Detection of equine rotavirus by reverse transcription loop-mediated isothermal amplification (RT-LAMP).
Abstract: Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was applied to detection of equine rotavirus. Because equine rotavirus of the single P genotype, P[12], is predominant in the equine population worldwide, an RT-LAMP primer set was designed to target the genotype P[12] sequence and thus detect equine rotavirus. The detection limit of the RT-LAMP assay was 10(3) copies of viral RNA, whereas that of semi-nested RT-PCR for genotype P[12] was 10(5) copies. The RT-LAMP assay specifically amplified genotype P[12] but did not amplify the other P genotype strains. The RT-LAMP assay did not amplify any pathogens related to equine intestinal disorder other than rotavirus. Using 96 diarrheal stools, the RT-LAMP assay detected equine rotavirus in 58 samples, whereas semi-nested RT-PCR only detected equine rotavirus in 25 samples. The RT-LAMP assay did not detect equine rotavirus with fecal samples collected from nine healthy foals. These results indicate that the RT-LAMP assay is specific for equine rotavirus and more sensitive than semi-nested RT-PCR. Because it is easy to manipulate without the need for a thermal cycler or gel electrophoresis, the RT-LAMP assay should be applicable to diagnosis of equine rotavirus infections in diagnostic laboratories.
Publication Date: 2010-02-16 PubMed ID: 20160420DOI: 10.1292/jvms.09-0446Google Scholar: Lookup
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- Journal Article
Summary
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The research focuses on the process of detecting equine rotavirus using reverse transcription loop-mediated isothermal amplification (RT-LAMP) instead of the traditional semi-nested reverse transcription polymerase chain reaction (RT-PCR). The study suggests that RT-LAMP is a more sensitive and specific approach than semi-nested RT-PCR for this purpose.
Overview of the Research Process
- The research spotlights the application of RT-LAMP in detecting equine rotavirus, a disease that has predominantly plagued the global horse population with a single P genotype, P[12].
- Researchers designed an RT-LAMP primer set to specifically target the P[12] genotype sequence in order to detect the equine rotavirus.
Comparison Between RT-LAMP and Semi-Nested RT-PCR
- The researchers discovered that the RT-LAMP testing method had a significantly lower detection limit (10^3 copies of viral RNA) compared to semi-nested RT-PCR (10^5 copies).
- It was found that the RT-LAMP test specifically amplified the P[12] genotype but did not react to other P genotypes.
- The RT-LAMP method did not detect any other pathogens related to equine intestinal disorders apart from the rotavirus. Consequently, its specificity for equine rotavirus is exceptional.
Testing Using Diarrhoeal Stool Samples
- By applying RT-LAMP to 96 diarrheal stool samples, the researchers were able to detect equine rotavirus in 58 samples, showing a superior detection rate compared to semi-nested RT-PCR, which only detected the virus in 25 samples.
- Furthermore, healthy fecal samples from nine foals were tested with the RT-LAMP assay, and all came back negative for equine rotavirus.
Implications and Applications of the Research
- Given its superior sensitivity and specificity compared to semi-nested RT-PCR, RT-LAMP emerges as a robust method for detecting equine rotavirus.
- The fact that RT-LAMP does not require a thermal cycler or gel electrophoresis adds to its advantages as it simplifies the detection process, making it highly valuable in diagnosis of equine rotavirus infections in diagnostic laboratories.
Cite This Article
APA
Nemoto M, Imagawa H, Tsujimura K, Yamanaka T, Kondo T, Matsumura T.
(2010).
Detection of equine rotavirus by reverse transcription loop-mediated isothermal amplification (RT-LAMP).
J Vet Med Sci, 72(6), 823-826.
https://doi.org/10.1292/jvms.09-0446 Publication
Researcher Affiliations
- Epizootic Research Center, Equine Research Institute, Japan Racing Association, Shiba, Shimotsuke, Tochigi, Japan. nemoto_manabu@epizoo.equinst.go.jp
MeSH Terms
- Animals
- Bacterial Infections / diagnosis
- Bacterial Infections / veterinary
- Base Sequence
- Capsid / immunology
- Capsid Proteins / immunology
- DNA Primers
- Diarrhea / veterinary
- Diarrhea / virology
- Gene Amplification
- Genome, Viral
- Horse Diseases / diagnosis
- Horse Diseases / virology
- Horses
- Intestinal Diseases / diagnosis
- Intestinal Diseases / microbiology
- Intestinal Diseases / veterinary
- Intestinal Diseases / virology
- Reverse Transcriptase Polymerase Chain Reaction / methods
- Reverse Transcription
- Rotavirus / genetics
- Rotavirus / isolation & purification
- Rotavirus Infections / diagnosis
- Rotavirus Infections / veterinary
Citations
This article has been cited 16 times.- Yasuura M, Nakaya Y, Ashiba H, Fukuda T. Investigation on the applicability of a long-range reverse-transcription quantitative polymerase chain reaction assay for the rapid detection of active viruses. BMC Microbiol 2022 Dec 12;22(1):300.
- Nemoto M, Matsumura T. Equine rotavirus infection. J Equine Sci 2021 Mar;32(1):1-9.
- da Silva SJR, Pardee K, Balasuriya UBR, Pena L. Development and validation of a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of ZIKV in patient samples from Brazil. Sci Rep 2021 Feb 18;11(1):4111.
- Nakaya Y, Fukuda T, Ashiba H, Yasuura M, Fujimaki M. Quick assessment of influenza a virus infectivity with a long-range reverse-transcription quantitative polymerase chain reaction assay. BMC Infect Dis 2020 Aug 6;20(1):585.
- Silva SJRD, Pardee K, Pena L. Loop-Mediated Isothermal Amplification (LAMP) for the Diagnosis of Zika Virus: A Review. Viruses 2019 Dec 23;12(1).
- Silva SJRD, Paiva MHS, Guedes DRD, Krokovsky L, Melo FL, Silva MALD, Silva AD, Ayres CFJ, Pena LJ. Development and Validation of Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) for Rapid Detection of ZIKV in Mosquito Samples from Brazil. Sci Rep 2019 Mar 14;9(1):4494.
- Uzal FA, Diab SS. Gastritis, Enteritis, and Colitis in Horses. Vet Clin North Am Equine Pract 2015 Aug;31(2):337-58.
- Nemoto M, Morita Y, Niwa H, Bannai H, Tsujimura K, Yamanaka T, Kondo T. Rapid detection of equine coronavirus by reverse transcription loop-mediated isothermal amplification. J Virol Methods 2015 Apr;215-216:13-6.
- Nemoto M, Oue Y, Morita Y, Kanno T, Kinoshita Y, Niwa H, Ueno T, Katayama Y, Bannai H, Tsujimura K, Yamanaka T, Kondo T. Experimental inoculation of equine coronavirus into Japanese draft horses. Arch Virol 2014 Dec;159(12):3329-34.
- Xie L, Xie Z, Zhao G, Liu J, Pang Y, Deng X, Xie Z, Fan Q, Luo S. A loop-mediated isothermal amplification assay for the visual detection of duck circovirus. Virol J 2014 Apr 29;11:76.
- Huang XY, Hu XN, Ma H, Du YH, Ma HX, Kang K, You AG, Wang HF, Zhang L, Chen HM, Dumler JS, Xu BL. Detection of new bunyavirus RNA by reverse transcription-loop-mediated isothermal amplification. J Clin Microbiol 2014 Feb;52(2):531-5.
- Bailey KE, Gilkerson JR, Browning GF. Equine rotaviruses--current understanding and continuing challenges. Vet Microbiol 2013 Nov 29;167(1-2):135-44.
- Nemoto M, Tsunemitsu H, Murase H, Nambo Y, Sato S, Orita Y, Imagawa H, Bannai H, Tsujimura K, Yamanaka T, Matsumura T, Kondo T. Antibody response in vaccinated pregnant mares to recent G3BP[12] and G14P[12] equine rotaviruses. Acta Vet Scand 2012 Nov 6;54(1):63.
- Xie Z, Fan Q, Liu J, Pang Y, Deng X, Xie Z, Liji X, Khan MI. Reverse transcription loop-mediated isothermal amplification assay for rapid detection of Bovine Rotavirus. BMC Vet Res 2012 Aug 15;8:133.
- Mukhopadhyay HK, Amsaveni S, Matta SL, Antony PX, Thanislass J, Pillai RM. Development and evaluation of loop-mediated isothermal amplification assay for rapid and sensitive detection of canine parvovirus DNA directly in faecal specimens. Lett Appl Microbiol 2012 Sep;55(3):202-9.
- Khumtong K, Rapichai W, Saejung W, Khamsingnok P, Meecharoen N, Ratanabunyong S, Dong HV, Tuanthap S, Rattanasrisomporn A, Choowongkomon K, Rungsuriyawiboon O, Rattanasrisomporn J. Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification with Xylenol Orange Targeting Nucleocapsid Gene for Detection of Feline Coronavirus Infection. Viruses 2025 Mar 14;17(3).
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