. L., SILVESTRE, R. V. D., DE SOUZA OLIVEIRA, D., LINHARES, A. C., ... & MASCARENHAS, J. D. A. P. (2012). Detection of avian group D rotavirus using the polymerase chain reaction for the VP6 gene. Journal of virological methods, 185(2), 189-192.
BOOM, R. C. J. A., SOL, C. J., SALIMANS, M. M., JANSEN, C. L., WERTHEIM-VAN DILLEN, P. M., & VAN DER NOORDAA, J. P. M. E. (1990). Rapid and simple method for purification of nucleic acids. Journal of clinical microbiology, 28(3), 495-503.
CHHABRA, P., GREGORICUS, N., WEINBERG, G. A., HALASA, N., CHAPPELL, J., HASSAN, F., ... & VINJÉ, J. (2017). Comparison of three multiplex gastrointestinal platforms for the detection of gastroenteritis viruses. Journal of Clinical Virology, 95, 66-71.
COSTA, L. C. P. N., TEIXEIRA, D. M., PORTELA, A. C. R., DE LIMA, I. C. G., DA SILVA BANDEIRA, R., SOUSA JÚNIOR, E. C., ... & GABBAY, Y. B. (2019). Molecular and evolutionary characterization of norovirus GII. 17 in the northern region of Brazil. BMC Infectious Diseases, 19, 1-11.
DEOL, P., KATTOOR, J. J., SIRCAR, S., GHOSH, S., BÁNYAI, K., DHAMA, K., & MALIK, Y. S. (2017). Avian group D rotaviruses: structure, epidemiology, diagnosis, and perspectives on future research challenges. Pathogens, 6(4), 53.
ELNIFRO, E. M., ASHSHI, A. M., COOPER, R. J., & KLAPPER, P. E. (2000). Multiplex PCR: optimization and application in diagnostic virology. Clinical microbiology reviews, 13(4), 559-570.
FALCONE, E., BUSI, C., LAVAZZA, A., MONINI, M., BERTOLETTI, M., CANELLI, E., ... & BONIOTTI, M. B. (2015). Molecular characterization of avian rotaviruses circulating in Italian poultry flocks. Avian Pathology, 44(6), 509-515.
FONSECA JR, A. A., GOUVEA, M. V., ZARONI, M. M. H., CARVALHO, L. B., & XAVIER, S. M. (2015). Manual de Verificação de Desempenho de Métodos para Diagnóstico Molecular de Doenças Infecciosas na Rede Nacional de Laboratórios Agropecuários. MAPA/ACS, Brasília. 57p.
International Committee on Taxonomy of Viruses (ICTV). Rotavirus. In: ICTV Virus Taxonomy: 2023 Release [Internet]. London (UK): ICTV; 2023 [cited 2025 May 29]. Available from:https://ictv.global/report/chapter/sedoreoviridae/sedoreoviridae/rotavirus
JOHNE, R., OTTO, P., ROTH, B., LÖHREN, U., BELNAP, D., REETZ, J., & TROJNAR, E. (2011). Sequence analysis of the VP6-encoding genome segment of avian group F and G rotaviruses. Virology, 412(2), 384-391.
KARIM, M. R., RUME, F. I., ALAM, M. M., & AHMED, M. U. (2007). Molecular epidemiologic study on avian rotavirus prevailing in Bangladesh. Bangladesh Journal of Veterinary Medicine, 43-48.
KEARSE, M., MOIR, R., WILSON, A., STONES-HAVAS, S., CHEUNG, M., STURROCK, S., ... & DRUMMOND, A. (2012). Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics, 28(12), 1647-1649.
KUSUMAKAR, A. L., MALIK, Y. P. S., & PRASAD, G. (2008). Detection and characterization of group A and D avian rotaviruses in India. Indian Journal of Biotechnology, 7, 554-556.
LAAMIRI, N., AOUINI, R., MARNISSI, B., GHRAM, A., & HMILA, I. (2018). A multiplex real-time RT-PCR for simultaneous detection of four most common avian respiratory viruses. Virology, 515, 29-37.
MASCARENHAS, J. D., BEZERRA, D. A., SILVA, R. R., SILVA, M. J., JÚNIOR, E. C. S., & SOARES, L. S. (2016). Detection of the VP6 gene of group F and G rotaviruses in broiler chicken fecal samples from the Amazon region of Brazil. Archives of virology, 161, 2263-2268.
MCNULTY, M. S., TODD, D., ALLAN, G. M., MCFERRAN, J. B., & GREENE, J. A. (1984). Epidemiology of rotavirus infection in broiler chickens: recognition of four serogroups. Archives of virology, 81(1-2), 113-121.
OTTO, P. H., AHMED, M. U., HOTZEL, H., MACHNOWSKA, P., REETZ, J., ROTH, B., ... & JOHNE, R. (2012). Detection of avian rotaviruses of groups A, D, F and G in diseased chickens and turkeys from Europe and Bangladesh. Veterinary Microbiology, 156(1-2), 8-15.
OTTO, P., LIEBLER-TENORIO, E. M., ELSCHNER, M., REETZ, J., LÖHREN, U., & DILLER, R. (2006). Detection of rotaviruses and intestinal lesions in broiler chicks from flocks with runting and stunting syndrome (RSS). Avian Diseases, 50(3), 411-418.
PANTIN-JACKWOOD, M. J., SPACKMAN, E., MICHAEL DAY, J., & RIVES, D. (2007). Periodic monitoring of commercial turkeys for enteric viruses indicates continuous presence of astrovirus and rotavirus on the farms. Avian diseases, 51(3), 674-680.
PAULY, M., ONI, O. O., SAUSY, A., OWOADE, A. A., ADEYEFA, C. A., MULLER, C. P., ... & SNOECK, C. J. (2017). Molecular epidemiology of Avian Rotaviruses Group A and D shed by different bird species in Nigeria. Virology journal, 14, 1-10.
PHAM, N. T. K., TRINH, Q. D., CHAN-IT, W., KHAMRIN, P., SHIMIZU, H., OKITSU, S., ... & USHIJIMA, H. (2010). A novel RT-multiplex PCR for detection of Aichi virus, human parechovirus, enteroviruses, and human bocavirus among infants and children with acute gastroenteritis. Journal of virological methods, 169(1), 193-197.
PHAN, T. G., VO, N. P., BOROS, A., PANKOVICS, P., REUTER, G., LI, O. T., ... & DELWART, E. (2013). The viruses of wild pigeon droppings. PloS one, 8(9), e72787.
POLANCO, G., GONZÁLEZ, M., MANZANO, L., CÁMARA, J., & PUERTO, M. (2004). Rotavirus en animales asintomáticos: Detección y clasificación antigénica. Archivos de medicina veterinaria, 36(1), 65-70.
RIBEIRO, E. L., OLIVEIRA, A. G., LAGUARDIA-NASCIMENTO, M., MATA, C. P. S. M. D., REIS, J. K. D., & FONSECA, A. A. (2016). Estudo comparativo e validação de três técnicas de PCR em tempo real (qPCR) para diagnóstico de Peste Suína Africana. Pesquisa Veterinária Brasileira, 36(06), 473-478.
RUBBENSTROTH, D., PEUS, E., SCHRAMM, E., KOTTMANN, D., BARTELS, H., MCCOWAN, C., ... & TESKE, L. (2019). Identification of a novel clade of group A rotaviruses in fatally diseased domestic pigeons in Europe. Transboundary and emerging diseases, 66(1), 552-561.
SCUARCIALUPI, L. N., HOCHHEIM, J. R., SOARES, R. M., & GREGORI, F. (2018). Genetic characterization of avian rotavirus groups F and G in Brazilian farms. Hosts and Viruses, 5(2), 12.
SILVA, R. R., BEZERRA, D. A. M., KAIANO, J. H. L., MANNO, M. C., DE SOUZA OLIVEIRA, D., SANTO SAGICA, F. D. E., ... & MASCARENHAS, J. D. A. P. (2013). Molecular epidemiology of avian rotavirus in fecal samples of broiler chickens in Amazon Region, Brazil, from August 2008 to May 2011. Revista Pan-Amazônica de Saúde, 4(2), 8-8.
SONG, D. S., KANG, B. K., OH, J. S., HA, G. W., YANG, J. S., MOON, H. J., ... & PARK, B. K. (2006). Multiplex reverse transcription-PCR for rapid differential detection of porcine epidemic diarrhea virus, transmissible gastroenteritis virus, and porcine group A rotavirus. Journal of veterinary diagnostic investigation, 18(3), 278-281.
TAMEHIRO, C. Y., ALFIERI, A. F., MÉDICI, C., & ALFIERI, A. A. (2003). Segmented double-stranded genomic RNA viruses in fecal samples from broiler chicken. Brazilian Journal of Microbiology, 34, 349-353.
THANTHRIGE-DON, N., LUNG, O., FURUKAWA-STOFFER, T., BUCHANAN, C., JOSEPH, T., GODSON, D. L., ... & AMBAGALA, A. (2018). A novel multiplex PCR-electronic microarray assay for rapid and simultaneous detection of bovine respiratory and enteric pathogens. Journal of Virological Methods, 261, 51-62.
URSU, K., PAPP, H., KISFALI, P., RIGÓ, D., MELEGH, B., MARTELLA, V., & BÁNYAI, K. (2011). Monitoring of group A rotaviruses in wild-living birds in Hungary. Avian Diseases, 55(1), 123-127.
YAN, H., NGUYEN, T. A., PHAN, T. G., OKITSU, S., LI, Y., & USHIJIMA, H. (2004). Development of RT-multiplex PCR assay for detection of adenovirus and group A and C rotaviruses in diarrheal fecal specimens from children in China. Journal of the Japanese Association for Infectious Diseases, 78(8), 699-709.
YUAN, X., LV, J., LIN, X., ZHANG, C., DENG, J., WANG, C., ... & WU, S. (2019). Multiplex detection of six swine viruses on an integrated centrifugal disk using loop-mediated isothermal amplification. Journal of Veterinary Diagnostic Investigation, 31(3), 415-425.
