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RT-LAMP assay: African Horse Sickness virus detection
Synonym(s): Reverse transcription loop-mediated isothermal amplification assay for the rapid detection of African Horse Sickness virus, Field test for the detection of African Horse Sickness virus
Overview
- Laboratory confirmation of clinical suspicion of African horse sickness (AHS) African horse sickness has been traditionally made by detection of AHS virus (ASHV) in blood or post-mortem tissues using classical virus isolation (VI), immunohistochemistry or antigen detection ELISA techniques.
- However these methods have now been superseded by the adoption of more rapid, sensitive and accurate molecular diagnostic techniques such as reverse-transcription-PCR (RT-PCR) and real-time RT-PCR (rRT-PCR).
- These assays are widely used in most AHSV diagnostic laboratories and are the recommended tests by the OIE (World Organisation for Animal Health) (OIE, 2012) not only for confirmation of clinical diagnosis but also for animal certification purposes, import/export testing and surveillance.
- Whilst current RT-PCR and rRT-PCR methods are accurate, rapid and sensitive, they are required to be performed in laboratory settings and samples must be transported under the appropriate conditions from the point of collection to the laboratory. This process can delay the confirmation of suspicion of AHSV infection and the implementation of adequate control measures.
- The development of rapid field-based diagnostic assays that can be used either in the laboratory or at the point of sample collection therefore enable the fast implementation of animal movement controls, since unlike rRT-PCR a result is obtained within 30 min.
- 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 AHSV.
- LAMP can be performed at a single temperature using either a fluorimeter, eg Genie® II device or PCR thermocycler, or be performed 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 nucleic acid copies with comparable analytical sensitivity to rRT-PCR.
- LAMP assays can be multiplexed to detect multiple pathogens in a single reaction.
Uses
Alone
- Within the literature there are an increasing number of LAMP assays for the detection of veterinary pathogens.
- The most common application of LAMP is as a simple screening assay to rapidly confirm disease (<30 min) by detecting very low amounts of circulating pathogen nucleic acid (DNA or RNA). This protocol describes exactly this method.
- However, in combination with the development of portable LAMP platforms or visualization of LAMP products by LFD, enables disease confirmation at the point of suspicion, eg on the stable yard.
- Quick confirmation of disease enables control measures to be implemented immediately.
In combination
- At present results generated from the use of the AHSV LAMP assay should still be supported by routine laboratory diagnostics as recommended by the OIE, eg:
- Classical virus isolation (VI).
- Immunohistochemistry Immunostaining: immunohistochemistry / immunocytochemistry - direct and indirect.
- Antigen detection ELISA techniques Enzyme linked immunosorbent assay (ELISA).
Other points
- LAMP can be performed in laboratory settings using automated nucleic acid extraction platforms, or can be performed in the field using manual extraction kits.
- LAMP assays are highly sensitive with comparable analytical sensitivity to rRT-PCR.
- LAMP assays are less prone to sample derived inhibition and can be performed on a wide range of clinical samples. Some LAMP assays have been validated for use without combining an extraction step.
Sampling
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Tests
Methodologies
- Extract RNA in duplicate from the sample (EDTA blood or tissue) using an appropriate extraction kit.
- Heat the extracted RNA at 95˚C/203˚F for 5 min.
- RT-LAMP is performed in a total reaction mixture of 25 µl containing: 15 µl isothermal master mix ISO-001, optimized primer concentrations African Horse Sickness: RT-LAMP amplification - oligonucleotide primers, 2 U (0.2 µl) 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/176˚F decreasing at 0.05˚C/39.09˚F per sec (using fluorescence detection) using the portable LAMP machine, eg Genie® II.
Availability
- The 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.
Validity
Sensitivity
- The AHSV RT-LAMP analytical sensitivity is comparable to rRT-PCR over a 4 log10 dilution range.
- The AHSV RT-LAMP diagnostic sensitivity is 97.4%.
Specificity
- The specificity of the AHSV RT-LAMP assay is 100% when assessed against related viruses such as Bluetongue virus (BTV) and Equine encephalosis virus (EEV).
Technique intrinsic limitations
- At present the test has only been validated for use on extracted RNA, therefore an extraction kit (either manual or automated) must be used in combination.
- At present the test must be performed and analysed on a fluorimeter platform, eg field based such as the Genie® II or any laboratory based thermocycler.
- Good quality pipettes are required due to the aliquotting of small volumes.
Technician extrinsic limitations
- Test should only be performed by technicians whom are familiar with working with exotic pathogens for the knowhow surrounding appropriate biosecurity Biosecurity and biosafety Laboratory: safety.
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.
- Fowler V L (2016) Development of a novel RT-LAMP assay for the rapid detection of African Horse Sickness Virus. Transbound Emerg Dis 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.
- 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.
- Guthrie A J (2013) Diagnostic accuracy of a duplex real-time reverse transcription quantitative PCR assay for detection of African horse sickness virus. J Virol Methods 189 (1), 30-35 PubMed.
- Quan M et al (2010) Development and optimisation of a duplex real-time reverse transcription quantitative PCR assay targeting the VP7 and NS2 genes of African horse sickness virus. J Virol Methods 167 (1), 45-52 PubMed.
- Fernandez-Pinero J et al (2009) Rapid and sensitive detection of African horse sickness virus by real-time PCR. Res Vet Sci 86 (2), 353-358 PubMed.
- Aguero M et al (2008) Real-time fluorogenic reverse transcription polymerase chain reaction assay for detection of African horse sickness virus. J Vet Diagn Invest 20 (3), 325-328 PubMed.
- Maree S & Paweska J T (2005) Preparation of recombinant African horse sickness virus VP7 antigen via a simple method and validation of a VP7-based indirect ELISA for the detection of group-specific IgG antibodies in horse sera. J Virol Methods 125 (1), 55-65 PubMed.
- Notomi T (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28 (12), E63 PubMed.
- Sailleau C (1997) Detection of African horse sickness virus in the blood of experimentally infected horses: comparison of virus isolation and a PCR assay. Res Vet Sci 62 (3), 229-232 PubMed.
- House J A (1996) A blocking ELISA for detection of antibody to a subgroup-reactive epitope of African horsesickness viral protein 7 (VP7) using a novel gamma-irradiated antigen. Ann N Y Acad Sci 791, 333-344 PubMed.
- Zientara S et al (1995) Application of the polymerase chain reaction to the detection of African horse sickness viruses. J Virol Methods 53 (1), 47-54 PubMed.
- Bremer C W, du Plessis D H & van Dijk A A (1994) Baculovirus expression of non-structural protein NS2 and core protein VP7 of African horsesickness virus serotype 3 and their use as antigens in an indirect ELISA. J Virol Methods 48 (2-3), 245-256 PubMed
- Zientara S et al (1993) Diagnosis and molecular epidemiology of the African horse sickness virus by the polymerase chain reaction and restriction patterns. Vet Res 24 (5), 385-395 PubMed.
- Chuma T et al (1992) Expression of the major core antigen VP7 of African horsesickness virus by a recombinant baculovirus and its use as a group-specific diagnostic reagent. J Gen Virol 73 (4), 925-931 PubMed.
- Laviada M D et al (1992) Detection of African horsesickness virus in infected spleens by a sandwich ELISA using two monoclonal antibodies specific for VP7. J Virol Methods 38 (2), 229-242 PubMed.
Other sources of information
- Office International des Epizzoties (2012) African Horse Sickness. In: OIE Manual of Standards for Diagnostic Tests and Vaccines. OIE, Paris, France.
- Office International des Epizzoties (2016) Terrestrial Animal Health Code. Website: www.oie.int/en/international-standard-setting/terrestrial-code/access-online. Last Accessed 17th November 2016.
Organisation(s)
- New England Biolabs Inc. Website: www.neb.com.
- OptiGene Ltd, UK. Website: www.optigene.co.uk
- The Pirbright Institute, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK. Website: www.pirbright.ac.uk.
- Thermo Fisher Scientific. Website: www.thermofisher.com