Equis ISSN 2398-2977

RT-LAMP assay: vesicular stomatitis virus detection

Synonym(s): Reverse transcription loop-mediated isothermal amplification assay for the rapid detection of vesicular stomatitis New Jersey virus, Field test for the detection of vesicular stomatitis New Jersey virus

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, an arbovirus, can affect humans and causes vesicular stomatitis (VS) in horses Vesicular stomatitis, 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 (FMD).
  • Loop-mediated isothermal amplification (LAMP) is an isothermal, autocyling, strand-displacement DNA amplification technique.
  • LAMP utilizes 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 VSNJV.
  • LAMP can be performed at a single temperature on a dedicated LAMP machine or using a heat block/water bath combined with simple, disposable visualization using molecular lateral-flow devices (LFD’s).
  • LAMP is a highly sensitive technique that allows for the detection of a very low number of RNA copies, with comparable analytical sensitivity to real-time reverse transcriptase PCR (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 straight into the RT-LAMP master-mix following a 1:10 dilution in nuclease free water.
  • Epithelial tissue: 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 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 protocol.
  • RT-LAMP is performed in a total reaction mixture of 25 µl containing: 15 µl isothermal master mix ISO-001, optimized primer concentrations Vesicular stomatitis: RT-LAMP amplification - oligonucleotide primers, 2 U AMV reverse transcriptase, 5 µl RNA template and made up to volume with nuclease-free water.
  • RT-LAMP reactions are run at 65°C/149°F for 30 min on a portable LAMP machine, eg Genie® II device.
  • Samples should be tested in duplicate.
  • Post-amplification anneal analysis should be performed on LAMP products by heating the LAMP reaction to 98°C/208.4°F for 1 min, then cooling to 80˚C/176oF decreasing at 0.05°C/32.09°F per sec (using fluorescence detection) using, eg the Genie® II.

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 lyophilized format already containing AMV and disease-specific primers.
  • Genotube swabs can be purchased from Thermo Fisher Scientific.

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, calibrated pipettes are required due to the requirement for aliquotting small volumes.

Technician (extrinsic) limitations

  • Test should only be performed by technicians who are familiar with working with exotic and zoonotic pathogens for the knowhow surrounding appropriate biosecurity Biosecurity and biosafety Laboratory: safety.
  • Test should only be performed by individuals who have a minimum understanding of the test protocol, pipette handling and test requirements.

Result Data

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Further Reading

Publications

Refereed Papers

  • Recent references from PubMed and VetMedResource.
  • Fowler V L 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. J Virol Methods 234, 123-131 PubMed.
  • Howson E L A et al (2015) Evaluation of two lyophilized molecular assays to rapidly detect foot-and-mouth disease virus directly from clinical samples in field settings. Transbound Emerg Dis PubMed.
  • Velazquez-Salinas L 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 449, 17-24 PubMed.
  • Waters R A 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), e105630 PubMed.
  • Rainwater-Lovett K et al (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 (7), 2042–2051 PubMed.
  • Notomi T (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28 (12), 2041-2051 PubMed.
  • Letchworth G J, Rodriguez L L & Barrera J C (1999) Vesicular stomatitis. Vet J 157 (3), 239-260 PubMed.

Other sources of information

  • Office International des Epizooties (2016) Terrestrial Animal Health Code. Website: www.oie.int. Last Accessed 17th November 2016.

Organisation(s)


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