title: Use of reverse transcription loop-mediated isothermal amplification combined with lateral flow dipstick for an easy and rapid detection of Jembrana disease virus.
abstract: Jembrana disease virus (JDV) is a viral pathogen that causes Jembrana disease in Bali cattle (Bos javanicus) with high mortality rate. An easy and rapid diagnostic method is essential for further control this disease. We used a reverse transcription loop-mediated isothermal amplification (RT-LAMP) combined with lateral flow dipstick (LFD), based on conserved tm subunit of Jembrana disease virus env gene. The RT-LAMP conditions were optimized by varying the concentration of MgSO4, betaine, dNTP, and temperature as well as the time and duration of reaction. The primers sensitivity for JDV was confirmed. The method was able to detect env-tm gene dilution which contained 2 × 10(-15) g of template. Comparatively, the sensitivity of RT-LAMP/LFD was 100-fold more sensitive than reverse transcription-polymerase chain reaction. The primers specificity for JDV was also confirmed using positive and negative controls. This work also showed that virus detection could be done not only on total RNA extracted from blood but various organs could also be analyzed for the presence of JDV using RT-LAMP/LFD method. The whole process, including the LAMP reaction and the LFD hybridization step only lasts approximately 75 min. Results of analysis can be easily observed with naked eyes without addition of any chemical or further analysis. The combination of RT-LAMP with LFD makes the method a more suitable diagnostic tool in conditions where sophisticated and expensive equipments are not available for field investigations on Jembrana disease in Bali cattle.
authors.affiliation: Department of Reproduction, Faculty of Veterinary Medicine, Gadjah Mada University, Jalan Fauna No. 2, Yogyakarta, 55281 Indonesia ; Center of Biotechnology Study, Gadjah Mada University, Jalan Teknika Utara, Yogyakarta, 55281 Indonesia.
authors.forename: Asmarani
authors.initials: A
authors.lastname: Kusumawati
authors.affiliation: Center of Biotechnology Study, Gadjah Mada University, Jalan Teknika Utara, Yogyakarta, 55281 Indonesia.
authors.forename: Issabellina Dwades
authors.initials: ID
authors.lastname: Tampubolon
authors.affiliation: Health Polytechnique of Yogyakarta, Ministry of Health of Indonesia, Jalan Tata Bumi No. 3, Yogyakarta, 55293 Indonesia.
authors.forename: Narendra Yoga
authors.initials: NY
authors.lastname: Hendarta
authors.affiliation: Center of Biotechnology Study, Gadjah Mada University, Jalan Teknika Utara, Yogyakarta, 55281 Indonesia ; Department of Clinical Pathology, Faculty of Veterinary Medicine, Gadjah Mada University, Jalan Fauna No. 2, Yogyakarta, 55281 Indonesia.
authors.forename: Siti Isrina Oktavia
authors.initials: SI
authors.lastname: Salasia
authors.affiliation: Faculty of Medicine, Sebelas Maret University, Jalan Ir. Sutami 36A, Surakarta, 57126 Indonesia.
authors.forename: Tenri Ashari
authors.initials: TA
authors.lastname: Wanahari
authors.affiliation: Faculty of Medicine, Sebelas Maret University, Jalan Ir. Sutami 36A, Surakarta, 57126 Indonesia.
authors.forename: Basofi Ashari
authors.initials: BA
authors.lastname: Mappakaya
authors.affiliation: Department of Internal Medicine, Faculty of Veterinary Medicine, Gadjah Mada University, Jalan Fauna No. 2, Yogyakarta, 55281 Indonesia.
authors.forename: Sri
authors.initials: S
authors.lastname: Hartati
keywords: Bovine disease; Env gene; Jembrana disease virus; Lateral flow immunoassay; Loop mediated isothermal amplification