Bovis ISSN 2398-2993

Vesicular stomatitis - testing using reverse transcriptase loop-mediated isothermal amplification

Synonym(s): molecular diagnostic field test

Contributor(s): Veronica Fowler , Valerie Mioulet

Overview

  • Vesicular Stomatitis New Jersey virus (VSNJV), in the family Rhabdoviridae, genus Vesiculovirus, is endemic in southern Mexico, Central America and northern regions of South America (Colombia, Venezuela, Ecuador and Peru).
  • VSNJV is an arbovirus that can affect humans and causes vesicular stomatitis (VS) in horses, cattle and pigs.
  • There have been a number of multiyear outbreaks of VSNJV in the United States which can cause heightened concern when the disease is observed in cattle or pigs due to the similarity in clinical presentation to Foot and mouth disease Foot and mouth disease (FMD).
  • Loop-mediated isothermal amplification (LAMP) is an isothermal, autocycling, strand-displacement DNA amplification technique.
  • LAMP utilises 4-6 primers which target 6-8 regions of the pathogen’s genome.
  • LAMP can combine a reverse transcription step (RT-LAMP) to detect RNA viruses such as VSV.
  • LAMP can be performed at a single temperature on a dedicated LAMP machine or be performed using a heat block/water bath combined with simple, disposable visualisation using molecular lateral-flow devices (LFD’s).
  • LAMP is a highly sensitive technique that allows for the detection of a very low number of copies of RNA molecules with comparable analytical sensitivity to rRT-PCR.
  • LAMP assays can be multiplexed to detect multiple pathogens in a single reaction.

Sampling

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Tests

Methodologies

Sample types
  • Vesicular fluid: 
    • This sample requires no sample preparation. 
    • RNA can be directly extracted from it or this sample can be added directly into the RT-LAMP assay following a 1:10 dilution in nuclease free water.
  • Epithelial tissue: 
    • Epithelial suspensions are prepared from epithelial tissue at 10% (w/v) in M25 phosphate buffer (35 mM Na2HPO4 , 5.7 mM KH2PO4, pH 7.6).
  • Swabs:  
    • Lesion swabs can be directly placed in 1ml phosphate buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4). 
    • RNA can be directly extracted from it or this sample can be added directly into the RT-LAMP assay following a 1:10 dilution in nuclease free water.
Protocol
  • Extract RNA in duplicate from the sample (epithelial suspensions or swabs) using an appropriate extraction kit.
  • RT-LAMP is performed in a total reaction mixture of 25 µl containing: 15 µl isothermal master mix ISO-001 (OptiGene Ltd, UK), optimised primer concentrations (Table 1), 2 U AMV reverse transcriptase (New England Biolabs, UK), 5 µl RNA template and made up to volume with nuclease-free water.
  • RT-LAMP reactions are run at 65°C for 30 minutes on a portable LAMP machine (eg Genie® II device, OptiGene Ltd, UK).
  • Samples should be tested in duplicate.
  • Post-amplification anneal analysis should be performed on LAMP products by heating the LAMP reaction to 98°C for 1 minute, then cooling to 80˚C decreasing at 0.05°C/s (using fluorescence detection) using the Genie® II (OptiGene Ltd, UK).

Availability

  • Genie® II can be purchased from OptiGene Ltd, UK.
  • ISO-001 can be purchased from OptiGene Ltd, UK in either a wet format for the user to add primers and AMV or in a lyophilised format already containing AMV and disease specific primers.

Validity

Sensitivity

  • The analytical sensitivity of the VSNJV RT-LAMP assay is 101 RNA copies. 

Specificity

  • The specificity of the VSNJV RT-LAMP assay is 100% when assessed against related (VS Indiana virus) and differential viruses such as FMDV and swine vesicular disease virus (SVDV).

Technique (intrinsic) limitations

  • Good quality pipettes are required for the dispensing of small volumes.

Technician (extrinsic) limitations

  • Test should only be performed by technicians who are familiar with working with exotic and zoonotic pathogens and have the knowhow surrounding appropriate biosecurity and biosafety procedures.

Result Data

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Further Reading

Publications

Refereed Papers

  • Recent references from PubMed and VetMed Resource.
  • Fowler V L, Howson E L A, Madi M, Mioulet V et al (2016) Development of a reverse transcription loop-mediated isothermal amplification assay for the detection of vesicular stomatitis New Jersey virus: Use of rapid molecular assays to differentiate between vesicular disease viruses. Journal of Virological Methods 234 pp 123-131.
  • Howson E L A, Armson B, Madi M, Kasanga C J et al (2015) Evaluation of Two Lyophilised Molecular Assays to Rapidly Detect Foot-And-Mouth Disease Virus Directly from Clinical Samples in Field Settings. Transboundary and Emerging Diseases 64 (3) pp 861-871.
  • Velazquez-Salinas L, Pauszek S J, Zarate S, Basurto-Alcantara F J et al (2014) Phylogeographic characteristics of vesicular stomatitis New Jersey viruses circulating in Mexico from 2005 to 2011 and their relationship to epidemics in the United States. Virology 20 (449) pp 17-24.
  • Waters R A, Fowler V L, Armson B, Nelson N et al (2014) Preliminary validation of direct detection of foot-and-mouth disease virus within clinical samples using reverse transcription loop-mediated isothermal amplification coupled with a simple lateral flow device for detection. PLoS one 9 (8).
  • Rainwater-Lovett K, Pauszek S J, Kelley W N & Rodriguez L L (2007) Molecular epidemiology of vesicular stomatitis New Jersey virus from the 2004–2005 US outbreak indicates a common origin with Mexican strains. J Gen Virol 88 pp 2042–2051.
  • Notomi T, Okayama H, Masubuchi H, Yonekawa T et al (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Research 28 (12) pp 63.
  • Letchworth G J, Rodriguez L L & Barrera J C (1999) Vesicular stomatitis. Vet J 157 pp 239–260.

Other sources of information

  • OIE (2015) Terrestrial Animal Health Code. [Online] Available at: www.oie.int/en.

Organisation(s)

  • The Pirbright Institute, Ash Road, Pirbright, Woking, Surrey, GU24 0NF, UK.


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