The recently developed microchip based real-time PCR offers accurate, robust and cost effective detection of pathogens in potato seed, crop, and soil which is essential for diagnosis, and surveillance for this important food source in addition to various other applications.1-3 The innovative features of this technology impart an edge over the currently used methods for potato pathogen detection as cost effective technology with high throughput screening capability (Table 1).
To meet its throughput, a simple, and fast nucleic acid isolation from potato tubers, seeds, crops, and soil with amenability to automation is also essential as an upstream method.4 To address this requirement, a variety of nucleic acid extraction methods are available along with commercial kits.5 Many of these methods involve time-consuming and labour-intensive complex steps such as centrifugation, precipitation, and ﬁltration. The choice of method has a profound impact on the time and effort required to process the screening of potato pathogens. The choice of method is also important when many samples need to be processed. Thus the existing methods are not always suitable while an innovative sample preparation can enhance chances of pathogen detection and imparts ease and speed to the detection process. This is especially important when lab staff has limited training and time.
Nucleic acid isolation and purification consists of three basic steps: lysis, removal of protein and contaminants, and recovery of DNA or RNA. Currently, commercial kits using a combination of solvents and solid phase support including paramagnetic beads, and spin columns are widely used to isolate and purify DNA and RNA.6 The paramagnetic beads are especially very popular by providing fast, efﬁcient puriﬁcation, and carrying pre-concentration of the isolated nucleic acid typically carried out in the physiological range.7-8 The paramagnetic DNA purification is an improvement upon centrifuge-dependent isolation techniques. The need to move the samples into and out of a centrifuge is inefficient for meeting high throughput demands. On the other hand, the use of paramagnetic beads being amenable to automation, allows the tubes to remain on its deck throughout the entire process.
In the present study, we have adapted the paramagnetic bead approach to potato samples by isolating the plasmid DNA spiked into the potato sample by replacing silica beads with paramagnetic beads and analyzing the isolated DNA by microchip real- time PCR.
Materials and methods
1. Procurement of materials: The silica bead based DNAsorb nucleic acid extraction kit was procured from AmpliSens, Bratislava, Slovak Republic and paramagnetic beads were purchased from Qiagen, Valencia, CA, USA. Recombinant plasmid pUC-35S gene of cauliflower mosaic virus (CAM virus) was custom synthesised from ATG Service-Gen, St.-Petersburg, Russia.
2. Potato sampling and processing: Sample piece was scooped out from the potato eye using a tiny spatula and placed into the cup of the squeezer ensuring the potato skin to squeeze first (Figure 1). The potato mesh was collected further into a 30 ml falcon tube and was further
crushed with spatula followed by weighing out 30 mg in individual 1.5 ml taper tubes for further process.
3. Spiking of the potato samples: To each tube containing 30 mg of the sample, 1μl (10 pg/μl) of plasmid pUC-35S was spiked using programmable automatic pipettor (Sartorius AG, Gottingen, Germany). It was followed by addition of 300 μl of lysis buffer of the Ribo-sorb kit and vortexed for 5 minutes. The tubes were then heated at 65°C for 5 minutes using dry bath heater (MK-20, China) with occasional manual mixing. The tubes were centrifuge for 5 sec at 5000×g to remove drops from internal surface of the lids.
4. Addition of mobile solid phase: To one of the tubes, 25 μl of silica beads suspension, and to the other tube 10 μl of paramagnetic beads suspension was added, respectively. The tubes were vortexed for 10 sec and incubated at room temperature for 60 sec and again mixed for 10 sec and incubated for 5 min at room temperature.
5. Separation of beads: The tubes containing silica beads were centrifuge for 30 sec at 10,000×g to pellet the beads for removing and discarding supernate without disturbing the bead pellet. The tubes containing paramagnetic beads were inserted into the magnet block for 1 minute to pellet the beads and remove the supernate.
6. Washing of beads: To each tube, 400 μl of Washing Solution 1 was added and the silica bead containing tubes were vigorously vortexed to fully resuspend the beads followed by centrifugation for 30 sec at 10,000×g. The paramagnetic bead containing tubes were subjected to magnet block for pelleting beads and removal of supernate. To each of the bead containing tubes, 500 μl of Washing Solution 2 was added followed by vigorous vortex. The silica bead tubes were centrifuge for 30 sec at 10,000×g. The supernate was carefully removed and discarded. The tubes containing paramagnetic beads were inserted into the magnet block for 1 minute to pellet the beads and remove the supernate. The procedure was repeated with this buffer.
7. Pre-drying of DNA bound beads: All the tubes were incubated with open caps for 12 min at 60°C for pre-drying in the heater ensuring no over-drying of beads.
8. Elution: The bead pellets were resuspended in 50 μl of the TE buffer and incubated for 3 min at 60°C with intermittent mixing.
9. Optimization studies: The sample prep was also attempted for using isopropanol for washing the paramagnetic beads during the nucleic acid isolation process.
10. Microchip real-time PCR:
a. The real-time PCR was set up for 35S gene of CAM virus. Master mix was prepared following the Table 1, and then 1.2 μl was dispensed into each of the well of the 6×5 format microchip as per the microchip map presented in Figure 1d. The microchip was filled with 620 μl of silicone oil that covered the dispensed test mix in wells to contain evaporation during thermocycling.
b. The amplification was performed with thermal condition: Hold 95°C for 120 sec followed by 45 cycles of 95°C for 1 sec and 60°C for 30 sec. For each well, a fluorescent light emission was recorded. Analysis was based on the Ct values of the PCR products. The Ct value is the PCR cycle at which the fluorescence measured between each cycle exceeds a threshold determined by background fluorescence at baseline and is placed in the exponential phase of the amplification curve. The threshold and baseline were set automatically by software or were user-defined.
Results & discussion
Numerous detection methodologies for pathogens are now available, but their acceptance to perform high throughput screening, faces an important question on sensitivity, accuracy, robustness, frequency of testing and the cost. Recently, a microchip based fast and simple polymerase chain reaction method has been developed for detection of pathogens in diverse biological and soil samples using miniaturized volumes of samples and reagents.1-3 The features of this technology detailed in Table 1, address these questions. The low running cost of this approach, makes it especially attractive for large-scale screening of crops, poultry, cattle, fish, and food for pathogen detection.
With the objective of providing an easy, fast and low cost sample prep method for isolation of bacterial, fungal, and viral pathogen nucleic acid to be used in the microchip, a paramagnetic beads based method is modified and optimized in the present studies.
Comparison of silica beads and paramagnetic beads: From 30 mg of potato tissues spiked with 1 μl of plasmid DNA, the DNA extracted using paramagnetic beads resulted amplification Ct values comparable to silica beads which are supplied as optimized reagent by the kit provider (Figure 2).
Optimization of paramagnetic beads: With the objective of optimizing the quantity of paramagnetic beads, a set of extractions were carried with 30 mg of potato spiked with 1 μl of plasmid DNA with addition of 5, 10, 15, and 20 μl of paramagnetic bead suspension. The results indicated that 20 μl of the beads was slightly better than the other samples even though there was not a significant difference among them (Figure 4).
In view of the importance of false negatives in the screening process, it is better to provide larger surface area to enhance the probability of catching pathogen nucleic acid, 20 μl of the beads should be used in this screen. The data also suggest that DNA extraction with both silica beads and paramagnetic beads resulted similar quantity of DNA in the presence and absence of 30 mg of potato tissue.
The extraction efficiency: The efficiency of DNA extraction does not seem affected by the 30 mg of potato tissue used in the extraction process. This observation is supported by the Ct values of the DNA extracted from the control (1 μl in 30 μl of TE buffer) using 10 μl of paramagnetic beads which is close to that of the 30 mg of potato extracted with 10 μl of the beads (Figure 4).
Optimization of wash process of paramagnetic beads: The sample prep was also attempted with isopropanol for washing the paramagnetic beads during the nucleic acid isolation process. The results indicated that two washes of 200 μl isopropanol were equivalent to the standard wash solution of the kit (Figure 5).
This suggests that sample prep can be simplified with isopropanol which in turn reduced the pre-drying step from 5-10 min to 1-2 min (n=5). The number of the ct values (29.7) for the extraction with isopropanol corresponds with that of the standard wash buffers used in the previous experiment while the control displayed the ct 20.46.
Total duration of protocols: The duration of each step has been presented in the Table 2 where addition and removal of liquid is not included for easy comparison of total duration of both sorbent beads and paramagnetic beads.
The latter technique is about five minutes shorter, easier to perform using magnet block rather than centrifugation, and also allows to avoid inconsistent removal of supernate due to pellet disturbance. Moreover, the use of paramagnetic beads is amenable to automation as magnet block can be used on the deck of automated workstations for high throughput isolations.4
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