PENGEMBANGAN KOMBINASI METODE ONE STEP REVERSE TRANSCRIPTASE-LOOP MEDIATED ISOTHERMAL AMPLIFICATION (RT-LAMP) DAN LATERAL FLOW DIPSTICK (LFD) UNTUK DETEKSI VIRUS PENYAKIT JEMBRANA
| Main Authors: | , Issabellina D. Tampubolon S.Si, , Dr. drh. Asmarani Kusumawati, MP. |
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| Format: | Thesis NonPeerReviewed |
| Terbitan: |
[Yogyakarta] : Universitas Gadjah Mada
, 2013
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| Subjects: | |
| Online Access: |
https://repository.ugm.ac.id/118166/ http://etd.ugm.ac.id/index.php?mod=penelitian_detail&sub=PenelitianDetail&act=view&typ=html&buku_id=57820 |
Daftar Isi:
- Jembrana disease is a viral disease in Bali cattle caused by a member of the Lentivirus called Jembrana disease virus (JDV), causing an acute and severe disease syndrome, even lead to death after a short incubation period. Since it is not only occur in Bali but also has spread to other areas in Indonesia, an early detection technique which can be applied simply but provides a specific and sensitive result is highly required. Development of one step Reverse Transcriptase Loop-mediated Isothermal Amplification (RT-LAMP) has been done and in this study it was developed by involving the use of lateral flow dipstick (LFD) as a method of specificity assay of the reaction and as an alternative to replace agarose gel electrophoresis for amplification reaction analysis. Optimization of LAMP has been carried out using pGEX-TM, a recombinant plasmid of pGEX-2T and env tm gene of JDV, showed that the reaction is spesific, only amplify DNA target i.e. env tm gene of JDV. This result was confirmed by the success of hybridization between LAMP amplicon and a 6-Fam (6-Carboxyfluorescein) labeled probe which was detected in the LFD up to concentration 1 fg/ Âμl, equivalent to 1,52x102 molekul/Âμl copy number. This technique gives a JDV positive result in organ samples of spleen, liver, lung and tongue which derived from cattle experimentally inoculated JDV. LAMP reaction is also sensitive, could amplifying the DNA concentration up to 0.1 fg/ul or 1,52x10 molekul/Âμl copy number, 100 time more sensitive than PCR which could amplifying only up to 0.01 pg/ul 1,52x102 molekul/Âμl copy number. The optimal LAMP reaction can be applied simply and shortly on one step RT-LAMP to detect the presence of JDV in field samples of blood.