Development of a Rapid Detection Method for Potato virus X by Reverse Transcription Loop-Mediated Isothermal Amplification

Abstract

The primary step for efficient control of viral diseases is the development of simple, rapid, and sensitive virus detection. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) has been used to detect viral RNA molecules because of its simplicity and high sensitivity for a number of viruses. RT-LAMP for the detection of Potato virus X (PVX) was developed and compared with conventional reverse transcription polymerase chain reaction (RT-PCR) to demonstrate its advantages over RT-PCR. RT-LAMP reactions were conducted with or without a set of loop primers since one out of six primers showed PVX specificity. Based on real-time monitoring, RT-LAMP detected PVX around 30 min, compared to 120 min for RT-PCR. By adding a fluorescent reagent during the reaction, the extra step of visualization by gel electrophoresis was not necessary. RT-LAMP was conducted using simple inexpensive instruments and a regular incubator to evaluate whether RNA could be amplified at a constant temperature instead of using an expensive thermal cycler. This study shows the potential of RT-LAMP for the diagnosis of viral diseases and PVX epidemiology because of its simplicity and rapidness compared to RT-PCR.

Species-specific loop-mediated isothermal amplification (LAMP) assay for identification of tissue of cattle origin by targeting mitochondrial gene sequences

“…Deoxynucleoside Triphosphate (dNTP) is source of nucleotides for amplification of target template DNA. Jeong et al (2015) cited that the amplified LAMP products were obtained using a concentration of dNTPs ranging from 0.4 to 1.4 mM, but no reaction products were obtained at 0.2 mM. Betaine (N,N,N-trimethyl glycine) is routinely used in LAMP amplification to lower the melting temperature, increase the specificity and facilitate the separation of DNA templates (En et al 2008).…”

Jasmonic acid and methyl dihydrojasmonate enhance saponin biosynthesis as well as expression of functional genes in adventitious roots of Panax notoginseng F.H. Chen

“…In this study, the combinations of low concentration IBA and NAA were effective for root growth, this was probably because IBA was the most effective treatment in causing root elongation, whereas NAA was effective for increasing the number of roots [19]. Meantime, it is reported that the optimum culture conditions for the growth of Panax ginseng adventitious roots were likely to be 3/4 medium salt strength, 6% sucrose, and 5 g/L inoculum density [23,24]. Medium salt strength is a critical determinant in controlling the growth of adventitious roots [25].…”

Major In Vitro Techniques for Potato Virus Elimination and Post Eradication Detection Methods. A Review

“…Considering the capability of TaqMan real-time RT-PCR to discriminate small sequence it was found to be the most useful, practicable technique (Singh et al 2013), and using a quadruplex real-time RT-PCR, PLRV, PVA, PVX, and PVY can be detected simultaneously (Jeong et al 2015). Optimum dilution, inhibition of RNAses, and the optimization of RT-PCR steps are critical factors that influence the detection of these four viruses from dormant potato tubers (Agindotan et al 2007).…”

“…Furthermore, for field testing, due to its closed-tube format, the LAMP has been found to be able to control substances that can inhibit PCR reaction so that allowing the technique to be used with simple protocols of genetic materials amplification (Kaneko et al 2007). Ju (2011) showed RT-LAMP as an extremely efficient, sensitive, accurate, simple, fast and reliable detection tool for (Jeong et al 2015) PLRV detection within potato tubers. After visual detection of PLRV by a LAMP with the Gene Finder™ dye, it has been an open door for plant pathology in general (Almasi et al 2013).…”