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Potatoes can be infected by many different viruses that can reduce yield and tuber quality. Potato virus X (PVX) is the most economically important virus in commercial potato production. The present study describes the first reverse transcription loop mediated isothermal amplification (RT-LAMP) assay to detect PVX. In this regard, all four RT-LAMP primers (i.e. F3, B3, FIP and BIP) together with RT-PCR primers (F and B) were designed on the basis of coat protein (CP) gene of virus. RT-LAMP method, on the whole, had the following advantages over the other mentioned procedures: (I) fascinatingly; (II) no requirement of expensive and sophisticated tools for amplification and detection; (III) no post-amplification treatment of the amplicons; (IV) a flexible and easy detection approach, which is visually detected by naked eyes using diverse visual dyes; (V) high sensitivity; (VI) high efficiency; (VII) safety; and (VIII) user friendly. We accordingly propose this assay as a highly reliable alternative viral recognition system regarding PVX recognition and probably other viral-based diseases.
Keywords Colorimetric assay · Immunocapture assay · LAMP assay · Potato virus X · Real-time quantitative Introduction
The potato is a tuberous crop from the perennial nightshade Solanum tuberosum L. It is the world’s fourth-largest food crop, following maize, wheat, and rice providing a good source of , protein, carbohydrate, vitamins and minerals (Neiderhauser 1993). Potato crop yields are determined by factors such as the crop breed, seed age and quality, crop management practices and the plant environment. Improvements in one or more of these yield determinants can be a major boost to food supply and farmer incomes in the developing world (Xu et al. 2011). The world dedicated 18.6 million hectares in 2010 for potato cultivation. The average world farm yield for potato was 17.4 tons per hectare, in 2010. Iran, with the yield of 5,240,000 tons, is assigned as the world’s number 12 potato producer (http://www.potato2010.org/en/world/asia.html). Good disease management is critical to the successful production of the potato. The potato plant is susceptible to at least 75 diseases and nonparasitic disorders, many of which consistently cause yield losses in potato production areas. Potatoes are a vegetatively propagated crop, and potato seed tubers can be an important source of disease inoculum. Several types of injury that can influence plant vigor and subsequent yields also occur on seed tubers. The management of many production problems depends on the identification of diseased or defective seed tubers (Agindotan et al. 2007; Almasi et al. 2012; Jones 1988). Potato virus X (PVX) is a plant pathogenic virus of the family Alphaflexiviridae and the order Tymovirales. It is the type species of the genus Potexvirus (Huisman et al. 1988; Skryabin et al. 1988). Potato Virus X (PVX) is one of the most widely distributed viruses of potatoes because no symptoms develop in some varieties (latent mosaic), the full extent of damage with PVX is not recognized. Potato virus X is very difficult to spot as there are very few symptoms however one clue to look out for is a reduction in yields (typically around 15%). Potato plants often do not exhibit symptoms, but the virus can cause symptoms of chlorosis, mosaic, decreased leaf size, and necrotic lesions in tubers. In tobacco it causes mottling or necrotic spotting, in tomato it causes mosaic and slight stunting. In all three hosts the severity of symptoms greatly increases in mixed infections with other viruses, e.g. potato virus Y (Singh and Nie 2003; Agindotan et al. 2007; Boonham et al. 2002). The virus infects more than 240 species in 16 families; the majority of hosts are in the Solanaceae. Tobacco, pepper, and tomato are additional hosts for this virus (Chapman et al. 1992; Boonham et al. 2002). Although the virus is spread mainly by either direct contact (between plants) or indirect contact (by man or machinery) transmission has been reported by the fungus Synchytrium endobioticum (Nienhaus and Stille 1965) and the grasshoppers Melanoplus differentialis (Walters 1952). There are no particular potato varieties particularly at risk of PVX; however a few are immune, including: Cara, King Edward, Maris Piper, Pentland Dell, Pentland Javelin, Pentland Squire and Sante. PVX can be controlled by disinfecting all clothes and machinery before entering tested and unaffected tuber stocks, and multiplication of seed stocks from virus-tested plants by specialist seed potato growers from good seed growing areas (Sonenberg et al. 1978; Manzer et al. 1978). PVX is a flexuous, rod-shaped virus that contains a linear single-stranded positive-sense 6.4 kb RNA genome (Skryabin et al. 1988). The genomic RNA of the virus has a 5′ cap, a 3′ poly (A) tail and five open reading frames (ORFs) (Huisman et al. 1988). Homologies with gene products of other viruses suggest that ORF 1 encodes an RNA replicase, the central regions of ORF 1 and ORF 2 encode NTP-binding helicase(s), ORF 3 and 4 encode membrane-bound proteins which may have a transport function and ORF 5 encodes a coat protein (Skryabin et al., 1988).
The development of sensitive methods for detecting viruses is therefore of importance to support seed certification schemes (Mumford et al. 2000). Standard methods of virus detection including mechanical inoculation to indicator hosts, electron microscopy (EM), enzyme linked immunosorbent assay (ELISA) and visual inspection are not adequate to reliably detect some potato viruses and might not be suitable for testing tuber samples (Barker et al. 1993; Singh and Singh 1998). The reverse transcription polymerase chain reaction (RT-PCR) assay offer the possibility of improved sensitivity and a more rapid diagnosis of individual viruses from various tissues of potato (Spiegel and Martin 1993; Singh et al. 1997; Walsh et al. 2001). However, detection of several individual viruses separately by RT-PCR reactions is expensive and time consuming. In order to reduce the labor and equipment costs for test, a multiplex RT-PCR (M-RT-PCR) protocol was developed for the detection of 5 viruses (PVA, PVS, PVX, PVY and PLRV) in potato tubers and simplified by using an oligo (dT) in the synthesis of cDNA for potato viruses RNAs (Mumford et al. 2000; Nie and Singh 2002). Multiplex assay accommodates several primer pairs in one reaction, saving time and expense, especially when large numbers of samples need to be tested (Singh et al. 1995; Rigotti and Gugerli 2007; Lorenzen et al. 2006). This method effectively detected multiple potato viruses in tubers, but interference from plant mRNA made it unsuitable for the detection of the same viruses in leaves (Hauser et al. 2000; Nie and Singh 2001). Loop mediated isothermal amplification (LAMP) is a single tube technique for the amplification of DNA. This may be of use in future as a low cost alternative to detect certain diseases. It may be combined with a reverse transcription step to allow the detection of RNA (Notomi et al. 2000). In contrast to the PCR technology in which the reaction is carried out with a series of alternating temperature steps or cycles, isothermal amplification is carried out at a constant temperature, and does not require a thermal cycler (Hadersdorfer et al. 2011). In LAMP, the target sequence is amplified at a constant temperature of 60-65 °C using either two or three sets of primers and a polymerase with high strand displacement activity in addition to a replication activity. Due to the specific nature of the action of these primers, the amount of DNA produced in LAMP is considerably higher than PCR based amplification (Nagamine et al. 2002). Detection of amplification product can be determined via photometry for turbidity caused by an increasing quantity of magnesium pyrophosphate precipitate in solution as a byproduct of amplification (Fukuta et al. 2003). Although a large number of studies have been accomplished using RT-LAMP including PVY (Nie 2005; Almasi and Dehabadi 2013), PLRV (Almasi et al. 2012; Almasi et al. 2013a; Almasi et al. 2013b; Ahmadi et al. 2012), because the technique has not been yet introduced for detection of PVX, here, an attempt was accordingly made to optimize a new protocol reverse transcription-LAMP (RT-LAMP) of it to detect PVX. Also, was developed colorimetric, immunocapture and real-time quantitative reactions with some minor modifications for detection of PVX from infected potato leaf tissues.
Materials and Methods
Survey studies were conducted from 11 separate potato fields in two provinces (Hamedan and Kordestan) where potato is commonly grown in Iran. A total of 38 fresh potato leaf samples infected dubiously with potato viruses on the basis of plant symptoms (see above) were collected in summer of 2014. The samples were sliced and rapidly desiccated in anhydrous calcium chloride and were kept at -80 °C at the laboratory.
Polyclonal PVX specific antibody was employed first to validate the existence of the virus in leaf extracts from each fresh sample (two duplicate wells of an ELISA plate). Table 1 shows the information of polyclonal specific antibody for PVX. Double antibody sandwich ELISA (DAS-ELISA) was carried out as described by Clark and Adams (1977) with some minor modifications, using a commercially available IgG and the alkaline phosphatase-conjugated IgG. These modifications included modest changes in the incubation time and temperature and two days to obtain results. Polystyrene microtiter plates were coated for 3 h at 37 °C, with 100 μl per well of IgG coating, in 50 mM carbonate buffer, pH 9.6. The plates were then incubated for 1 h at 37 °C with phosphate buffer saline (PBS) (10 mM phosphate buffer, pH 7.2, 0.8 % NaCl and 0.02 % KCl). After that, the plates were washed three times using washing buffer (0.8 % NaCl, pH 7.2 and 0.05 % Tween 20). The leaf extracts of each fresh sample were ground in ten volumes (w/v) of PBS buffer pH 7.2, containing 0.2 % polyvinylpyrrolidone (PVP) and 2 % of egg albumin (Sigma A5253). The infected preparations were serially diluted (five-fold dilution) at the same buffer. Aliquots of 100 μl of prepared samples and controls were added to each well (with two duplicate), and the plates were incubated overnight at 4 °C. Plates were then washed three times with washing buffer, incubated for 4 h at 37 °C, with 100 μl per well of alkaline phosphatase-conjugated IgG diluted in sample buffer, washed again, and incubated lastly for 1 h at 37 °C, with p-nitrophenyl-phosphate (1 mg/ml), in 10 % diethanolamine, pH 9.8.
Total RNA was isolated from virus-infected leaves of potato for PVX as described previously by Rowhani and Stace-Smith (1979). 2 ml of extraction buffer (21.7 g K2HPO4.3H2O, 1.4 g KH2PO4, pH 7.4, 100 g sucrose, 1.5 g BSA, 20 g PVP and 5.3 g ascorbic acid) was transferred into a mortar containing 0.2 g of leave samples. Next, two successive centrifugations were performed, 10 min (1100 g) and 20 min (16800 g) at 4 °C, respectively. Then, the resultant precipitate was mixed with 0.2 ml TE buffer (10 mM EDTA, 50 mM Tris-HCl, pH 8 and 0.1 % mercaptoethanol) and 10 % Sodium Dodecyl Sulphate (SDS). Afterwards, 80 μl of 5 M acetate potassium was added and the solution incubated for 10 min at 60 °C and for overnight at 4 °C, respectively. Then, the tubes were centrifuged at 4 °C for 15 min (16800 g). The aqueous phase was harvested and 30 μl acetate sodium 3 M was added followed by isopropanol. After incubation at -20 °C for 2 h, the last centrifugation was performed at 4 °C for 20 min (16800 g). The resultant pellet was washed with 70 % ethanol, dried under a vacuum, and dissolved in a total volume of 15 μl of double distilled water.
Design of Primers
Molecular assays were developed using specific primers designed on the basis of coat protein (CP) gene of PVX by the oligo7 (for RT-PCR) and Primer Explorer V.4 software (specific for RT-LAMP) (Table 2). At LAMP assay, a set of four primers recognizing six distinct regions in the target sequence were used, including F3, B3, FIP and BIP. Two of the oligonucleotides (FIP and BIP) each possess two sequences. Unlike primers that are used by other techniques, such as the PCR oligonucleotides, FIP and BIP are complementary to sequences from both strands of the targeted DNA that have close location to each other (designated as F1 and F2c for FIP and B1 and B2c for BIP, respectively) (Hadersdorfer et al. 2011; Tomita et al. 2008). The other two oligonucleotides (F3 and B3) are like ordinary PCR primers. As a first step, a stem-loop DNA structure, in which the sequences of both DNA ends are derived from the inner primers, is constructed as starting material (Parida et al. 2011). Subsequently, one inner primer hybridizes to the loop on the product in the LAMP cycle and initiates strand displacement DNA synthesis, yielding the original stem-loop DNA and a new stem-loop DNA with a stem twice as long (Tsai et al. 2009; Dukes et al. 2006). The final products are a mixture of stem-loop DNAs with various stem lengths and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target sequence in the same strand. The structures of the cycling inter-mediate and final products are schematically illustrated in ladder like DNA form (Hill et al. 2008; Ren et al. 2009). Fig. 1 shows the schematic position of primers within the CP gene of PVX. Finally, the primers were tested for similarities with other sequences available in the GenBank databases using the BLASTN algorithm.
Initially, cDNA was synthesized as described previously by Rowhani and Stace-Smith (1979). Extracted RNA (5 μl) of virus infected leaves (positive samples), a free virus sample (negative sample), positive control and negative control were incubated at 75 °C for 3 min and chilled on ice for 3 min. Then, 20 pmol B primer, 50 mM Tris-HCL, pH 8.3, 10 mM dithiothreithol (DTT), 2.5 mM MgCl2, 10 mM of each dNTP, 5 U of RNasin Ribonuclease Inhibitor (Fermentas Co, Cat. No EO0381), and 1.25 U of avian myeloblastosis virus (AMV) reverse transcriptase (Fermentas Co, cat. no. EP0641) were added in a 25 μl volume to tube. Afterwards, mixtures were incubated at 60 °C for 1 h. lastly; PCR reaction was performed on a Thermal Cycler (iCycler, BIO RAD, CA, USA) in a 25 μl volume containing 1 × PCR buffer (10 mM Tris-HCl, pH 8.3 and 50 mM KCl), 1.5 mM MgCl2, 0.2 mM of each dNTP, 20 pmol of each F and B primers, 0.625 U of Taq DNA polymerase (Cinagen Co, Cat. No TA7505C) and 2 μl cDNA. Subsequently, master-mix were amplified at 94 °C for 3 min, for 35 cycles followed by for 1 min at 94 °C, 1 min at 54 °C and 1 min at 72 °C. A final extension was accomplished for 10 min at 72 °C. Finally, Amplified products (5 μl) were electrophoresed on 1.5 % agarose gel, stained with ethidium bromide and photographed under UV light using Gel Documentation System (GELDOC 2000, Bio-Rad, USA).
The IC-RT-PCR was developed using F and B primers designed on the basis of virus coat protein (CP) gene. The protocol, to generate IC-RT-PCR products, was divided into two successive sections. At section 1, same as DAS-ELISA method, polystyrene microtiter plate was first coated with PVX specific IgG diluted in coating buffer and incubated for 3 h in 37 °C. Plate, in the following, was washed with washing buffer (see DAS-ELISA assay section). The extractions of positive samples, negative sample, positive control and negative control were added to IgG-coated wells and kept overnight at 4 °C. Plate, the next day, was washed using washing buffer, dried and employed for next section. In section 2, 20 pmol B primer, 50 mM Tris-HCL, pH 8.3, 10 mM dithiothreithol (DTT), 2.5 mM MgCl2, 10 mM of each dNTP, 5 U of RNasin Ribonuclease Inhibitor (Fermentas Co, Cat. No EO0381), and 1.25 U of AMV reverse transcriptase (Fermentas Co, cat. no. EP0641) were added to plate and incubated at 60 °C for 1 h in water bath. 2 μl of product was harvested and transported to tube, then amplification was performed in a 50 μl volume containing 1 × PCR buffer (10 mM Tris-HCl, pH 8.3 and 50 mM KCl), 1.5 mM MgCl2, 0.2 mM of each dNTP, 20 pmol of each F and B primers, 0.625 U of Taq DNA polymerase (Cinagen Co, Cat. No TA7505C) on a Thermal Cycler. master-mix were amplified at 94 °C for 3 min, for 35 cycles followed by for 1 min at 94 °C, 1 min at 54 °C and 1 min at 72 °C. A final extension was accomplished for 10 min at 72 °C. The products were lastly analyzed by gel electrophoresis and visualized under UV light.
RT-LAMP reaction was carried out by using obtained RNA which was previously described. The effects of temperatures and times as well as the concentrations of dNTP, Bst DNA polymerase and Betaine were examined and the reaction optimized. Initial, RNA (5 μl) was incubated were incubated at 75 °C for 3 min and chilled on ice for 3 min and it was served as a template in RT-LAMP reaction. The RT-LAMP was performed in a total volume of 50 μl containing 10 mM DTT, 5 U of RNase Ribonuclease Inhibitor (Fermentas Co., cat. no. EO0381), 20 mM Tris–HCl, pH 8.8, 10 mM KCl, 10 mM (NH4)2SO4, 0.1 % Triton X-100, 2 mM Betaine (Sigma-Aldrich, Oakville, ON, Canada), 10 mM each dNTP, 0.2 μM each of F3 and B3, 0.8 μM each of primer FIP and BIP, 1.25 U of AMV reverse transcriptase (Fermentas Co., cat. no.EP0641) and 8 U of Bst DNA polymerase (New England Biolabs Inc.). The mixture was incubated at 60 °C for 60 min in a water bath and the products from the reaction were separated in the electrophoresis of agarose gel as described above.
Even though the principles of the first section of immunocapture RT-LAMP (IC-RT-LAMP) assay exactly followed the IC-RT-PCR with no RNA extraction step, in the second part, a different methodology was employed, leading to a significant reduction in the time as well as the cost. Section 1 same as the section 1 of IC-RT-PCR procedure (see above). In section 2, master-mix in total volume of 100 μl containing 10 mM DTT, 5 U of RNase Ribonuclease Inhibitor (Fermentas Co., cat. no. EO0381), 20 mM Tris–HCl, pH 8.8, 10 mM KCl, 10 mM (NH4)2SO4, 0.1 % Triton X-100, 2 mM Betaine (Sigma-Aldrich, Oakville, ON, Canada), 10 mM each dNTP, 0.2 μM each of F3 and B3, 0.8 μM each of primer FIP and BIP, 1.25 U of AMV reverse transcriptase (Fermentas Co., cat. no.EP0641) and 8 U of Bst DNA polymerase (New England Biolabs Inc.) were added to plate and incubated at 60 °C for 60 min in water bath. An agarose gel electrophoresis system under UV illumination could be also employed to visualize positive reactions as described above.
Colorimetric RT-LAMP Assay
The validations of positive RT-LAMP reactions were justified by means of five staining approaches. To visually detect the LAMP products, several dyes were added to the RT-LAMP reaction as described by Almasi et al. (2015). Prior to amplification, 2 μl magnesium sulphate (8 mM MgSO4), 1 μl of GeneFinderTM (diluted to 1:10 with 6× loading buffer) (Biov. Bio. Xiamen, China), 1 μl of SYBR Green I (diluted to 1:10 with 6× loading buffer) (Invitrogen, Sydney, Australia), 1 μl of the hydroxynaphthol blue dye (HNB) (3 mM, Lemongreen, Shanghai, China) and 1 μl of phenol red (diluted to 1:10 with 6× loading buffer) (Sigma-Aldrich Chemie GmbH) was separately were added from a stock solution to the master mix. The tubes containing MgSO4, GeneFinderTM, SYBR Green I, HNB and phenol red dyes were easily monitored for colour change by simply looking at them in daylight.
Colorimetric IC-RT-LAMP Assay
To visually detect the IC-RT-LAMP products, prior to amplification four dyes including GeneFinderTM, SYBR Green I, HNB and phenol red were added to master mix as described in section 2 of IC-RT-LAMP assay. The plate incubated at 60 °C for 60 min in water bath and was easily monitored for colour change by simply looking at them in daylight.
Real-time Quantitative RT-LAMP Assay
The turbidimeter is detecting only the by-product of the DNA synthetic reaction, the pyrophosphate ions as the insoluble magnesium pyrophosphate. For this reason, we determined the relationship between the amount of amplified DNA and the turbidity measured by the real-time turbidimeter. The real-time RT-LAMP reactions were carried out in 25 μl reaction mixture containing the following reagents as described in RT-LAMP assay. The reaction was carried out and monitored at 63 °C for 60 min in a Loopamp real-time turbidimeter (LA500-C, Eiken Chemical Co., Ltd., Tokyo, Japan). Real-time RT-LAMP caused by the accumulation of magnesium pyrophosphate was monitored spectrophotometrically at 650 nm.
LAMP is comparable to PCR in terms of sensitivity, but is less affected by presence of non-targeted DNA and inhibitory molecules. Some researchers even report specific amplification with LAMP without prior extraction procedure, by directly adding reaction mixture to swab specimens or sera (Almasi et al. 2012; Moradi et al. 2012; Moradi et al. 2014). To evaluate sensitivity of the different assays, a ten dilution series of leaf extract (for DAS-ELISA) and RNA of the positive sample (for other assays), was prepared from 1×1010 to 1×101. The DAS-ELISA results carried out both visual detection and a DYNEX MRX microplate reader. Similarly, the detection limit of the other assays was approved by electrophoresis on 1.5 % agarose gel and visual detection.
On the whole 2 out of 38 (5.3 %) leaf samples suspicious of having infection with PVX showed positive responses against DAS-ELISA assay. Interestingly, due to observing a clear colour change in the wells containing positive reactions (green colour as an indicator), no attempt was accordingly made to employ an ELISA Reader (Fig. 2). All two samples, subsequent to nomination as PVX 10 and PVX 30, were utilized lastly for further analyses. As regards RT-PCR, following provide RNA template, the amplification occurred via both backward and forward primers to generate ultimate products. The method, overall, could successfully identify three aforementioned positive samples. As expected, a fragment with the size band of 400 bp was detected when the RT-PCR products were run on 1.5 % agarose gel and stained with ethidium bromide, but there are no amplification products appear in all evaluated negative samples and negative controls (Fig. 3a). The new IC-RT-PCR protocol could successfully identify positive samples, interestingly with no attempt to RNA extraction (Fig. 3b).
The RT-LAMP protocol could identify positive samples with use of RNA in a water bath directly. RT-LAMP amplicons were finally electrophoresed on a 1.5 % agarose gel (as an optional system), and a large number of fragments (a ladder-like pattern) were eventually visualized (Fig. 3c). The reaction produces a mixture of stem-loop and multi-loop cauliflower-like structures that are all constructed from multiple repeats of only the targeted genomic sequence. The IC-RT-LAMP protocol could identify positive samples with use of RNA in a water bath directly. IC-RT-LAMP amplicons were finally electrophoresed on a 1.5 % agarose gel (as an optional system), and a large number of fragments (a ladder-like pattern) were eventually visualized (Fig. 3d). The reaction produces a mixture of stem-loop and multi-loop cauliflower-like structures that are all constructed from multiple repeats of only the targeted genomic sequence. RT-LAMP amplicons were able to be detected with the naked eye by adding different visual dyes followed by colour changing in the solutions. In this regard, all used visual components could successfully make a clear distinction between positive and negative ones. It is noticeable that positive results by MgSO4, GeneFinderTM, SYBR Green I, HNB and phenol red were turbidity, green, green, sky blue and red respectively (Fig. 3e). The same as colorimetric RT-LAMP, our new colorimetric IC-RT-LAMP protocol could identify positive samples with the naked eye (Fig. 3f).
First, we validated the real-time RT-LAMP designed for this study. Fig. 3g shows the results of real-time turbidity measurements conducted in 4 wells containing the same template RNA. For best amplification the optimum temperature was 63 °C for the activation of Bst DNA polymerase. It required 15 min for initiation of amplification to cause a change in the turbidity by magnesium pyrophosphate. These data demonstrate that the temperature control and turbidity measurements of the 4 wells are sufficiently uniform. Therefore, we concluded that there were essentially no variations in measurement results attributable to well position and thus conducted the following experiments.
Our results, interestingly, indicated that RT-LAMP (by different method) can produce reliable products even under lower RNA concentration (1×102 or more), whilst DAS-ELISA, RT-PCR and IC-RT-PCR require higher level of DNA (1×104, 1×105 and 1×104 or more respectively (Fig. 4). The results showed that RT-LAMP higher sensitivity for detection of PVX in comparison with DAS-ELISA, RT-PCR and IC-RT-PCR (100-fold, 1000-fold and 100-fold respectively) (Table 3). The real-time RT-LAMP results showed that the assay is detectable up to 1×102 (Fig. 4i). It was observed that with an increase in the quantity of initial template RNA, it will shorten the threshold time. A standard curve was generated plotting Tt the log of the initial template concentration (Fig. 4k). These results revealed that the calibration curve of the quantity of initial template RNA obtained using the real-time turbidity measurements has a high linearity and good reproducibility. This observation indicates that the quantity of the template RNA of an unknown concentration can be determined by comparing the Tt value with the Tt values of the template RNA of known concentrations.
Potato is a versatile, carbohydrate-rich food highly popular worldwide, prepared and served in a variety of ways. In Iran, potato, after wheat and rice, plays a fundamental role in a daily food chain that is extremely used around the country (Almasi et al. 2012). Unfortunately, during the last few years, virus activity commercially has led to a noteworthy diminution in the total yield of potato. The same as some viral diseases, even though presumptive diagnosis of potato viruses can be relatively simple when typical symptomatology is evident, symptoms in plants are not always specific and can be confused with those caused by other biotic or abiotic agents (Almasi et al. 2013a; Almasi et al. 2013b; Ahmadi et al. 2012). On the other hand, detection of deleterious viruses in symptomless plant material for preventive control is a compulsory task but can be extremely difficult since low populations with uneven distribution of the pathogen can occur; developing fast, easy, highly sensitive, cost-effective and reliable diagnostic protocols to make an accurate discrimination is accordingly required (Ahmadi et al. 2012; Almasi et al. 2012; Clark and Adams 1977). Potato virus X (PVX) is widely recognized as a serious threat to potato production in Iran and several countries. This has increased the need for accurate identification of this virus. Genetic analysis is preferable in testing a patient for infectious diseases because it can yield detailed genetic information about the virulence and antibiotic resistance of a particular microbial. However, the results of genetic analysis take more than 1 h to obtain (Ren et al. 2009; Ahmadi et al. 2012; Almasi et al. 2012; Moradi et al. 2014; Almasi et al. 2015). Nucleic amplification is one of the most important tools for many investigators, including molecular biologists. In particular, in application oriented fields such as clinical medicine, genetic diagnosis is used for monitoring infectious diseases, genetic disorders and genetic traits (Van der Linden et al. 2004; Moradi et al. 2014). LAMP is a gene amplification method with a variety of characteristics and applications in a wide range of field and has several advantages over PCR. First, the specificity of LAMP is high because the LAMP method uses multiple primers, recognizing six distinct sequences in the target DNA. Second, the method is both rapid and simple; only (30-90 min) are needed to amplify the target sequences. Third, the cost of the equipment is low compared with PCR, which is one of the major reasons why PCR diagnostics have not been more widely utilized (Hadersdorfer et al. 2011; Mori and Notomi 2009; Nagamine et al. 2002; Fukuta et al. 2003). On the other hand, LAMP can amplify genes isothermally and the amplification reaction can be carried out with a simple heater. There is no need for the special device used for polymerase chain reaction (PCR) to rapidly control the temperature. This characteristic greatly facilitates detection of the LAMP reaction (Nie 2005; Almasi and Dehabadi 2013; Ahmadi et al. 2012; Almasi et al. 2012; Almasi et al. 2014; Van der Linden et al. 2004; Moradi et al. 2014; Almasi et al. 2015).
In this study, as a result, several detection methods were assessed to explore positive and negative aspects of each one, followed by introducing the best one regarding PVX detection. Even though all five techniques had enough potential to make differentiation and detect infected plant samples accurately, colorimetric IC-RT-LAMP proved to be much more useful as some factors including time, safety, simplicity, cost and being user friendly (Table 4). This, in turn, would simplify the detection procedure and result in saving of significant time needing for separating of the amplified products on the gel and the analyzing of the data which are commonly used in the other PCR-based methods. HNB, SYBR Green I, GeneFinderTM and phenol red dyes were used in this study to decrease contamination and intensify color stability and better visibility of the reaction results. These dyes can be used to create a visible color change that can be seen with naked eyes without the need for expensive equipment, or a response that can more accurately be measured by instrumentation. Dye molecules intercalate or directly label the DNA, and in turn can be correlated to the number of copies initially present. The introduction of an in-tube or in-plate color indicator such as these dyes eliminates the need for additional staining, and makes visual detection easier and more rapid since post-amplification processes are obviated. There is no need for the use of UV light which is irritating to the eyes.
In turbidity method, it is important to have a stable baseline in quantifying the amount of initial template nucleic acid by checking the amplification curve in real-time. This is because a stable baseline permits the specification of the threshold in the earlier phase of amplification, resulting in an improvement in the reproducibility of the quantitative results. In contrast to the real-time fluorometry measuring the gradual increase in the signal intensity, it is easier to obtain a stable baseline by the real-time turbidimetry measuring the decrease in the signal intensity (Mori et al. 2004; Parida et al. 2004). For the RNA quantification experiments using the real-time turbidimeter designed as a prototype in this study, this factor may account for the high degree of linearity and good reproducibility of the calibration curve. This method for the quantification of nucleic acids takes advantages of two important features of the RT-LAMP method, namely the high specificity that eliminates the need to check the amplification product, and the ease with which turbidimetry can detect the amplification product. So, this method can quantify template RNA without any reagent for detection. This important feature of real-time RT-LAMP enables us to develop more cost-effective reagent for quantitative analysis of RNA than that for real-time RT-PCR method, which usually needs expensive fluorescence probe for ensuring specificity of reaction (Mori et al. 2004).
Therefore, colorimetric IC-RT-LAMP was appropriate for field laboratories without thermal cyclers or other sophisticated facilities for identification of PVX. The advantages of method make it a suitable field test tool for diagnosis of emerging and reemerging diseases. As it is relatively cheap and reliable most probably colorimetric IC-RT-LAMP will be more widely integrated into routine use of molecular techniques.
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