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Novel Isothermal Reverse Transcription Polymerase Spiral Reaction (RT-PSR) Assay for the Detection of Newcastle Disease Virus in Avian Species

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Abstract

Newcastle disease in avian species is caused by Newcastle disease virus (NDV). It is responsible for large economic losses to the poultry industry. The early diagnosis of NDV infection is important for epidemiologic control and clinical management. The present study aimed to develop reverse transcription polymerase spiral reaction (RT-PSR) assay that accurately detects the NDV at isothermal temperature. The RT-PSR assay was developed using specifically designed primers targeting the conserved fusion gene of NDV. The assay could be performed within 2 h at an isothermal temperature of 65 °C. The developed assay is highly sensitive and capable to detect as low as 1.08 fg of template. It was found to be 1000-fold more sensitive than conventional RT-PCR and was comparable to real-time RT-PCR assay. The developed assay was found to be specific as it did not detect closely related infectious bronchitis virus and Merck disease virus. Further, the developed assay was evaluated with clinical samples and it has shown > 99% concordance with RT-PCR methods. To the best of our knowledge this is the first report of isothermal detection of NDV without the need of high-end equipment like thermal cycler. Moreover, this assay has the potential to detect NDV under field conditions. In conclusions, the results of this study indicated that the RT-PSR assay is a simple, rapid, sensitive, and specific assay for the detection of NDV that may improve diagnostic potential in clinical laboratories or can be used at diagnostic laboratories with the least infrastructure or even at field condition.

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Abbreviations

ECEs:

Embryonated chicken eggs

SPF:

Specific Pathogen free

NDV:

Newcastle disease virus

F:

Fusion

HA:

Haemagglutination assay

MDV:

Marek’s disease virus

ND:

Newcastle disease

NDV:

Newcastle disease virus

OIE:

World organisation for animal health

PCR:

Polymerase chain reaction

PSR:

Polymerase spiral reaction

RT:

Reverse transcriptase

References

  1. Bello MB, Yusoff KM, Ideris A, Hair-Bejo M, Peeters BP, Jibril AH, Omar AR (2018) Genotype diversity of Newcastle disease virus in Nigeria: disease control challenges and future outlook. Adv Virol 2018:1–17

    Article  Google Scholar 

  2. Wan H, Chen L, Wu L, Liu X (2004) Newcastle disease in geese: natural occurrence and experimental infection. Avian Pathol 33:216–221

    Article  PubMed  Google Scholar 

  3. Xu Q, Sun J, Gao M, Zhao S, Liu H, Zhang T, Liu S (2017) Genetic, antigenic, and pathogenic characteristics of Newcastle disease viruses isolated from geese in China. J Vet Diagn Invest 29:489–498

    Article  CAS  PubMed  Google Scholar 

  4. Lindh E, Ek-Kommonen C, Väänänen VM, Alasaari J, Vaheri A, Vapalahti O, Huovilainen A (2012) Molecular epidemiology of outbreak-associated and wild-waterfowl-derived Newcastle disease virus strains in Finland, including a novel class I genotype. J Clin Microbiol 50:3664–3673

    Article  PubMed  PubMed Central  Google Scholar 

  5. Ganar K, Das M, Sinha S, Kumar S (2014) Newcastle disease virus: current status and our understanding. Virus Res 184:71–81

    Article  CAS  PubMed  Google Scholar 

  6. Kim SH, Chen Z, Yoshida A, Paldurai A, Xiao S, Samal SK (2017) Evaluation of fusion protein cleavage site sequences of Newcastle disease virus in genotype matched vaccines. PLoS ONE 12:e0173965

    Article  PubMed  PubMed Central  Google Scholar 

  7. Shofa M, Wibawan IWT, Zarkasie K, Setiyaningsih S (2018) Molecular analysis of Newcastle disease virus isolated from a vaccinated layer farm in Indonesia. IOP Conf Ser 197:012044

    Article  Google Scholar 

  8. Yadav S, Prasad M, Singh N (2023) Biosensor: an emerging technological tool for microorganisms and its disease diagnosis. Indian J Microbiol 63:1–3

    Article  Google Scholar 

  9. Haque MH, Hossain MT, Islam MT, Zinnah MA, Khan MSR, Islam MA (2010) Isolation and detection of Newcastle disease virus from field outbreaks in broiler and layer chickens by reverse transcription polymerase chain reaction. Bangladesh J Vet Med 8:87–92

    Article  Google Scholar 

  10. Liu W, Dong D, Yang Z, Zou D, Chen Z, Yuan J et al (2015) Polymerase spiral reaction (PSR): a novel isothermal nucleic acid amplification method. Sci Rep 5:12723. https://doi.org/10.1038/srep12723

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Liu L, Benyeda Z, Zohari S, Yacoub A, Isaksson M, Leijon M, Belak S (2016) Assessment of preparation of samples under the field conditions and a portable real-time RT-PCR assay for the rapid on-site detection of Newcastle disease virus. Transbound Emerg Dis 63:e245–e250

    Article  CAS  PubMed  Google Scholar 

  12. Ji J, Xu X, Wang X, Zuo K, Li Z, Leng C, Bi Y (2019) Novel polymerase spiral reaction assay for the visible molecular detection of porcine circovirus type 3. BMC Vet Res 15:322

    Article  PubMed  PubMed Central  Google Scholar 

  13. Kumar A, Maan S, Mahajan NK, Rana VP, Jindal N, Batra K, Maan NS (2013) Detection and molecular characterization of Newcastle disease virus in peafowl (Pavo cristatus) in Haryana state, India. Indian J Virol 24:380–385

    Article  PubMed  PubMed Central  Google Scholar 

  14. OIE (2019) Newcastle disease (infection with Newcastle disease virus). In: Manual of diagnostic tests and vaccines for terrestrial animals: mammals, birds and bees. Office international des epizooties, Paris, 964–983

  15. Taylor LH (2014) OIE manual of diagnostic tests and vaccines for terrestrial animals

  16. da Silva AP, Aston EJ, Chiwanga GH, Birakos A, Muhairwa AP, Kayang BB, Gallardo RA (2020) Molecular characterization of Newcastle disease viruses isolated from chickens in Tanzania and Ghana. Viruses 12:916

    Article  PubMed  PubMed Central  Google Scholar 

  17. Peeters BP, de Leeuw OS, Koch G, Gielkens AL (1999) Rescue of Newcastle disease virus from cloned cDNA: evidence that cleavability of the fusion protein is a major determinant for virulence. J Virol 73:5001–5009

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Pham HM, Nakajima C, Ohashi K, Onuma M (2005) Loop-mediated isothermal amplification for rapid detection of Newcastle disease virus. J Clin Microbiol 43:1646–1650

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. El-Tholoth M, Branavan M, Naveenathayalan A, Balachandran W (2019) Recombinase polymerase amplification–nucleic acid lateral flow immunoassays for Newcastle disease virus and infectious bronchitis virus detection. Mol Biol Rep 46:6391–6397

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Milton AAP, Momin KM, Ghatak S, Thomas SC, Priya GB, Angappan M, Kandpal BK (2021) Development of a novel polymerase spiral reaction (PSR) assay for rapid and visual detection of staphylococcus aureus in meat. LWT 139:110507

    Article  CAS  Google Scholar 

  21. Jiang X, Dong D, Bian L, Zou D, He X, Ao D, Huang L (2016) Rapid detection of candida albicans by polymerase spiral reaction assay in clinical blood samples. Front Microbiol 7:916

    Article  PubMed  PubMed Central  Google Scholar 

  22. Wang X, Xu X, Hu W, Zuo K, Li Z, Kan Y et al (2019) Visual detection of porcine epidemic diarrhea virus using a novel reverse transcription polymerase spiral reaction method. BMC Vet Res 15:116. https://doi.org/10.1186/s12917-019-1851-7

    Article  PubMed  PubMed Central  Google Scholar 

  23. Malla JA, Chakravarti S, Gupta V, Chander V, Sharma GK, Qureshi S, Nandi S (2018) Novel polymerase spiral reaction (PSR) for rapid visual detection of bovine herpesvirus 1 genomic DNA from aborted bovine fetus and semen. Gene 644:107–112

    Article  CAS  PubMed  Google Scholar 

  24. Gupta V, Chakravarti S, Chander V, Majumder S, Bhat SA, Gupta VK, Nandi S (2017) Polymerase spiral reaction (PSR): a novel, visual isothermal amplification method for detection of canine parvovirus 2 genomic DNA. Adv Virol 162:1995–2001

    CAS  Google Scholar 

  25. Tomar PS, Kumar JS, Patel S, Sharma S (2020) Polymerase spiral reaction assay for rapid and real time detection of west nile virus from clinical samples. Front Cell Infect Microbiol 10:426

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Woźniakowski G, Frączyk M, Kowalczyk A, Pomorska-Mól M, Niemczuk K, Pejsak Z (2017) Polymerase cross-linking spiral reaction (PCLSR) for detection of African swine fever virus (ASFV) in pigs and wild boars. Sci Rep. https://doi.org/10.1038/srep42903

    Article  PubMed  PubMed Central  Google Scholar 

  27. Wu Q, Xu X, Chen Q, Zuo K, Zhou Y, Zhang Z, Xie Q (2019) Rapid and visible detection of Mycoplasma synoviae using a novel polymerase spiral reaction assay. Poult Sci 98:5355–5360

    Article  CAS  PubMed  Google Scholar 

  28. Louhrasby V, Mirhosseini SA, Golmohammadi R (2022) A novel and rapid isothermal nucleic acid based detection assay of vibrio cholerae by polymerase spiral reaction (PSR) in emergency situations. Iran J Pub Health 51:1364–1370

    Google Scholar 

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Acknowledgements

The authors are thankful to Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, Haryana, India and DBT–Government of India for providing funds and infrastructure for conducting the study.

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Authors and Affiliations

  1. Lab No 202 Microbial Biotechnology, Department of Bio and Nano Technology, Guru Jambheshwar University of Science and Technology (GJUSAT), Hisar, Haryana, 125001, India

    Anubha Sharma & Namita Singh

  2. Department of Animal Biotechnology, Lala Lajpat Rai University of Veterinary and Animal Sciences (LUVAS), Hisar, Haryana, 125004, India

    Anubha Sharma, Aman Kumar & Sushila Maan

Authors
  1. Anubha Sharma
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  2. Aman Kumar
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  3. Namita Singh
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  4. Sushila Maan
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Contributions

AK, NS conceptualised the study, AS carried out the experiments to generate data and drafted the manuscript. AK, AS analysed the data. AK, NS and SM edited the draft manuscript. All authors have read the manuscript.

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Correspondence to Aman Kumar or Namita Singh.

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Sharma, A., Kumar, A., Singh, N. et al. Novel Isothermal Reverse Transcription Polymerase Spiral Reaction (RT-PSR) Assay for the Detection of Newcastle Disease Virus in Avian Species. Indian J Microbiol (2024). https://doi.org/10.1007/s12088-024-01268-9

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