Loop-mediated isothermal amplification assays for detection of Equid herpesvirus 1 and 4 and differentiating a gene-deleted candidate vaccine strain from wild-type Equid herpesvirus 1 strains.
Abstract: Loop-mediated isothermal amplification (LAMP) is a novel method for the rapid and sensitive detection of DNA without the need for expensive equipment. In the present study, LAMP assays were developed for the specific detection of Equid herpesvirus 1 and 4 (EHV-1 and EHV-4, respectively) and for the differentiation of glycoprotein E (gE)-deleted EHV-1 (DeltagE) strain, a candidate strain for a live vaccine, from field EHV-1 strains. Specific primer sets were designed for the gC and gE genes of EHV-1 and for the gC gene of EHV-4. The analytical sensitivities of the LAMP assays were compared with those of polymerase chain reaction (PCR). The detection limits of LAMP for EHV-1 gC and gE and PCR for EHV-1 gC were 1 plaque-forming unit (PFU)/tube, and those of LAMP and PCR for EHV-4 gC were 0.1 PFU/tube. The DeltagE strain could be differentiated from wild-type EHV-1 strains based on the reactivity in the LAMP for EHV-1 gC in combination with the LAMP for EHV-1 gE. The analytical specificities of the LAMP for EHV-1 and EHV-4 were examined by using several equine pathogens, and no cross-reactions were observed. The LAMP detection abilities for EHV-1 and EHV-4 on nasal swab samples collected from experimentally infected horses were in good agreement with that of PCR for EHV-1 and EHV-4, respectively. The LAMP assays developed in the current study were sensitive and specific for EHV-1 and EHV-4, and should provide a valuable alternative to PCR for use in clinical laboratories in the field.
Publication Date: 2010-01-23 PubMed ID: 20093679DOI: 10.1177/104063871002200105Google Scholar: Lookup
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Summary
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This research involves developing a Loop-mediated isothermal amplification (LAMP) method for specific detection of Equid herpesvirus 1 and 4 (EHV-1 and EHV-4) and differentiating a specific gene-deleted strain of EHV-1 used as a live vaccine from wild strains of the virus. This method provides an effective alternative to expensive equipment and was found to be sensitive, specific, and efficient in detection.
Introduction to Loop-mediated isothermal amplification (LAMP)
- LAMP is a novel method for rapid and sensitive detection of DNA without the need for expensive equipment.
- It is becoming popular due to its high sensitivity, specificity, and rapidness compared to traditional methods.
Focus on Equid herpesvirus 1 and 4 (EHV-1 and EHV-4)
- The researchers developed LAMP assays specifically for the detection of Equid herpesvirus (EHV) strains 1 and 4, common viruses in equine species.
- Specific primer sets were designed for the gC and gE genes of EHV-1 and for the gC gene of EHV-4.
- The LAMP detection abilities for EHV-1 and EHV-4 on nasal swab samples collected from experimentally infected horses were validated against PCR test results.
Differentiating Vaccine Strain from Wild-type Virus
- One goal of the research was to differentiate a specific glycoprotein E (gE)-deleted EHV-1 strain, a candidate strain for a live vaccine, from wild-type EHV-1 strains.
- The differentiation was achieved based on the reactivity in the LAMP for EHV-1 gC in combination with the LAMP for EHV-1 gE.
Comparing LAMP and PCR Methods
- The researchers also compared the analytical sensitivities of the LAMP and PCR methods.
- The detection limits of LAMP for EHV-1 gC and gE and PCR for EHV-1 gC were found to be similar, while the LAMP and PCR for EHV-4 gC were ten times more sensitive.
- Both LAMP and PCR methods were able to successfully detect EHV-1 and EHV-4 on nasal swab samples from experimentally infected horses, indicating their potential use in clinical laboratories in the field.
Conclusion
- The LAMP assays developed in this study were sensitive and specific for EHV-1 and EHV-4.
- They could differentiate between vaccine and wild strains and therefore, provide a valuable alternative to PCR for use in clinical laboratories.
Cite This Article
APA
Nemoto M, Tsujimura K, Yamanaka T, Kondo T, Matsumura T.
(2010).
Loop-mediated isothermal amplification assays for detection of Equid herpesvirus 1 and 4 and differentiating a gene-deleted candidate vaccine strain from wild-type Equid herpesvirus 1 strains.
J Vet Diagn Invest, 22(1), 30-36.
https://doi.org/10.1177/104063871002200105 Publication
Researcher Affiliations
- Epizootic Research Center, Equine Research Institute, Japan. nemoto_manabu@epizoo.equinst.go.jp
MeSH Terms
- Animals
- DNA, Viral / isolation & purification
- Herpesviridae Infections / prevention & control
- Herpesviridae Infections / veterinary
- Herpesviridae Infections / virology
- Herpesvirus 1, Equid / isolation & purification
- Herpesvirus 4, Equid / isolation & purification
- Herpesvirus Vaccines / immunology
- Horse Diseases / prevention & control
- Horses
- Nucleic Acid Amplification Techniques / veterinary
- RNA, Viral / genetics
- Sensitivity and Specificity
Citations
This article has been cited 6 times.- Knox A, Zerna G, Beddoe T. Current and Future Advances in the Detection and Surveillance of Biosecurity-Relevant Equine Bacterial Diseases Using Loop-Mediated Isothermal Amplification (LAMP).. Animals (Basel) 2023 Aug 18;13(16).
- Ning Y, Huang Y, Wang M, Cheng A, Yang Q, Wu Y, Tian B, Ou X, Huang J, Mao S, Sun D, Zhao X, Zhang S, Gao Q, Chen S, Liu M, Zhu D, Jia R. Alphaherpesvirus glycoprotein E: A review of its interactions with other proteins of the virus and its application in vaccinology.. Front Microbiol 2022;13:970545.
- Jelocnik M, Nyari S, Anstey S, Playford N, Fraser TA, Mitchell K, Blishen A, Pollak NM, Carrick J, Chicken C, Jenkins C. Real-time fluorometric and end-point colorimetric isothermal assays for detection of equine pathogens C. psittaci and equine herpes virus 1: validation, comparison and application at the point of care.. BMC Vet Res 2021 Aug 19;17(1):279.
- Knox A, Beddoe T. Isothermal Nucleic Acid Amplification Technologies for the Detection of Equine Viral Pathogens.. Animals (Basel) 2021 Jul 20;11(7).
- Kinoshita Y, Takechi M, Uchida-Fujii E, Miyazawa K, Nukada T, Niwa H. Ten cases of Mycobacterium avium subsp. hominissuis infections linked to equine abortions in Japan, 2018-2019.. Vet Med Sci 2021 May;7(3):621-625.
- Murase H, Miyazawa M, Harada T, Ozawa M, Sato F, Hada T. Aborted fetal sizes of Thoroughbred horses in Hidaka, Japan, between 2005 and 2015.. J Equine Sci 2017;28(2):47-53.
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