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The abaca industry in the Philippines has been severely hampered by the prevalence of viral diseases. BBrMV has been reported as one of the most destructive viruses due to its effects on fiber yield and quality resulting in significant economic losses to the industry. Efforts have been done to manage the disease by providing virus-free planting materials to local farmers but local agencies do not have extensive resources to conduct ELISA or RT-PCR for routine detection of planting materials and on-site field testing. To establish an effective disease management program, a simple, reliable and efficient fast detection method is needed to identify disease-free planting materials and monitoring disease incidence in the field. LAMP has intrinsic characteristics that satisfy the criteria of an ideal detection method.
LAMP can amplify target nucleic acids under isothermal conditions (Notomi et al., 2000) and can be modified to amplify RNA targets by the addition of reverse transcriptase (Nagamine et al., 2002; Fukuta et al., 2003; Varga and James, 2006). Although LAMP was originally designed to amplify DNA targets, the addition of a reverse transcriptase enzyme in a LAMP reaction allows for the simultaneous completion of reverse transcription and loop mediated isothermal amplification process in a single tube. This reduces the time and cost to conduct routine detection of RNA viruses in large amounts of samples. The reverse transcriptase chosen must be functional in isothermal conditions for the LAMP amplification to work. Three reverse transcriptase enzymes, AMV, M-MLV and WarmStart® RTx, were initially evaluated to determine their success in reverse transcribing RNA templates in the one-step RT-LAMP reaction under isothermal conditions. The three reverse transcriptase enzymes tested has been extensively used for one-step RT-LAMP detection of plant RNA viruses (Fukuta et al, 2003; Nie, 2005; Varga and James, 2006; Boubourakas et al., 2009; Zhang et al., 2011; Elvira-González, et al., 2017). Although M-MLV has optimum enzyme activity at 42oC (Nie, 2005), published works on one-step RT-LAMP detection of plant pathogens have proven that the M-MLV enzyme activity is still active at temperature range of 60oC-65oC (Zhang et al., 2011; Zhou et al., 2012; Shen et al., 2014; Keizerweerd et al., 2015). Figure 1 shows that all the three enzymes can be successfully used in the one-step RT-LAMP reaction regardless of reverse transcriptase enzyme concentrations. M-MLV was therefore chosen for subsequent one-step RT-LAMP reactions due to its relatively lower cost compared to the other two enzymes.
In LAMP, The use of a high-strand displacing enzyme under single temperature removes the need for sophisticated equipment thereby reducing diagnostic cost per sample (Notomi et al., 2000). As shown in Figures 2 and 4, the use of Bst polymerase allows the amplification of ladder-like patterns under temperature ranges of 60oC to 65oC within as little as 30 minutes. The fast reaction time is a result of the use of four primers recognizing six regions within the target sequence and the use of loop primers that accelerate the LAMP reaction (Nagamine et al., 2002). In addition, the use of isothermal conditions reduces the loss of time caused by constant temperature cycling inherent in PCR reactions. Published studies on one-step RT-LAMP detection of plant RNA viruses also report on the completion of reactions under 60min (Boubourakas et al., 2009; Zhang et al., 2011; Fukuta et al., 2013). Moreover, the ability of LAMP reactions to successfully progress in a temperature range (60oC to 65oC) allows it to be applied in setups wherein simple water bath or heat block can maintain a temperature range of 60oC to 65oC. The short amount of time is also advantageous when a limited amount of time and energy is an issue in the area of testing.
The use of six primers that target eight different regions in the sequence allows increased specificity of the assay thereby preventing cross-amplification from other viruses (Notomi et al., 2000; Nagamine et al., 2002). When using the designed primers for the one-step RT-LAMP detection of BBrMV, no amplicons or ladder-like patterns were produced when RNA from healthy, ABTV-, BBTV- or SCMV-Ab-infected abaca samples was supplied as templates (Figure 5). The high level of sensitivity is also attributed to the use of six primers as well as the high amounts of DNA produced by the reaction due to its inherently high amplification efficiency (Nagamine et al., 2002; Varga and James, 2006; Boubourakas, et al. 2009). The developed one-step RT-LAMP assay was more sensitive than the conventional RT-PCR by 1000-fold. Published studies on the detection of plant RNA viruses via one-step RT-LAMP also report the higher levels of sensitivities of the one-step RT-LAMP ranging from 10-fold to 1000-fold (Boubourakas et al., 2009; Zhang et al., 2011; Zhou et al., 2012; Fukuta et al., 2013; Shen et al., 2014). The high amounts of DNA products amplified also enables the results of LAMP reaction to be directly observed with the use of fluorescent intercalating dyes that bind to the DNA products (Woźniakowski et al., 2012; Fishbach et al., 2015). The results show that both the use of GelRedTM or SYBRGreenTM produces discernible results between positive and negative results. The use of SYBRGreenTM provides further advantage by eliminating the use of portable UV lamps to view the results.
The use of crude RNA extracts did not hinder the progress of the reaction and did not adversely affect the visualization of results (Figure 7). This allows the developed one-step RT-LAMP technique to be applied in sites where there is an absence of sophisticated equipment for ELISA, RNA extraction and RT-PCR. The use of an easy visualization technique allows the local agencies to conduct routine detection of planting materials without the need to conduct electrophoresis and use of gel documentation systems which saves time and cost.
The developed one-step RT-LAMP is simple, fast and low-cost detection method for BBrMV in abaca samples and is amenable for an easy visualization method in determining positive results. The protocol only requires a simple water bath as an incubation equipment. Depending on availability of fluorescent intercalating dyes and commercially available UV lamps, both GelRedTM and SYBR GreenTM can be employed to visualize the results. With the simplicity and straightforwardness of the one-step RT-LAMP detection method, the technique can be applied by local agencies and even farmers for sensitive and specific fast detection of BBrMV-infected samples under field conditions.