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 MgSO 4 , 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 9 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.

Rapid and sensitive detection of Senecavirus A by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick method

“…Compared with the calcein RT-LAMP method, the method established in this experiment eliminates human error caused by visual observation of color, and the test is completed in a closed container, thus eliminating false-positive amplification caused by additional contamination of fluorescent dye in the RT-LAMP product. In some studies, only one labeled primer was used in the LAMP reaction[36, 37], and another labeled probe was added to the LAMP products to form a double-labeled detectable product, which easily caused contamination of the LAMP product. In our study, the two internal primers were labeled with biotin isothiocyanate and fluorescein isothiocyanate respectively.…”

Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms

“…Lateral flow dipstick (LFD) was developed as well for simultaneous detection of pathogenic Leptospira spp. (Nurul Najian et al 2016), measles virus (Xu et al 2016), animal babesiosis, caused by Babesia bovis and Babesia bigemina , foot-and-mouth diseases (Waters et al 2014), Japanese encephalitis (Deng et al 2015), P. vivax, and P. falciparum (Yongkiettrakul et al 2014;Kongkasuriyachai et al 2017), Jembrana disease virus (Kusumawati et al 2015), canine parvovirus (Sun et al 2014), Vibrio alginolyticus (Plaon et al 2015), Macrobrachium rosenbergii nodavirus and shrimp yellow head virus (Khunthong et al 2013).…”

Serotype Specific Sequence for Multi Test Line Nucleic Acid Lateral Flow Development

“…al., 2021). Research on the use of LFD for NALFIA / NALF has been carried out for detecting pathogenic diseases in both animals and humans (Ding et al, 2010;Nimitphak et al, 2010;Kusumawati et al, 2015, Kusumawati andFatimah, 2018;Ajie et. al., 2021).…”