Rapid and Sensitive Detection of Lettuce Necrotic Yellows Virus and Cucumber Mosaic Virus Infecting Lettuce (Lactuca sativa L.) by Reverse Transcription Loop-Mediated Isothermal Amplification

Abstract

Cucumber mosaic virus (CMV) is damaging to the growth and quality of lettuce crops in Lanzhou, China. Recently, however, for the first time an isolate of lettuce necrotic yellows virus (LNYV) has been detected in lettuce crops in China, and there is concern that this virus may also pose a threat to lettuce production in China. Consequently, there is a need to develop a rapid and efficient detection method to accurately identify LNYV and CMV infections and help limit their spread. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays were developed to detect the nucleoprotein (N) and coat protein (CP) genes of LNYV and CMV, respectively. RT-LAMP amplification products were visually assessed in reaction tubes separately using green fluorescence and gel electrophoresis. The assays successfully detected both viruses in infected plants without cross reactivity recorded from either CMV or LNYV or four other related plant viruses. Optimum LAMP reactions were conducted in betaine-free media with 6 mM Mg 2+ at 65°C for LNYV and 60°C for 60 min for CMV, respectively. The detection limit was 3.5 pg/ml and 20 fg/ml using RT-LAMP for LNYV and CMV plasmids, respectively. Detection sensitivity for both RT-LAMP assays was greater by a factor of 100 compared to the conventional reverse transcription polymerase chain reaction assays. This rapid, specific, and sensitive technique should be more widely applied due to its low cost and minimal equipment requirements.

Development of a reverse transcription loop‐mediated isothermal amplification assay for rapid detection of strawberry crinkle virus

“…We found a similar pattern in other experiments according to incomplete statistics that the DNA was amplified only in few particular temperatures (He et al, 2016; Iftikhar et al, 2020). Furthermore, different temperatures may also promote the formation of different band patterns (Zhang et al, 2020). The reasons for this phenomenon are complex, of which the most important one is that temperature is a prerequisite affecting enzyme activity.…”

Development of immunocapture reverse transcription loop‐mediated isothermal amplification assays to detect both lily symptomless virus and cucumber mosaic virus

“…The concentrations of MgSO 4 and betaine have been found important for the IC-RT-LAMP assay. However, from our past work with assays developed to detect Japanese hornwort mosaic virus (JHMV) (Zhang, Wang, et al, 2020a), lettuce necrotic yellows virus (LNYV) and cucumber mosaic virus (CMV) (Zhang, Xie, et al, 2020) and detect piper yellow mottle virus (PYMoV) (Bhat, Siljo, & Deeshma, 2013), we found the optimum MgSO 4 concentration was 6 mM. Furthermore, when using Bst DNA polymerase (New England Biolabs), the concentration of MgSO 4 is also recommended at 6 mM.…”

“…However, the lily isolate of CMV has a narrow host range. This isolate can infect cucumber, Datura stramonium and Phaseolus vulgaris, but not typical CMV indicator hosts such as Nicotiana tabacum, Nicotiana glutinosa and Chenopodium amaranticolor (Liang, Wei, & Xu, 2008;Zhang et al, 2020).…”

“…Moreover, amplicons can be readily visualised by adding SYBR Green I, ethidium bromide or calcein to the reaction mixture (Jia, Guo, & Xia, 2016;Parida, Sannarangaiah, Dash, & Rao, 2008;Waliullah et al, 2020;Zhang et al, 2020a;Zhang et al, 2020b;Zhang, Xie, et al, 2020). This hybrid diagnostic technique improves an assay's sensitivity and specificity, while mitigating the problems caused by substances that inhibit PCR and provides a cheaper and more convenient method of preparing amplification templates (Gambley, Geering, & Thomas, 2009).…”

The Potential Use of Isothermal Amplification Assays for In-Field Diagnostics of Plant Pathogens

“…For RNA viruses, LAMP is combined with reverse transcription. Some LAMP tests propose SGI detection without UV light, making the tests cheaper and easier [10,[153][154][155][156].…”