Veterinary Research Forum
Vet Res Forum

Development of a multiplexed Luminex assay for simultaneous detection of enteric viruses in cattle

Document Type : Original Article

Authors

Monika Punia 1
Sushila Maan 2
Kanisht Batra 2
Aman Kumar 2
Narender Singh Maan 2
Suresh Kumar Gahlawat 1

1 Department of Biotechnology, Faculty of Life Sciences, Chaudhary Devi Lal University, Sirsa, India

2 Department of Animal Biotechnology, College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, India

https://doi.org/10.30466/vrf.2023.2005728.3925
Abstract
Viral and bacterial gastroenteritis and diarrhea have long been a problem in livestock with devastating effects on animal health and production causing a heavy financial burden on producers. Therefore, the bead-based multiplex detection assay was created for simultaneous detection of three livestock viral diarrheic agents viz. bovine rotavirus (BRV), bovine coronavirus (BCoV) and bluetongue virus (BTV). The primers and probes for triplex MAGPIX assay for simultaneous detection of three enteric viruses were designed and the assay was optimized for hybridization temperature, primer-probe and bead concentrations. The newly developed MAGPIX assay was used to determine the prevalence of these diarrhea-associated viruses by testing 200 fecal samples collected from Haryana state of India during 2018-2019. The limit of detection of the developed triplex assay was 1 × 105, 1 × 104, and 1 × 105 RNA copies for BRV, BCoV, and BTV, respectively, being lower than the reverse transcription-quantitative polymerase chain reaction (RT-qPCR). However, it was higher than the conventional RT-PCR, showing it to be more sensitive. The newly developed MAGPIX assay was a rapid, cost-effective and high throughput diagnostic tool for identification of three major entero-pathogenic diarrhea associated viruses, either alone or in tandem, with the aim to prevent and control viral diarrhea in animals.

Keywords

Bluetongue virus
Bovine coronavirus
Enteric viruses
Luminex assay
Rotavirus

Subjects

  1. Singh P, Metgud SC, Roy S, et al. Evolution of diarrheagenic Escherichia coli pathotypes in India. J Lab Physicians 2019; 11(4): 346-351.
  2. Geletu US, Usmael MA, Bari FD. Rotavirus in calves and its zoonotic importance. Vet Med Int 2021; 2021: 6639701. doi: 10.1155/2021/6639701.
  3. Singh S, Singh R, Singh KP, et al. Immunohistochemical and molecular detection of natural cases of bovine rotavirus and coronavirus infection causing enteritis in dairy calves. Microb Pathog 2020; 138:103814. doi: 10.1016/j.micpath.2019.103814.
  4. Amoroso MG, Lucifora G, Uberti BD, et al. Fatal interstitial pneumonia associated with bovine coronavirus in cows from southern Italy. Viruses 2020; 12(11): 1331. doi: 10.3390/v12111331.
  5. Frucchi APS, Agnol AMD, Bronkhorst DE, et al. Bovine coronavirus co-infection and molecular characterization in dairy calves with or without clinical respiratory disease. Front Vet Sci 2022; 9: 895492. doi: 10.3389/fvets.2022.895492.
  6. Bakhshesh M, Otarod V, Mehrabadi MHF. Large-scale seroprevalence and risk factors associated with bluetongue virus in Iran. Prev Vet Med 2020; 179: 104994. doi: 10.1016/j.prevetmed.2020.104994.
  7. Rojas JM, Rodríguez-Martín D, Martín V, et al. Diagnosing bluetongue virus in domestic ruminants: current perspectives. Vet Med (Auckl) 2019; 10: 17-27.
  8. Maan S, Maan NS, Belaganahalli MN, et al. Full-genome sequencing as a basis for molecular epidemiology studies of bluetongue virus in India. PLoS One 2015; 10(6): e0131257. doi: 10.1371/journal.pone.0131257.
  9. Shi Y, Li B, Tao J, et al. The complex co-infections of multiple porcine diarrhea viruses in local area based on the Luminex xTAG multiplex detection method. Front Vet Sci 2021; 8: 602866. doi: 10.3389/fvets. 2021.602866.
  10. Chen R, Yu X-L, Gao X-B, et al. Bead-based suspension array for simultaneous differential detection of five major swine viruses. Appl Microbiol Biotechnol 2015; 99(2): 919-928.
  11. Perry MD, Corden SA, Howe RA. Evaluation of the Luminex xTAG Gastrointestinal Pathogen Panel and the Savyon Diagnostics Gastrointestinal Infection Panel for the detection of enteric pathogens in clinical samples. J Med Microbiol 2014; 63(Pt 11): 1419-1426.
  12. Hamza IA, Jurzik L, Wilhelm M. Development of a Luminex assay for the simultaneous detection of human enteric viruses in sewage and river water. J Virol Methods 2014; 204: 65-72.
  13. Pavliakova D, Giardina PC, Moghazeh S, et al. Development and validation of 13-plex Luminex-based assay for measuring human serum antibodies to Streptococcus pneumoniae capsular polysaccharides. mSphere 2018; 3(4): e00128-18. doi: 10.1128/ mSphere.00128-18.
  14. Wang H, Cong F, Guan J, et al. Development of a sensitive and specific xMAP assay for detection of antibodies against infectious laryngotracheitis and bronchitis viruses. Virol J 2018; 15(1): 146. doi: 10.1186/s12985-018-1048-x.
  15. Maan S, Batra K, Chaudhary D, et al. Detection and genomic characterization of Senecavirus from Indian pigs. Indian J Anim Res 2023; 57(10): 1344-1350.
  16. Maan S, Punia M, Batra K, et al. Multiplexed Luminex MAGPIX Assay for concurrent detection of Bluetongue virus, Bovine Coronavirus and Rotavirus in India. Indian Patent Application No.: 202111022744 online 28th February, 2022.
  17. Brunauer M, Roch F-F, Conrady B. Prevalence of worldwide neonatal calf diarrhoea caused by bovine rotavirus in combination with bovine coronavirus, Escherichia coli K99 and Cryptosporidium: a meta-analysis.Animals (Basel) 2021; 11(4): 1014. doi: 10.3390/ani11041014.
  18. Cassedy A, Parle-McDermott A, O’Kennedy R. Virus detection: a review of the current and emerging molecular and immunological methods. Front Mol Biosci 2021; 8: 637559. doi: 10.3389/fmolb. 2021.637559.
  19. Shrinet G, Chhabra R, Sharma A, et al. High throughput Luminex beads based multiplex assay for identification of six major bacterial pathogens of mastitis in dairy animals. Front Cell Infect Microbiol 2023; 13: 1125562. doi: 10.3389/fcimb.2023.1125562.
  20. Wu M, Zhu Y, Cong F, et al. Rapid detection of three rabbit pathogens by use of the Luminex x-TAG assay. BMC Vet Res 2018; 14: 1-6.
  21. Wu M, Ma L, Cong F, et al. High-throughput Luminex xMAP assay for simultaneous detection of antibodies against rabbit hemorrhagic disease virus, Sendai virus and rabbit rotavirus. Arch Virol 2019; 164(6): 1639-1646.
  22. Ko DH, Kim HS, Hyun J, et al. Comparison of the Luminex xTAG respiratory viral panel Fast v2 assay with Anyplex II RV16 detection kit and AdvanSure RV real-time RT-PCR assay for the detection of respiratory viruses. Ann Lab Med 2017; 37(5): 408-414.
Veterinary Research Forum
Volume 15, Issue 1
January 2024
Pages 13-19

  • Receive Date 03 July 2023
  • Revise Date 01 September 2023
  • Accept Date 23 September 2023

Home

Guide for Authors

Submit Manuscript

Reviewers

Contact Us