Name: reverse transcription loop-mediated isothermal amplification (rt-lamp) assay for the specific and rapid detection of tilapia lake virus
Uploaded: 2020-05-18T14:00:00
Description: Watch this Scientific Journal Video about Reverse Transcription Loop-Mediated Isothermal Amplification RT-LAMP Assay for the Specific and Rapid Detection of Tilapia Lake Virus at JoVE.com

Projektet er finansieret af Thailand Research Fund (TRF) tilskud nummer RDG6050078 og Center for Advanced Studies for Landbrug og Fødevarer, Institut for Avancerede Studier, Kasetsart University, Bangkok, Thailand under Higher Education Research Promotion og National Research University Project of Thailand, Office of the Higher Education Commission, Ministry of Education, Thailand. Forskningen er delvist støttet af Graduate Program Scholarship fra Graduate School, Kasetsart University. Forfatterne vil gerne takke Dr. Kwanrawee Sirikanchana for den fortælling, der taler om videoen og Piyawatchara Sikarin for redigering af videoen.

Dette eksperiment, som omfattede anvendelse af animalsk væv, blev godkendt af Institutional Animal Care and Use Committee of Kasetsart University, Bangkok, Thailand (protokolnummer ACKU61-VET-009).
1. Vævsindsamling
<ol><li>Euthanize tilapia fisk ved hjælp af en overdosis af fed olie (dvs. mere end 3 ml/L). Tricaine methanesulfonat kan bruges som et alternativ til fed olie.</li>
	<li>Brug steril mayo saks og pincet til at skære åbne maven af postmortem tilapia og punktafgifter ca 30-50 ...

<ol>
	<li>Surachetpong, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outbreaks+of+Tilapia+Lake+Virus+Infection,+Thailand,+2015-2016.">Outbreaks of Tilapia Lake Virus Infection, Thailand, 2015-2016.</a> Emerging Infectious Diseases. 23 (6), 1031-1033 (2017).</li><li>Eyngor, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+novel+RNA+virus+lethal+to+tilapia.">Identification of a novel RNA virus lethal to tilapia.</a> Journal of Clinical Microbiology. 52 (12), 4137-4146 (2014).</li><li>Adams, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+to+taxonomy+and+the+International+Code+of+Virus+Classification+and+Nomenclature+ratified+by+the+International+Committee+on+Taxonomy+of+Viruses+(2017).">Changes to taxonomy and the International Code of Virus Classification and Nomenclature ratified by the International Committee on Taxonomy of Viruses (2017).</a> Archives of Virology. 162 (8), 2505-2538 (2017).</li><li>Bacharach, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+Novel+Orthomyxo-like+Virus+Causing+Mass+Die-Offs+of+Tilapia.">Characterization of a Novel Orthomyxo-like Virus Causing Mass Die-Offs of Tilapia.</a> MBio. 7 (2), 00431 (2016).</li><li>Jaemwimol, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Susceptibility+of+important+warm+water+fish+species+to+tilapia+lake+virus+(TiLV)+infection.">Susceptibility of important warm water fish species to tilapia lake virus (TiLV) infection.</a> Aquaculture. 497, 462-468 (2018).</li><li>Giews, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+Information+and+Early+Warning+System+On+Food+And+Agriculture.">Global Information and Early Warning System On Food And Agriculture.</a> Food and Agriculture Organization of the United Nations. , (2017).</li><li>Al-Hussinee, L., Subramaniam, K., Ahasan, M. S., Keleher, B., Waltzek, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+Genome+Sequence+of+a+Tilapia+Lake+Virus+Isolate+Obtained+from+Nile+Tilapia+(Oreochromis+niloticus).">Complete Genome Sequence of a Tilapia Lake Virus Isolate Obtained from Nile Tilapia (Oreochromis niloticus).</a> Genome Announcements. 6 (26), (2018).</li><li>Thammatorn, W., Rawiwan, P., Surachetpong, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+risk+of+tilapia+lake+virus+transmission+via+frozen+tilapia+fillets.">Minimal risk of tilapia lake virus transmission via frozen tilapia fillets.</a> Journal of Fish Diseases. 42 (1), 3-9 (2019).</li><li>Fathi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Tilapia+Lake+Virus+in+Egypt+in+Nile+tilapia+affected+by+'summer+mortality'+syndrome.">Identification of Tilapia Lake Virus in Egypt in Nile tilapia affected by 'summer mortality' syndrome.</a> Aquaculture. 473, 430-432 (2017).</li><li>Tattiyapong, P., Sirikanchana, K., Surachetpong, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+a+reverse+transcription+quantitative+polymerase+chain+reaction+for+tilapia+lake+virus+detection+in+clinical+samples+and+experimentally+challenged+fish.">Development and validation of a reverse transcription quantitative polymerase chain reaction for tilapia lake virus detection in clinical samples and experimentally challenged fish.</a> Journal of Fish Diseases. 41 (2), 255-261 (2018).</li><li>Waiyamitra, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+TaqMan+RT-qPCR+assay+for+tilapia+lake+virus+(TiLV)+detection+in+tilapia.">A TaqMan RT-qPCR assay for tilapia lake virus (TiLV) detection in tilapia.</a> Aquaculture. 497, 184-188 (2018).</li><li>Kembou Tsofack, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Tilapia+Lake+Virus+in+Clinical+Samples+by+Culturing+and+Nested+Reverse+Transcription-PCR.">Detection of Tilapia Lake Virus in Clinical Samples by Culturing and Nested Reverse Transcription-PCR.</a> Journal of Clinical Microbiology. 55 (3), 759-767 (2017).</li><li>Dong, H. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+of+tilapia+lake+virus+in+Thailand+and+an+alternative+semi-nested+RT-PCR+for+detection.">Emergence of tilapia lake virus in Thailand and an alternative semi-nested RT-PCR for detection.</a> Aquaculture. 476, 111-118 (2017).</li><li>Notomi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+of+DNA.">Loop-mediated isothermal amplification of DNA.</a> Nucleic Acids Research. 28 (12), 63 (2000).</li><li>Mori, Y., Notomi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+(LAMP):+a+rapid,+accurate,+and+cost-effective+diagnostic+method+for+infectious+diseases.">Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases.</a> Journal of Infection and Chemotherapy. 15 (2), 62-69 (2009).</li><li>Mori, Y., Nagamine, K., Tomita, N., Notomi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+loop-mediated+isothermal+amplification+reaction+by+turbidity+derived+from+magnesium+pyrophosphate+formation.">Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation.</a> Biochemical and Biophysical Research Communications. 289 (1), 150-154 (2001).</li><li>Caipang, C. M., Haraguchi, I., Ohira, T., Hirono, I., Aoki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+of+a+fish+iridovirus+using+loop-mediated+isothermal+amplification+(LAMP).">Rapid detection of a fish iridovirus using loop-mediated isothermal amplification (LAMP).</a> Journal of Virological Methods. 121 (2), 155-161 (2004).</li><li>Soliman, H., El-Matbouli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+inexpensive+and+rapid+diagnostic+method+of+Koi+Herpesvirus+(KHV)+infection+by+loop-mediated+isothermal+amplification.">An inexpensive and rapid diagnostic method of Koi Herpesvirus (KHV) infection by loop-mediated isothermal amplification.</a> Virology Journal. 2, 83 (2005).</li><li>Gunimaladevi, I., Kono, T., Venugopal, M. N., Sakai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+koi+herpesvirus+in+common+carp,+Cyprinus+carpio+L.,+by+loop-mediated+isothermal+amplification.">Detection of koi herpesvirus in common carp, Cyprinus carpio L., by loop-mediated isothermal amplification.</a> Journal of Fish Diseases. 27 (10), 583-589 (2004).</li><li>Gunimaladevi, I., Kono, T., Lapatra, S. E., Sakai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+loop+mediated+isothermal+amplification+(LAMP)+method+for+detection+of+infectious+hematopoietic+necrosis+virus+(IHNV)+in+rainbow+trout+(Oncorhynchus+mykiss).">A loop mediated isothermal amplification (LAMP) method for detection of infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss).</a> Archives of Virology. 150 (5), 899-909 (2005).</li><li>Kono, T., Savan, R., Sakai, M., Itami, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+white+spot+syndrome+virus+in+shrimp+by+loop-mediated+isothermal+amplification.">Detection of white spot syndrome virus in shrimp by loop-mediated isothermal amplification.</a> Journal of Virological Methods. 115 (1), 59-65 (2004).</li><li>Soliman, H., El-Matbouli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+transcription+loop-mediated+isothermal+amplification+(RT-LAMP)+for+rapid+detection+of+viral+hemorrhagic+septicaemia+virus+(VHS).">Reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of viral hemorrhagic septicaemia virus (VHS).</a> Veterinary Microbiology. 114 (3-4), 205-213 (2006).</li><li>Yeh, H. Y., Shoemaker, C. A., Klesius, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+loop-mediated+isothermal+amplification+method+for+rapid+detection+of+channel+catfish+Ictalurus+punctatus+important+bacterial+pathogen+Edwardsiella+ictaluri.">Evaluation of a loop-mediated isothermal amplification method for rapid detection of channel catfish Ictalurus punctatus important bacterial pathogen Edwardsiella ictaluri.</a> Journal of Microbiological Methods. 63 (1), 36-44 (2005).</li><li>Yeh, H. Y., Shoemaker, C. A., Klesius, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+and+rapid+detection+of+Flavobacterium+columnare+in+channel+catfish+Ictalurus+punctatus+by+a+loop-mediated+isothermal+amplification+method.">Sensitive and rapid detection of Flavobacterium columnare in channel catfish Ictalurus punctatus by a loop-mediated isothermal amplification method.</a> Journal of Applied Microbiology. 100 (5), 919-925 (2006).</li><li>Sun, Z. F., Hu, C. Q., Ren, C. H., Shen, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+and+rapid+detection+of+infectious+hypodermal+and+hematopoietic+necrosis+virus+(IHHNV)+in+shrimps+by+loop-mediated+isothermal+amplification.">Sensitive and rapid detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in shrimps by loop-mediated isothermal amplification.</a> Journal of Virological Methods. 131 (1), 41-46 (2006).</li><li>Shivappa, R. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+spring+viraemia+of+carp+virus+(SVCV)+by+loop-mediated+isothermal+amplification+(LAMP)+in+koi+carp,+Cyprinus+carpio+L.">Detection of spring viraemia of carp virus (SVCV) by loop-mediated isothermal amplification (LAMP) in koi carp, Cyprinus carpio L.</a> Journal of Fish Diseases. 31 (4), 249-258 (2004).</li><li>Wei, X. N., Zheng, Z. J., Zhang, L. H., Qu, F., Huang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+and+rapid+detection+of+Aeromonas+caviae+in+stool+samples+by+loop-mediated+isothermal+amplification.">Sensitive and rapid detection of Aeromonas caviae in stool samples by loop-mediated isothermal amplification.</a> Diagnostic Microbiology and Infectious Disease. 60 (1), 113-116 (2008).</li><li>Chinabut, S., Puttinaowarat, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+choice+of+disease+control+strategies+to+secure+international+market+access+for+aquaculture+products.">The choice of disease control strategies to secure international market access for aquaculture products.</a> Developmental Biology. 121, 255-261 (2005).</li><li>Soto, E., Yun, S., Surachetpong, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Susceptibility+of+Tilapia+Lake+Virus+to+buffered+Povidone-iodine+complex+and+chlorine.">Susceptibility of Tilapia Lake Virus to buffered Povidone-iodine complex and chlorine.</a> Aquaculture. 512, 734342 (2019).</li><li>Jaemwimol, P., Sirikanchana, K., Tattiyapong, P., Mongkolsuk, S., Surachetpong, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virucidal+effects+of+common+disinfectants+against+tilapia+lake+virus.">Virucidal effects of common disinfectants against tilapia lake virus.</a> Journal of Fish Diseases. 42 (10), 1383-1389 (2019).</li><li>Jansen, M. D., Dong, H. T., Mohan, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tilapia+lake+virus:+a+threat+to+the+global+tilapia+industry.">Tilapia lake virus: a threat to the global tilapia industry.</a> Reviews in Aquaculture. 11 (3), 725-739 (2019).</li><li>Yin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+simple+and+rapid+reverse+transcription-loopmediated+isothermal+amplification+(RT-LAMP)+assay+for+sensitive+detection+of+tilapia+lake+virus.">Development of a simple and rapid reverse transcription-loopmediated isothermal amplification (RT-LAMP) assay for sensitive detection of tilapia lake virus.</a> Journal of Fish Diseases. 42 (6), 817-824 (2019).</li><li>Savan, R., Kono, T., Itami, T., Sakai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification:+an+emerging+technology+for+detection+of+fish+and+shellfish+pathogens.">Loop-mediated isothermal amplification: an emerging technology for detection of fish and shellfish pathogens.</a> Journal of Fish Diseases. 28 (10), 573-581 (2005).</li><li>Phusantisampan, T., Tattiyapong, P., Mutrakulcharoen, P., Sriariyanun, M., Surachetpong, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+of+tilapia+lake+virus+using+a+one-step+reverse+transcription+loop-mediated+isothermal+amplification+assay.">Rapid detection of tilapia lake virus using a one-step reverse transcription loop-mediated isothermal amplification assay.</a> Aquaculture. 507, 35-39 (2019).</li><li>Liamnimitr, P., Thammatorn, W., U-thoomporn, S., Tattiyapong, P., Surachetpong, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-lethal+sampling+for+Tilapia+Lake+Virus+detection+by+RT-qPCR+and+cell+culture.">Non-lethal sampling for Tilapia Lake Virus detection by RT-qPCR and cell culture.</a> Aquaculture. 486, 75-80 (2018).</li><li>Yin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+simple+and+rapid+reverse+transcription-loopmediated+isothermal+amplification+(RT-LAMP)+assay+for+sensitive+detection+of+tilapia+lake+virus.">Development of a simple and rapid reverse transcription-loopmediated isothermal amplification (RT-LAMP) assay for sensitive detection of tilapia lake virus.</a> Journal of Fish Diseases. 42 (6), 817-824 (2019).</li><li>Khan, R. S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+of+infectious+bursal+disease+by+loop-mediated+isothermal+amplification+for+field+analysis.">Rapid detection of infectious bursal disease by loop-mediated isothermal amplification for field analysis.</a> Iranian Journal of Veterinary Research. 19 (2), 101-107 (2018).</li><li>Nagamine, K., Hase, T., Notomi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+reaction+by+loop-mediated+isothermal+amplification+using+loop+primers.">Accelerated reaction by loop-mediated isothermal amplification using loop primers.</a> Molecular and Cellular Probes. 16 (3), 223-229 (2002).</li><li>Debode, F., Marien, A., Janssen, E., Bragard, C., Berben, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+amplicon+length+on+real-time+PCR+results.">The influence of amplicon length on real-time PCR results.</a> Biotechnology, Agronomy, Society and Environment. 21 (1), 3-11 (2017).</li></ol>

Akvakulturindustrien trues konstant af virusinfektioner , der forårsager betydelige økonomiske tab9,23,28. F.eks. udgør den nye TiLV en stor trussel mod tilapiaproducerende lande i mange dele af verden1,6,9. Indtil nu har der ikke været nogen specifik terapeutisk til rådighed for at forhindre TiLVD. Mens udviklingen af en vaccine e...

Tilapia sø virus sygdom (TiLVD) er en virussygdom i tilapia(Oreochromis spp.), der efter sigende forårsager tilapia dødsfald i mange regioner i verden, herunder Asien1,2,Afrika og Amerika. Sygdommen blev først anerkendt under massedødeligheden af tilapia i 2009 i Israel, hvor antallet af vilde tilapia i Lake Kinneret styrtdykkede dramatisk fra 257 til 8 tons om året2. Sygdommen er forårsaget af tilapia sø virus (TiLV), som er blevet tildelt familien Amnoonviridae som en ny slægt Tilapinevirus og en ny art Tilapia tilapinevirus3. Genetisk karakterisering af TiLV viste, at virussen er en roman indhyllet, negativ-sans, enkeltstrenget RNA-virus, der har 10 segmenter kodning 10 proteiner1,2,4. Forskellige arter af tilapia i slægten Sarotherodon, Oreochromis, og Tilapin og andre varmt vand fisk (f.eks kæmpe gourami (Osphronemus goramy)) har vist sig at være modtagelige for TiLV2,5. I øjeblikket, denne virus fortsætter med at sprede sig globalt, muligvis gennem flytning af inficerede levende fisk6,7, mens risikoen for viral transmission via frosne tilapia eller dens produkt er begrænset8. Betydelig dødelighed som følge af TiLV-infektion har potentiale til at have en betydelig skadelig økonomisk indvirkning på tilapiaindustrien. For eksempel, de økonomiske konsekvenser af sommerdødelighed syndrom i Egypten i forbindelse med TiLV infektion blev beregnet til at være US $ 100 millioner9. Det er derfor vigtigt at udvikle en hurtig og korrekt diagnostisk metode til at lette kontrollen med denne sygdom i dambrug.
Indtil nu har diagnosen TiLVD været baseret på molekylære analyser, viral isolation og histopatologisk. Forskellige PCR protokoller og primere er blevet udviklet til TiLV diagnose10,11. For eksempel, en SYBR grøn-baseret omvendt transskription kvantitative PCR (RT-qPCR) metode med følsomhed til at opdage så få som to kopier / μL af virus er blevet udviklet og valideret for TiLV påvisning10. Andre PCR-metoder til TiLV-registrering omfatter TaqMan quantitative PCR11, RT-PCR2, indlejret RT-PCR12og semi-indlejret RT-PCR13. Disse metoder kræver imidlertid avanceret laboratorieudstyr og relativt længere tid til at give resultater på grund af reaktionernes kompleksitet, hvilket gør dem uegnede til anvendelse i marken.
Den loop-medieret isotermisk forstærkning (LAMP) assay er en hurtig, enkel og praktisk for-felt anvendelse14,15. Teknikken anvender princippet om en streng forskydning reaktion, mens forstærkning reaktion løber under isomale forhold uden en sofistikeret og dyr termisk cycler14,15. Derfor analyseres forstærkede LAMP-produkter eller RT-LAMP-produkter i stigelignende bånd ved hjælp af agarose gel elektroforese med en fluorescerende plet til enten sikker visualisering af DNA eller RNA14 eller observation med det blotte øje for tilstedeværelsen af turbiditet eller en hvid bundfald16,17,18. Denne teknik er derfor blevet anvendt til påvisning på stedet af forskellige fiskepatogener17,18,19,20,21,22,23,24,25,26,27. Formålet med denne undersøgelse var at etablere en hurtig, følsom og nøjagtig RT-LAMP-analyse til tilvdetektion. RT-LAMP-analysen tilbyder screening for TiLV i fiskeprøver inden for 30 min. Teknikken kan anvendes til diagnosticering og overvågning af TiLVD.

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			<td height="21" style="height:21px;width:255px;">Tissue collection:</td>
			<td style="width:224px;"></td>
			<td style="width:188px;"></td>
			<td style="width:295px;"></td>
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			<td height="21" style="height:21px;width:255px;">Clove oil</td>
			<td style="width:224px;">Better Pharma</td>
			<td style="width:188px;">N/A</td>
			<td></td>
		</tr>
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			<td height="22" style="height:22px;width:255px;">Tricaine methanesulfonate</td>
			<td style="width:224px;">Sigma-Aldrich</td>
			<td style="width:188px;">E10521</td>
			<td>An alternative option to clove oil</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">RNA extraction:</td>
			<td style="width:224px;"></td>
			<td style="width:188px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Acid guanidinium-phenol based reagent (TRIzol reagent)</td>
			<td style="width:224px;">ThermoFisher Scientific Corp.</td>
			<td style="width:188px;">15596026</td>
			<td style="width:295px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Acid guanidinium-phenol based reagent (GENEzol reagent)</td>
			<td style="width:224px;">Geneaid</td>
			<td style="width:188px;">GZR100</td>
			<td style="width:295px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Direct-zol RNA Kit:</td>
			<td style="width:224px;">Zymo Research</td>
			<td style="width:188px;">R2071</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">- Direct-zol RNA PreWash</td>
			<td style="width:224px;"></td>
			<td style="width:188px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">- RNA Wash Buffer</td>
			<td style="width:224px;"></td>
			<td style="width:188px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">- DNase/RNase-free water</td>
			<td style="width:224px;"></td>
			<td style="width:188px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">- Zymo-spin IIICG columns</td>
			<td style="width:224px;"></td>
			<td style="width:188px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">- Collection Tubes</td>
			<td style="width:224px;"></td>
			<td style="width:188px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">RT-LAMP:</td>
			<td style="width:224px;"></td>
			<td style="width:188px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">1x SD II reaction buffer</td>
			<td style="width:224px;">Biotechrabbit</td>
			<td style="width:188px;">BR1101301</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:255px;">Magnesium sulfate (MgSO4)</td>
			<td style="width:224px;">Sigma-Aldrich</td>
			<td style="width:188px;">7487-88-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">dNTP set</td>
			<td style="width:224px;">Bioline</td>
			<td style="width:188px;">BIO-39053</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Betaine</td>
			<td style="width:224px;">Sigma-Aldrich</td>
			<td style="width:188px;">B2629</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">Calcein mixture</td>
			<td style="width:224px;">Merck</td>
			<td style="width:188px;">1461-15-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Bst DNA polymerase</td>
			<td style="width:224px;">Biotechrabbit</td>
			<td style="width:188px;">BR1101301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">AMV reverse transcriptase</td>
			<td style="width:224px;">Promega</td>
			<td style="width:188px;">M510A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Nuclease-free water</td>
			<td style="width:224px;">Invitrogen</td>
			<td style="width:188px;">10320995</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">Elite dry bath incubator, single unit</td>
			<td style="width:224px;">Major Science</td>
			<td style="width:188px;">EL-01-220</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">Gel electrophoresis:</td>
			<td style="width:224px;"></td>
			<td style="width:188px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Agarose</td>
			<td style="width:224px;">Vivantis Technologies</td>
			<td style="width:188px;">PC0701-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tris-borate-EDTA (TBE) buffer</td>
			<td style="width:224px;">Sigma-Aldrich</td>
			<td style="width:188px;">SRE0062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tris-acetic-EDTA (TAE) buffer:</td>
			<td style="width:224px;"></td>
			<td style="width:188px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">- Tris</td>
			<td style="width:224px;">Vivantis Technologies</td>
			<td style="width:188px;">PR0612-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">- Acetic acid (glacial), EMSURE</td>
			<td style="width:224px;">Merck Millipore</td>
			<td style="width:188px;">1000632500</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:255px;">- Disodium Ethylenediaminetetraacetate dihydrate (EDTA), Vetec</td>
			<td style="width:224px;">Sigma-Aldrich</td>
			<td style="width:188px;">V800170-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Neogreen</td>
			<td style="width:224px;">NeoScience Co., Ltd.</td>
			<td style="width:188px;">GR107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">DNA gel loading dye (6X)</td>
			<td style="width:224px;">ThermoFisher Scientific Corp.</td>
			<td style="width:188px;">R0611</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">DNA ladder and markers</td>
			<td style="width:224px;">Vivantis Technologies</td>
			<td style="width:188px;">PC701-100G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Mini Ready Sub-Cell GT (Horizontal electrophoresis system)</td>
			<td style="width:224px;">Bio-Rad</td>
			<td style="width:188px;">1704487</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">PowerPac HC power supply</td>
			<td style="width:224px;">Bio-Rad</td>
			<td style="width:188px;">1645052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Gel Doc EZ System</td>
			<td style="width:224px;">Bio-Rad</td>
			<td style="width:188px;">1708270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">UV sample tray</td>
			<td style="width:224px;">Bio-Rad</td>
			<td style="width:188px;">1708271</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">N&#945;BI imager</td>
			<td style="width:224px;">Neogene Science</td>
			<td style="width:188px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">cDNA synthesis:</td>
			<td style="width:224px;"></td>
			<td style="width:188px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">ReverTra Ace qPCR RT Kit</td>
			<td style="width:224px;">Toyobo</td>
			<td style="width:188px;">FSQ-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Viva cDNA Synthesis Kit</td>
			<td style="width:224px;">Vivantis Technologies</td>
			<td style="width:188px;">cDSK01</td>
			<td>An alternative option for cDNA synthesis</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">NanoDrop2000 (microvolume spectrophotometer)</td>
			<td style="width:224px;">ThermoFisher Scientific Corp.</td>
			<td style="width:188px;">ND-2000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">T100 Thermal Cycler</td>
			<td style="width:224px;">Bio-Rad</td>
			<td style="width:188px;">1861096</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">RT-qPCR:</td>
			<td style="width:224px;"></td>
			<td style="width:188px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">iTaq Universal SYBR Green Supermix</td>
			<td style="width:224px;">Bio-Rad</td>
			<td style="width:188px;">1725120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Nuclease-free water, sterile water</td>
			<td style="width:224px;">MultiCell</td>
			<td style="width:188px;">809-115-CL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">8-tube PCR strips, white</td>
			<td style="width:224px;">Bio-Rad</td>
			<td style="width:188px;">TLS0851</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Flat PCR tube 8-cap strips, optical</td>
			<td style="width:224px;">Bio-Rad</td>
			<td style="width:188px;">TCS0803</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">CFX96 Touch Thermal Cycler</td>
			<td style="width:224px;">Bio-Rad</td>
			<td style="width:188px;">1855196</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">General equipment and materials:</td>
			<td style="width:224px;"></td>
			<td style="width:188px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Mayo scissors</td>
			<td style="width:224px;"></td>
			<td style="width:188px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Forceps</td>
			<td style="width:224px;"></td>
			<td style="width:188px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Vortex Genie 2 (vortex mixer)</td>
			<td style="width:224px;">Scientific Industries</td>
			<td style="width:188px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Microcentrifuge LM-60</td>
			<td style="width:224px;">LioFuge</td>
			<td style="width:188px;">CM610</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Corning LSE mini microcentrifuge</td>
			<td style="width:224px;">Corning</td>
			<td style="width:188px;">6765</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Pipettes</td>
			<td style="width:224px;">Rainin</td>
			<td style="width:188px;">Pipete-Lite XLS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">QSP filtered pipette tips</td>
			<td style="width:224px;">Quality Scientific Plastics</td>
			<td style="width:188px;">TF series</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Corning Isotip filtered tips</td>
			<td style="width:224px;">Merck</td>
			<td style="width:188px;">CLS series</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:255px;">Nuclease-free 1.5 mL microcentrifuge tubes, NEST</td>
			<td style="width:224px;">Wuxi NEST Biotechnology</td>
			<td style="width:188px;">615601</td>
			<td></td>
		</tr>
	</tbody>
</table>

reverse transcription loop-mediated isothermal amplification (rt-lamp) assay for the specific and rapid detection of tilapia lake virus

Tilapia sø virus sygdom (TiLVD), en spirende virussygdom i tilapia forårsaget af tilapia sø virus (TiLV), er en vedvarende udfordring i akvakulturindustrien, der har resulteret i masse sygelighed og dødelighed af tilapia i mange dele af verden. Det er derfor nødvendigt med en effektiv, hurtig og nøjagtig diagnostisk analyse for TiLV-infektion for at påvise den oprindelige infektion og forhindre spredning af sygdommen i akvakulturbrug. I denne undersøgelse, en meget følsom og praktisk omvendt transskription loop-medieret isotermisk forstærkning (RT-LAMP) metode præsenteres for at opdage tilapia sø virus i fiskevæv. En sammenligning af RT-qPCR- og RT-LAMP-analyserne af inficerede prøver viste positive resultater i 63 (100 %) og 51 (80,95 %) prøver. Desuden viste en analyse af ikke-inficerede prøver, at alle 63 ikke-inficerede væv gav negative resultater for både RT-qPCR- og RT-LAMP-analyserne. Krydsreaktivitet med fem patogener i tilapia blev evalueret ved hjælp af RT-LAMP, og alle test viste negative resultater. Både lever- og slimprøver fra inficerede fisk viste sammenlignelige resultater ved hjælp af RT-LAMP-metoden, hvilket tyder på, at slim kan anvendes i RT-LAMP som en ikke-dødelig analyse for at undgå at dræbe fisk. Resultaterne viste, at den præsenterede RT-LAMP-analyse giver en effektiv metode til TiLV-detektion i tilapiavæv inden for 1 time. Metoden anbefales derfor som screeningsværktøj på bedrifterne med henblik på hurtig diagnosticering af TiLV.

I denne undersøgelse blev der udviklet en RT-LAMP-analyse til påvisning af TiLV-infektion i tilapia. De testede prøver blev indsamlet fra 14 gårde i forskellige dele af Thailand mellem 2015 og 2016. De inficerede og ikke-inficerede fisk var primært grupperet baseret på fysiske diagnoser og optrædener af symptomatisk TiLVD. TiLV-infektion blev efterfølgende bekræftet ved hjælp af RT-PCR efter indsamlingsprocessen. Agarose gel elektroforese og påvisning af en selvlysende grøn farve blev valgt som evalueringsmet...

Watch this Scientific Journal Video about Reverse Transcription Loop-Mediated Isothermal Amplification RT-LAMP Assay for the Specific and Rapid Detection of Tilapia Lake Virus at JoVE.com

Reverse Transcription Loop-Mediated Isothermal Amplification RT-LAMP Assay for the Specific and Rapid Detection of Tilapia Lake Virus

Vi præsenterer en RT-LAMP analyse til påvisning af TiLV i tilapia fisk ved hjælp af enkle instrumenter over en relativt kort periode i forhold til konventionelle RT-PCR teknikker. Denne protokol kan bidrage til at kontrollere den epidemiske spredning af TiLVD, især i udviklingslandene.

Omvendt transskription Loop-medieret Isothermal Amplification (RT-LAMP) Assay for den specifikke og hurtige påvisning af Tilapia Lake Virus

Tilapia lake virus disease (TiLVD), an emerging viral disease in tilapia caused by the tilapia lake virus (TiLV), is a persistent challenge in the ...

Tilapia søvirus er en spirende virus og smitsomme patogener, der forårsager høj dødelighed i tilapia og dens hybride arter. Da tilapia er en vigtig proteinkilde for mange udviklingslande, har den nye virusinfektion en stor indvirkning på tilapia-akvakulturen og truer også verdens fødevaresikkerhed. Udviklingen af en hurtig, præcis og følsom metode til at opdage virus i fiskevæv, vil hjælpe sygdomsbekæmpelse, og reducere virkningen af virus.I denne video vil vi vise den omvendte transskription loop-medieret isotermisk forstærkning, eller RT-LAMP assay, til hurtig påvisning af tilapia sø virus i fisk prøver. Del to, RNA-ekstraktion. For det første tilsættes 3250 milligram levervæv i et sterilt 1,5 milliliter mikrocentrifugerør, indeholdende 600 til 1000 mikroliters syre guanidinium-phenol base reagens, og hakket indtil homogen.Centrifuge ved 10.000 RCF i 30 sekunder, og overfør supernatanten til et nyt rør. Tilsæt et tilsvarende volumen på 95% ethanol til røret, og bland grundigt. Overfør opløsningen til en spin kolonne, placeret i en samling rør, derefter centrifuge ved 10, 000 RCF i 30 sekunder.Kassér flow-through, og overfør kolonnen til et nyt opsamlingsrør. Tilsæt 400 mikroliter RNA PreWash i kolonnen, og centrifuge ved 10.000 RCF i 30 sekunder. Kassér flow-through, og gentag dette trin.Der tilsættes 700 mikroliter RNA-vaskebuffer i kolonnen, og centrifugeres ved 10.000 RCF i to minutter. Derefter overføres kolonnen til et sterilt 1,5 milliliter mikrocentrifugerør. Endelig eluterer RNA i kolonnematrixen med en 100-mikroliter, nukleasefrit vand og centrifuge ved 10.000 RCF i 30 sekunder.Del tre, primer design. PrimerExplorer Version 4 er det mest almindelige bioinformatikværktøj til at designe effektive LAMP- eller RT-LAMP-primere, hvor gode manualer til softwaren er tilgængelige på deres hjemmeside. Her blev fire specifikke primere, der består af to interne og to eksterne primere, designet til at genkende seks forskellige regioner i segment 3 af TiLV, stammer fra Thailand.Når du har indtastet hjemmesiden, hvor linket er vist i videoen, skal du klikke på PrimerExplorer V4. Vælg den fil, der indeholder sekvensen af segment 3 af TiLV, i FASTA-format, hentet fra GenBank-adgang nummer, KX631923, eller tilapia sø virus TV1 segment 3, og klik derefter Primer Design knap. Når du klikker på Generer knappen, vil softwaren behandle, og resultaterne vil dukke op. Efter optimering af sekvenserne for LAMP forstærkning, i alt, et par eksterne primere, og to par interne primere, blev udvalgt til denne undersøgelse, at opdage TiLV infektion.Dette sæt af primere er vist her. Del fire, demonstration af RT-LAMP. Master mix for RT-LAMP specifikke for tilapia søvirus, består af følgende kemikalier.Forbered en master mix ved hjælp af de kemikalier, der er vist i tabellen. Dispenseres 22 mikroliter af master mix i et sterilt 1,5 milliliter mikrocentrifugerør. Derefter tilsættes tre mikroliter RNA-skabelon i reaktionsrøret.For den negative kontrol, bruge destilleret vand i stedet for RNA materialer, og bland godt. Inkuber reaktionerne ved 65 grader Celsius i 60 minutter, efterfulgt af 80 grader Celsius i 10 minutter. Efter inkubationen i 30 minutter observeres de kalorimetriske ændringer visuelt med nøgne øjne.Hvor det er relevant, kan vi bekræfte RT-LAMP assay resultater, ved at udføre gel elektroforese på 1,5% agarose gel, der kører med en 100-Volt konstant, i 40 minutter, hvorved en-kilobase-par molekylær størrelse DNA Ladder er indlæst sammen som en markør til at bestemme fragment størrelse. Del fem, repræsentative resultater og fortolkning. Efter inkubationen i 30 minutter blev kornfluorescensreaktionen, der antyder den positive forstærkning af TiLV i fiskevæv, tydeligvis observeret i inficerede prøver under UV-lys ved en bølgelængde på 365 nanometer, mens de negative kontrolmeter havde en klar og let farveløs opløsning.Tilsvarende blev forstærkningen af RT-LAMP-produkter bekræftet på agarosegelelektroforese, der viste forskellige størrelser af de mange forstærkninger af omvendte DNA-gentagelser, der danner stængelsløjfstrukturer under UV-lampe i TiLV-inficerede prøver, mens der ikke blev vist bånd i brøndene i den negative kontrol, hvor resultaterne var i overensstemmelse med resultaterne af RT-LAMP-analysen. Så for at reducere spredningen af tilapia sø virus, en øjeblikkelig screening af disse inficerede fisk, ved hjælp af en effektivt diagnostisk metode, er en af de vigtige nøgler. Så den nyudviklede RT-LAMP analyse bør tilbyde en hurtig og følsom diagnostisk værktøj, der skal anvendes i processen med biosikkerhed foranstaltning.Analysen kan anvendes enten i laboratoriet eller på stedet. Vi håber, at metoden vil blive gennemført for at screene inficerede fisk befolkning, der kan føre til en reduktion af spredningen af tilapia sø virus.

Research

JoVE Journal

Immunology and Infection

Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for the Specific and Rapid Detection of Tilapia Lake Virus

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

High-throughput Detection Method for Influenza Virus

High-throughput Detection Metode til Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Hurtig screening af HIV revers transkriptase og Integrase hæmmere

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Loop-medieret Isotermisk Amplification (lampe) assays til Art-specifik påvisning af<em&gt; Eimeria</em&gt; Der inficerer Kyllinger

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Nasal Wipes for influenza A-virus Detection og Isolering fra svin

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Udviklingen af ​​Frysetørret Loop-medierede Isotermiske Amplification reagenser til påvisning af<em&gt; Coxiella burnetii</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

In Vivo Assay til påvisning af Antigen-specifikke T-celle cytolytisk funktion ved hjælp af en Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Zika Virus Specific Diagnostic Epitope Discovery

Zika Virus specifikke diagnostiske epitop opdagelse

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for ...