Introduction
Many present diagnostic tests and drug discovery application requires analysis of large numbers of biomolecules simultaneously. These analyses should be versatile for high throughput and small sample volumes. This is traditionally achieved by surface-based microarray technologies. The comparison of bead-based suspension arrays and microarrays showed the former has numerous advantages.
- Smaller sample volumes
- Improved binding kinetics
- Acts as a support for the synthesis of large numbers of different molecules (probes and drugs) in both surface and mix synthesis.
- Probe molecules coated with different microparticles act as platforms for the analysis of biological samples. For each particle, a unique code is present which helps in its detection after a biological response. Particle florescence is the major used detection parameter.
- Availability of various coding methods and readout platforms is in use.
The application of Suspension Array Technology for the detection of viroids is discussed in this article. Smallest plant pathogens are classified as viroids. They possess characters of autonomous replication, unencapsidated infectious RNAs. These RNAs moe through an infected plant in a systematic manner. Their genomic size ranges from 250–400 nucleotides (nt) and it is circular in nature. These nucleotide sequences do not code any specific protein. Viroid species are classified into two families Pospiviroidae and Avsunviroidae. Pospiviroidae contains five genera and Avsunviroidae contains three genera.
There are ten known species of Pospiviroidae.
- Chrysanthemum stunt viroid (CSVd)
- Citrus exocortis viroid (CEVd)
- Columnea latent viroid (CLVd)
- Iresine viroid 1 (IrVd-1)
- Mexican papita viroid (MPVd)
- Pepper chat fruit viroid (PCFVd)
- Potato spindle tuber viroid (PSTVd)
- Tomato apical stunt viroid (TASVd)
- Tomato chlorotic dwarf viroid (TCDVd)
- Tomato planta macho viroid (TPMVd)
A recent discovery of conspecificity of MPVd and TPMVd has to lead to the reclassification of these species to new genera namely TPMVd. The economic importance of the Pospiviroids is that they are responsible for major diseases of horticultural and agricultural crops. List of plants attacked by this Pospiviroids are tomato (Solanum lycopersicum), potato (Solanum tuberosum), pepper (Capsicum annuum), citrus (Citrus spp.) and ornamental chrysanthemum (Chrysanthemum morifolium). Tomatoes are infected by all pospiviroid species, except IrVd-1. The symptoms which are produced due to this infection is also similar. Stunting, epinasty, leaf distortion, smaller fruit and reduced yield are the general symptoms of these infections. These diseases are also widespread in different climatic zones and environment. Thus it became a worldwide concern of phytosanitary. In EU, PSTVd and CSVd have an explicit quarantine pest status. The member states of EU ave also have a statutory obligation to control these viroids. To preserve open trade the control of these pospiviroids is of great economic importance.
The source of infection of these pospiviroids is linked to infected planting material, including infected seed lots, and also to asymptomatic viroid-infected ornamental plants. The most efficient method of control and management of pospiviroid relies on the indexing of planting material and asymptomatic ornamental hosts as part of quarantine and certification schemes. It has effectively controlled, to an extent, the introduction and spread of the pathogen. The indexing procedure can be carried out using conventional RT-PCR methods with the use of degenerate primers. Several pospiviroids can be detected by real-time RT-PCR methods.
An effective and improved method of pospiviroid detection can be achieved by using multiplexed detection methods. This may result in faster response to incursion events improved biosecurity outcomes. An approach to simultaneous detection of multiple nucleic acid sequences in a single reaction can greatly reduce the time, cost and labor associated with conventional single reaction detection technologies. Such a technique has been developed recently. The multiplexed detection of pathogens has been proved in clinical settings. It employs 6.5 mm carboxylated, superparamagnetic polystyrene microspheres. These microspheres are internally labeled with a spectrally distinct fluorescent dye. An anti-MagPlex-TAG oligonucleotide sequence is also pre-coupled with these microspheres. Spectral address aids in the classification of the different microsphere. When they are coupled simultaneous detection of 150 different nucleic acid sequence targets in a single reaction.
The first step in the Luminex MagPlex-TAG microsphere system is a generic multiplexed RT-PCR step. This is followed by multiplexed asymmetric PCR step. This step is termed as Target Specific Primer Extension (TSPE). During this step, if there is a sequence match, hybridization of the internal primer of the multiplexed amplification occurs. This internal primer is also extended in this step. The resultant TSPE products are biotinylated and labeled with complementary MagPlex-TAG sequences at their 5′ end. The next step is the hybridization of TSPE products and MagPlex-TAG microsphere mixture (MagPlex-TAG/anti-MagPlex-TAG hybridization). The detection of the incorporated biotin is done by a fluorescent reporter molecule. The bead-TSPE product complexes are then detected on the Luminex instrument.
A threshold for positivity is required to determine whether a sample is positive or negative in a particular assay. This threshold value is also termed as a cut-off value in certain application/ studies. Manufactures of these bead-based arrays for nucleic acid detection defines the threshold for positivity for all assays within an array that exceeds that of the background (noise) by an arbitrary value or factor. But this approach cannot be applied to every application. This approach simply does not consider the unique properties of each assay, the complexity of these assays and multiplexed array. A suitable statistical method is lacking for this purpose. Generally, variability exhibited in median fluorescence intensity (MFI) values are considered. This arbitrarily defined threshold for positivity has no statistical meaning. It does not or cannot provide information on the percentage of false positives attributed to samples analyzed using that method.
In this article, the development of a multiplex PCR-Luminex MagPlex-TAG bead suspension array for the generic and individual detection of all nine currently recognized species in the genus Pospiviroid is described. The array is an 11-plex module with a hierarchical assay design with the incorporation of a near-universal assay to detect all pospiviroid species. Co-amplification assay of the plant mRNA, targeting the NADH dehydrogenase subunit 5 (nad5) gene was used as an internal control assay. It aids in exclusion of false negative results.
Materials and Methods
Plant Samples and Nucleic Acid Extractions
Leaves of viroid-infected plants used for assay development and validation were procured and stored at 2200C. Total RNA was extracted from 100 mg of frozen leaf material using the RNeasy Plant Mini Kit according to the manufacturer’s protocol and stored at 2200C. In order to simulate a plant infected with more than one viroid species, total RNA extracts from separately infected plants were mixed prior to testing.
Multiplexed Array Design
For the design of pospiviroid species-specific and universal assays, all 642 available full-length pospiviroid sequences were retrieved from the National Center for Biotechnology Information (NCBI) GenBank database. Sequences were aligned using ClustalW. Based on these multiple sequence alignments, candidate regions were identified that best demonstrated (i) sequence conservation within a species whilst exhibiting sequence divergence between species for the design of species-specific assays, and (ii) sequence conservation across all species for the design of the universal assay, excluding the sequence divergent CLVd species. For reference, in silico analysis was performed to predict the number of isolates likely to be detected with each assay. Multiplex RT-PCR primers and target specific primer extension (TSPE) primers were designed to these regions according to established guidelines for real-time PCR assays, with primer characteristics, checked using Free Online Tools and specificity checked against sequence data available on GenBank using BLAST (NCBI). The plant internal control assay (PlantIC), specific for mRNA of the mitochondrial NADH dehydrogenase subunit 5 (nad5) gene, was adapted from a real-time PCR assay. Each TSPE primer was designed with a unique MagPlex-TAG sequence appended to the 5′ end, complementary to the anti- MagPlex-TAGs displayed on the surface of the corresponding MagPlex-TAG bead address. Final primers were synthesized.
Multiplex Two-step RT-PCR and Post-PCR Purification
To initiate cDNA synthesis, a mixture containing 2 mL of RNA extract, 300 nM each of the reverse primers PospR1, PospR2 and Nad5R and nuclease-free distilled water (dH20) to a final volume of 10 mL was incubated at 800C for 10 min and snapcooled on ice. The cDNA synthesis reverse transcription (RT) the reaction mixture was then added, which contained 4 mL of 5 X first strand buffer , 10 mM dithiothreitol (Invitrogen), 0.5 mM dNTPs (Invitrogen), 10 U RNase OUT (Invitrogen), 50 U Superscript III (Invitrogen), and dH20 to a final volume of 10 mL, and incubated at 500C for 45 min, then 70oC for 15 min. Following cDNA synthesis multiplexed PCR was done using seven PCR primers PospF1, PospF2, PospF3, PospR1, PospR2, Nad5F, Nad5R, which were designed to amplify all pospiviroid sequences and the plant mRNA internal control. Each 25 mL reaction contained 3 mL cDNA template, 1 U Platinum Taq DNA Polymerase, 250 nM of each PCR primer, 100 mM of each dNTP, 1.5 mM MgCl2 and 1XPCR buffer (supplied). Cycling conditions for the multiplex PCR were 940C for 1 min, 30 cycles of 940C for 30 s, 580C for 30 s, and 72oC for 30 s, with a final extension of 72oC for 3 min. All PCRs were performed in 96 well plates using a Veriti thermal cycler Amplicons were purified using PCR clean-up filter plates to remove excess primers and dNTPs. Purified amplicons were used as templates for target-specific primer extension (TSPE) reactions utilizing target specific MagPlex-TAG primers. During initial experiments, multiplexed RT-PCR products were verified by 2% agarose gel electrophoresis with GelRed staining.
Multiplex TSPE
Linear TSPE reactions contained 5 mL of purified multiplex PCR amplicons, 0.75 U Platinum GenoTYPE Tsp DNA polymerase, 25 nM of each of the 11 TSPE primers, 5 mM each of dATP, dGTP, dTTP and biotin-dCTP, 1.25 mM MgCl2, 1XPCR buffer. Cycling conditions were 96oC for 2 min, 30 cycles of 94oC for 30 s, 58oC for 1 min, and 74oC for 1 min.
Hybridization and Detection
Microsphere hybridization reactions were performed according to the manufacturer’s recommendations. All washing steps were performed using a magnetic plate separator. To prepare the mixture of 11 optically distinct Luminex MagPlex- TAG beads, each bottle of MagPlex-TAG microspheres was vortexed vigorously for 1 min, then sonicated for 30 s. Bead mixtures were prepared in Eppendorf LoBind tubes, diluted with 2XTm hybridization buffer (0.2 M Tris-HCl pH 8.0, 0.4 M NaCl, 0.16% Triton X-100) to yield a final concentration of approximately 1250 beads of each bead address per 25 mL. The bead mixture was vortexed again for 30 s, then aliquoted in 25 mL portions into 96-well skirted microplates. Single- stranded biotinylated linear amplicons (5 mL) were added per well, with the total reaction volume adjusted to 50 mL per reaction with dH20. For negative (no template) control wells, 25 mL of dH20 was added to the bead mixture. Reactions were denatured at 96oC for 60 s, then hybridized at 37oC for 30 min. After hybridization, reactions were shaken for 5 min using a microplate shaker, then washed twice in 75 mL of 1XTm Buffer and resuspended in 50 mL of 1XTm Buffer containing 2 mg/mL streptavidin-R-phycoerythrin conjugate. Plates were incubated at 37oC for 15 min, then washed once in 75 mL of 1XTm Buffer, resuspended in 75 mL of 1XTm Buffer and shaken for 5 min. Samples were analyzed and microsphere complexes detected using a Luminex FlexMAP 3D instrument (Luminex). The MFI values for biotinylated extension products attached to 100 microspheres of each individual bead address were measured for each assay, and each sample tested in triplicate. The average MFI from three template-free control samples was also determined to monitor background fluorescence, and background MFI subtraction was not used.
Sensitivity and Inter-assay Reproducibility
To determine the lower limit of detection (LOD) of the multiplexed array, six ten-fold serial dilutions of total RNA extracted from a naturally infected PSTVd isolate #N were prepared, ranging from 268 ng/mL to 268 pg/mL in a background of healthy tomato RNA (1/500 dilution of neat extract), and tested in triplicate. Total RNA concentrations were quantified by spectrophotometry using an Eppendorf Biophotometer. To evaluate the inter-assay reproducibility of the array, one neat extract of PSTVd isolate was tested in ten independent reactions over separate days, with mean ln(MFI) values and error bars. The variability of the ln(MFI) values was assessed by one sample t-tests, with p-values ,0.05 considered statistically significant.
Blind Testing of Single and Mixed Infections
To further assess the performance of the Luminex MagPlex-TAG Pospiviroid array, 11 ‘‘blind’’ samples of unknown identity were tested by an independent operator at an independent facility. The sample panel included both single and simulated mixed infections, in addition to healthy tomato controls. All blind samples were previously characterized using DNA sequence analysis and were provided to the operator as total nucleic acid extracts. Samples were tested using the multiplexed array according to the conditions described in this study, except that microsphere hybridization reactions were detected using a Luminex MAGPIX instrument.
PCR assay
The two parallel reactions were performed using the RNA-to-CTTM 1-Step Kit. The reaction mixture of the generic reaction consisted of 12.5 ml 2× TaqMan RT-PCR mix, 0.6 ml 1× TaqMan RT enzyme mix, 0.75 ml forward primers TCR-F 1-1, TCR-F 1-3, TCR-F 1-4, TCR-F IrVd, TCR-F PCFVd and nad5-F and reverse primers TR-R1, TR-R CEVd, TR-R6 and nad5-R (0.3 mM each), 0.25 ml TaqMan probe pUCCR (0.1 mM) and 0.5 ml TaqMan probe nad5-P (0.2 mM). Sterile water and 2 ml RNA template were added to a final volume of 25 ml. For the CLVd reaction the target-specific primers and probe were replaced by 0.75 ml forward primers CLVd-F, CLVd- F2, reverse primer CLVd-R (0.3 mM each) and 0.75 ml TaqMan probe CLVd-P (0.1 mM). Real-time RT-PCRs were carried out in 96-well plates on a Real-Time PCR system, 15 min 48◦C, 10 min 95oC, followed by 40 cycles 15 s 95oC and 1 min 60oC, or on an Sequence Detection System, 30 min 48oC, 10 min 95oC, followed by the same cycling programme. Each plate included a positive control (PSTVd reference strain) and a negative control (no template).
Performing the reactions with One-Step RT-PCR master mix reagents revealed that the choice of reagents can be critical, because Ct values increased by 8–10 in comparison to the RNA-to-CTTM 1-Step Kit.
Data were analyzed with Bio-Rad CFX manager 2.0 software, applying a manual threshold of 200 RFU (relative fluorescent units) for VIC and FAM. Raw data were analyzed with Sequence Detection System (SDS) software applying an automatic baseline setting and a manual threshold setting of 0.2. Ct values were interpreted qualitatively with Ct values below 32 considered positive, between 32 and <37 dubious (needs confirmation) and ≥37, negative. These values are chosen such that false negatives are minimized. For both reactions the same values were used, even though the reactions may differ in sensitivity. Further fine tuning of these values may be required as more experience of the assay is gained. In addition, laboratories will need to verify cycle cut off values when implementing the assay under their conditions. Overall the result of the assay is based on results of the generic and CLVd, reaction and the nad5 control, and is positive when either the generic or CLVd reaction has a positive result.