Detection of Plant Quarantine Pathogen Ralstonia solanacearum Race 3 Biovar 2 with Portable POCKIT™ and BLItz® Systems
Published Date: September 15, 2016
Detection of Plant Quarantine Pathogen Ralstonia solanacearum Race 3 Biovar 2 with Portable POCKIT™ and BLItz® Systems
Di R1*, Huang Q2, Stulberg MJ2, Zhao L1, Levy L3
1Department of Plant Biology and Pathology, Rutgers, the State University of New Jersey, New Brunswick, NJ 08901, USA
2Floral and Nursery Plants Research Unit, Agricultural Research Service, United States Department of Agriculture, Beltsville, MD 20705, USA
3Center for Plant Health Science and Technology, Plant Protection and Quarantine, Animal and Plant Health Inspection Service, United States Department of Agriculture, Riverdale, MD 20747, USA
*Corresponding Author: Rong Di, Department of plant Biology and Pathology, Rutgers, the State University of New Jersey, New Brunswick, NJ 08901, USA. Tel: 848-932-6350; Fax: 732-932-9377; E-mail: email@example.com.
Citation: Di R, Huang Q, Stulberg MJ, Zhao L, Levy L (2016) Detection of Plant Quarantine Pathogen Ralstonia solanacearum Race 3 Biovar 2 with Portable POCKIT™ and BLItz® Systems. J Plant Heal 1(1): 103.
Ralstonia solanacearum (Rs) race 3 biovar 2 (r3b2) is designated as a quarantine pathogen in many countries and additionally as a Select Agent in the United States. Rapid, sensitive and accurate detection methods are urgently needed. We report here the development of two portable platforms for r3b2 detection, the TaqMan probe qPCR (quantitative, real-time polymerase chain reaction)-based POCKIT™ and the Rs monoclonal antibody (MAb)-coupled sensor BLI (bio-layer interferometry)-based BLItz®. Our data indicate that combining the palm sized POCKIT™ with the previously published TaqMan-based qPCR primers and probe set RsSA2, which targets a non-phage r3b2-unique sequence annotated as a probable n-6 adenine-specific DNA methylase, can detect as low as 10 CFU of the r3b2 strain UW551 in 32 minutes. The RsSA2/POCKIT™ system can specifically detect heat-inactivated Rs r3b2 strains, and such cells in spiked geranium stem sections, as well as in UW551-infected symptomatic and asymptomatic geranium plants. Additionally, we demonstrate that the BLI-based BLItz® instrument with the Forsite Rs MAb, which is more reactive to UW551 strain than other Rs species complex members, can detect as low as 2.5 × 105 CFU/ml of UW551 in spiked geranium extract in a few minutes. The Rs MAb/BLItz® system has comparable rapidity and sensitivity to the commercial ImmunoStrip® with the advantage of higher r3b2 specificity. Moreover, the Rs MAb-coupled sensors are sensitive to Rs r3b2 after three months of room temperature storage, although the detection sensitivity was reduced by 28%. These two independent, portable systems have the potential to facilitate Rs r3b2 detection at the ports of entry and in field settings.
Keywords: Ralstonia solanacearum; Quarantine Pathogen Detection; POCKIT™, BLItz®; Portable qPCR
Rs: Ralstonia Solanacearum; r3b2: Race 3 Biovar 2; qPCR: Quantitative Real-Time Polymerase Chain Reaction; Mab: Monoclonal Antibody; CFU: Colony Forming Unit; ELISA: Enzyme-Linked Immunosorbent Assay; FSAP: Federal Select Agent Program; PCR: Polymerase Chain Reaction; iiPCR: isothermal Polymerase Chain Reaction; SPR: Surface Plasmon Resonance; TNT: Trinitrotoluene; BLI: Bio-Layer Interferometry; CPHST: Center for Plant Health Science and Technology; Ps: Pseudomonas syringae.
Ralstonia solanacearum (Rs) causes bacterial wilt in many crops, resulting in significant economic losses worldwide. Rs strains historically have been classified into five races, based on host range, and five biovars, based on their differential ability to metabolize a panel of carbohydrates. Rs strains are also grouped into four phylotypes, which correspond to the strains' geographic origins, and they are sub-classified into sequevars based on partial endoglucanase sequence . Rs race 3 biovar 2 (r3b2) belongs to the phylotype IIB and sequevar 1 and 2 clusters (IIB1&2) . Rs r3b2, which was thought to originate in South America, is found to be better adapted to temperate climates than other races and biovars . Since Rs r3b2 causes the destructive potato brown rot, it has been designated as a quarantine pathogen in Europe and North America and is additionally listed as a Select Agent in the U.S. [2,3]. Due to the lack of resistant potato cultivars and effective control measures, the best management practice in the U.S. is quarantine and exclusion of Rs r3b2. The ability to detect Rs r3b2 at ports of entry and in field environments with adequate sensitivity and speed will support exclusion and mitigation practices.
As with many plant pathogens, Rs can be detected in symptomatic plant tissues by immunodiagnostic assays such as commercially available Rs immunoflow devices (vertical and lateral designs) and the enzyme-linked immunosorbent assay (ELISA) such as the Rs PathoScreen Kit (Agdia Inc. Elkhart, IN). Although immuno-detection is rapid, both immunoflow and the PathoScreen Kit cannot distinguish different biovars of Rs, and their sensitivity levels are limited to about 105 colony forming units (CFU)/ml, as claimed by the manufacturer (www.agdia.com). With a double-antibody sandwich indirect ELISA coupled with an enrichment step, Caruso et al. reported that 1 to 10 CFU of Rs per milliliter of potato extract could be detected . As the ELISA assay includes several incubation steps, a 72-hours enrichment step only further lengthens the assay time. Additionally, the monoclonal antibody (MAb) used in this assay appeared to interact with all typical Rs biovars, 1 to 5. Presently, there have been very few reports of r3b2-specific monoclonal antibodies that can be used to distinguish the different Rs biovars. Rs is a USDA-APHIS-PPQ regulated select agent at the species level and the regulation includes all races and biovars (http://www.ecfr.gov/cgi-bin/retrieveECFR?gp=1&SID=b9126e9fba23e3e7933354a1d2630d72&ty=HTML&h=L&n=7y22.214.171.124.9&r=PART#se7.5.331_13). Non-r3b2 strains can be excluded from select agent regulation only if written identification data is submitted to the Federal Select Agent Program (FSAP) for approval demonstrating that the Rs strain detected is not r3b2. The ability to detect Rs and also quickly and accurately identify r3b2 is needed, and the use of multi-level test protocols increases confidence of the identification. As indicated by Huang et al. , the standard carbohydrate-utilization biovar test using bromothymol blue as a pH indicator takes weeks to complete due to the time needed for the indicator to change colors. Huang et al. improved biovar test used phenol red as the pH indicator but the test still required four days to produce results .
As quantitative, real-time PCR (qPCR) is increasingly used to detect and differentiate microorganisms, it has become the method of choice for Rs detection. Combining PCR analysis with the use of fluorogenic TaqMan probes, Weller et al. reported that their designed broad-range probe detected all Rs biovars, and their B2 probe specifically detected biovar 2 A with sensitivity of ³ 102 cells/ml in pure cultures . Since the draft genome of Rs r3b2 strain UW551 is known and the unique open reading frames and sequences specific to r3b2 have been identified , more specific TaqMan probes have been designed to distinguish different Rs races and biovars. Stulberg and Huang  recently reported the specific detection of Rs IIB-1 & 2 r3b2 strains by TaqMan-based qPCR using a RsSA1 primers and probe set targeting a non-phage sequence of the predicted ferric siderophore receptor, and a RsSA2 set targeting another non-phage sequence annotated to code for a probable n-6 adenine-specific DNA methylase, with the Bio-Rad CFX96 thermo-cycler (Hercules, CA).
However, the requirement for a sophisticated qPCR instrument and expensive reagents limits the application of qPCR for in-field Rs identification. Therefore, the first objective of this study was to investigate the portable POCKIT™ Nucleic Acid Analyzer system by GeneReach Biotechnology Corporation (Taichung, Taiwan) for sensitive and specific detection of Rs r3b2 using TaqMan probes in the qPCR platform. This simple, palm-size device (63 mm W × 50 mm H × 152 mm L, 380 g) is powered by 120-240 V, 50/60 Hz, 0.2 A with the power output of DC 5 V 1 A. It allows rapid reaction and simple readouts of fluorescent probe-based insulated isothermal PCR (iiPCR) in an individual plastic capillary R-tube™ that is specially designed with a thermal baffle at the bottom. iiPCR is based on Rayleigh-Benard convection PCR . The POCKIT system has been used to detect yellow head virus , white spot syndrome virus , and canine distemper virus .
In order to rapidly detect and identify r3b2 strains of Rs and distinguish them from non-r3b2 strains combining two different, independent methods, we were prompted to evaluate other non-PCR based portable platforms. In the past decade, label-free, surface plasmon resonance (SPR)-based methods with various biosensors have been used to detect plant pathogens including Fusarium culmorum , Phytophthora infestans  and Phakopsora pachyrhizi . Most of these SPR-based detection methods use expensive and sophisticated instrumentation such as the Biacore instrument from GE Healthcare (Piscataway, NJ). Since its development, the portable, SPR-based SPIRIT™ system (Seattle Sensor Systems Corp., Seattle, WA) has been used to successfully detect Staphylococcus aureus enterotoxin B, cortisol, domoic acid, trinitrotoluene (TNT), viruses, bacteria and spores [14-17] with antibody-coupled biosensors. Both the Biacore and SPIRIT™ systems were evaluated previously, and it was found that the stringency of the microfluidic system in the SPR-based platforms limits their use in handling complex, diseased plant samples (data not shown). Another label-free, optical sensing-based technology relying on bio-layer interferometry (BLI) has recently been developed by Pall ForteBio Inc. (Menlo Park, CA). BLI-based instruments have been used to analyze protein-liposome interactions , to study the mode of action of the type 2 diabetes drug metformin , to screen hybridomas for the identification of high-affinity antibodies , and to detect fungal toxin deoxynivalenol  and food-contaminating domoic acid . The easy Dip-and-Read format and various biosensors for coupling antibodies available from Pall ForteBio led to our second objective of this study which was to investigate the possibility of using the BLI-based BLItz® instrument to detect Rs r3b2 using a Rs monoclonal antibody from Forsite Diagnostics Ltd (Sand Hutton, UK).
Samples and Reagents
Following USDA-APHIS FSAP approval, devitalized bacterial cells of Rs strains were provided by USDA-APHIS-PPQ-CPHST (Center for Plant Health Science and Technology) and USDA-ARS laboratories in Beltsville, MD. UW551 strain is a select agent categorized as r3b2 and is also designated, in current nomenclature, as biovar 2; phylotype IIB-sequevar 1 . PD1958 is a r3b2T (phylotype IIB-sequevar 2 ) strain. F26 and JJRs-2 (r1b1 strains) and Rs08-01 (unknown race, biovar 3) are non-select agents. UW349 (biovar 2T; phylotype IIB-sequevar 27), P673 (biovar 1; phylotype IIB-sequevar 4), GMI1000 (biovar 3; phylotype I- sequevar18), Pss51 (biovar 4; phylotype I- sequevar 15) and Molk2 (biovar 1; phylotype IIB- sequevar 3) are also non-select agents . Two non-r3b2 strains Rs C and Rs J and Pseudomonas syringae (Ps Avr strain) were kindly provided by Dr. D. Kobayashi and Dr. E. Lam at Rutgers University. E. coli DH5a is a common laboratory strain for gene cloning. Rs strains C and J, P. syringae and E. coli DH5a bacterial cells were grown on nutrient agar solid media, picked by inoculation loops, and suspended in 1 × PBS. They were subsequently heat-inactivated (killed), pelleted by centrifugation, and shipped frozen to Rutgers University. The ImmunoStrip® for Rs and Rs positive control (r1b1) were purchased from Agdia Inc. (Elkhart, IN). The freeze dried negative control geranium and potato plant extracts [(-) Ctr] were also purchased from Agdia Inc. (Elkhart, IN) and resuspended in 1 × PBS buffer (phosphate buffered saline, Thermo Fisher Scientific, Pittsburg, PA) to 0.3 mg/ml concentrations. An IgM monoclonal antibody (Mab) against Rs (#PM/BROT/021208) was obtained from Forsite Diagnostics Ltd. (Sand Hutton, York). All protein concentrations were determined with a ThermoFisher NanoDrop spectrophotometer (Pittsburgh, PA).
In accordance with FSAP regulations, geranium (Pelargonium x hortorum “zonal geranium”) was grown, inoculated with the r3b2 strain UW551 or the non-r3b2 strain K60, and stem samples prepared as described before . Total DNA from healthy and Rs-inoculated geranium samples was extracted using Qiagen’s Plant Kit according to manufacturer’s instructions (Valencia, CA).
Rs detection by qPCR with the portable POCKIT™ instrument
The four sample-formatted POCKIT™ system based on the principle of TaqMan probes and iiPCR was acquired from GeneReach Biotechnology Corporation (Taichung, Taiwan), along with the single sample Enzyme/dNTP Premix supplied in the lyophilized powder form and the 2 × Premix Buffer B.
Heat-inactivated Rs bacterial cells with known concentrations were spiked into three 1 mm stem sections of healthy geranium plant in a 1.7 ml microfuge tube, to which 300 ml extraction buffer  (100 mM Tris-HCl, 150 mM NaCl, 1 M Betaine monohydrate, 10% polyvinyl-pyrrolidone, and 5% Triton X-100, pH 9.3,) was added. Total genomic DNA (gDNA) was extracted by incubating the tubes with spiked samples in a MultiTherm Shaker (Benchmark Scientific, Edison, NJ) at 65°C with 1100 rpm shaking for 10 minutes. One microliter (1 ml)-samples were used for detection of Rs r3b2 by POCKIT™ and confirmed by qPCR using the Applied Biosystems/ThermoFisher Scientific (Foster City, CA) StepOnePlus instrument when indicated.
The r3b2 (phylotype IIB, sequevar-1 & 2)-specific TaqMan probe RsSA2 (6FAM-5’CCAAGAAGAGAATCATGGAGCCGTTGTCC-3’MGBNFQ) with fluorescent 6FAM-labeling at the 5’ end and MGBNFQ (minor groove binder/nonfluorescent quencher)-labeling at the 3’ end was synthesized by Applied Biosystems/Thermo Fisher Scientific (Pittsburg, PA). This probe targets the non-phage gene in the Rs chromosome annotated to code for a probable n-6 adenine-specific DNA methylase . The RsSA2 forward (5’-GTTATGGACGGTGGAAGTCTCTG-3’) and reverse primers (5’-CGTTCCAAGTAGTGGGCAATCAA-3’)  were synthesized by Sigma Aldrich (St. Louis, MO). The qPCR detection reaction was assembled as follows: 1 ml DNA template, 5 ml each of 10 mM forward and reverse primers (final 1 mM), 2.5 ml of 10 mM RsSA2 TaqMan probe (final 0.5 mM), 25 ml 2 × Premix Buffer B, and water up to 50 ml. The 50 ml mixture was added into the single tube containing the lyophilized Enzyme/dNTP Premix. The complete reaction mix was transferred to the R-tube (GeneReach Biotechnology Corporation). The R-tubes were centrifuged briefly to settle the qPCR reaction mixes and placed into the portable POCKIT™ instrument to start the 32-minutes reaction run. The results were shown as “+” or “-”, displayed on the screen monitor, and recorded by digital photography.
Validation of Rs qPCR detection using the ABI StepOnePlus thermocycler
The RsSA2 TaqMan probe designed by Stulberg and Huang  was used in a BioRad CFX96 thermocycler for Rs r3b2 detection. In this study, a StepOnePlus thermo-cycler and reagents by Applied Biosystems/ThermoFisher Scientific were used to validate the detection signals of Rs at different concentrations by the RsSA2 primers and probe set. Each 10 ml qPCR reaction consisted of 1 ml Rs gDNA, 1 ml each of 10 mM RsSA2-F and RsSA2-R primers (final 1 mM), 0.5 ml of 10 mM RsSA2-Pprobe (final 0.5 mM), and 5 ml 2 × TaqMan PCR master mix. The program started with 1 cycle of 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Each sample reaction had three replicates. The experiment was repeated three times.
Rs detection by Forsite Rs MAb-coupled sensor with the portable BLItz® instrument
The single sample-formatted BLItz® system, which is based on the principle of BLI, was acquired from Pall ForteBio (Menlo Park, CA). The ProL sensor with embedded protein L was used to couple the Forsite Rs IgM MAb. The detection protocol was set using the “Advanced Kinetics” program as follows: hydrate the sensor in 200 ml PBS buffer in a 250 ml microtube for 10 minutes; initialize the sensor in PBS (250 ml in a black 0.5 ml microtube) for 10 seconds; couple the Forsite Rs IgM MAb (5.5 mg/ml) onto the ProL sensor by dipping the sensor into 4 ml of the antibody in the “Drop” for 80 seconds; set baseline (washing) in PBS for 30 seconds; bind sample pathogen onto the antibody-coupled sensor by dipping the MAb-coupled sensor into 4 ml of sample solution in the “Drop” for 120 seconds; set baseline (washing) in PBS for 20 seconds; amplify detection signal by dipping the sensor into 4 ml Forsite Rs MAb in the “Drop” for 90 seconds. The total run time was 350 seconds (approximately 6 minutes). The pathogen-antibody interaction signal was recorded as “binding (nm)” every 0.2 second, and was exported to Excel and analyzed with binding (nm) on the Y-axis and time(s) on the X-axis.
RsSA2 primers and TaqMan probe set detects 10 CFU of Rs r3b2 strain UW551 in 32 minutes with the POCKIT™ system
Since live Rs r3b2 bacterial cells cannot leave the USDA-APHIS-approved quarantine facilities of USDA-APHIS-PPQ or USDA-ARS, the heat-killed samples were transported to Rutgers University under FSAP-approval for the development of detection methods. Thus, the conventional unit of CFU/ml could not be determined for the concentration of Rs cells. Instead, the unit of mg/ml was used, which was determined by measuring the total protein concentrations of Rs samples with a ThermoFisher NanoDrop spectrophotometer at 280 nm. It has been shown that 1 mg of Pseudomonas aeruginosa bacteria is equivalent to approximately 108 CFU . In order to determine the relationship between CFU and milligram (mg) for Rs, two non-r3b2 Rs strains (C and J) were obtained and grown in nutrient medium to the log phase, serially diluted and plated on nutrient agar plates with at least three different dilutions. The protein concentrations of serially diluted bacterial samples were determined by absorbance at 280 nm with the NanoDrop spectrophotometer. This experiment was repeated three times with both Rs C and Rs J separately. Our results indicated that, consistently, 1 mg of Rs is equivalent to 0.38-1 × 108 CFU.
It was previously shown that the RsSA2 TaqMan probe detected as low as 4 CFU Rs UW551 with Cq values of 35.46 and 35.85 under uniplex and multiplex conditions with the BioRad CFX96 thermo-cycler . As a positive control, gDNA was isolated from pure UW551 bacterial cells by the Promega Wizard® Genomic DNA Purification Kit (Madison, WI). Our data showed that when 1 ng gDNA from pure UW551 cells was tested in the RsSA2 qPCR detection reaction in the StepOnePlus thermocycler, the average Ct (cycle threshold) value was 20.59 from three independent experiments (Figure 1). After spiking different amounts (in mg/ml) of heat-killed UW551 bacterial cells into geranium stem sections, total gDNA was extracted with 300 ml extraction buffer. One microliter (1 ml) gDNA samples, equivalent to 2.2 × 104, 1 × 104, 1 × 103, 1 × 102, and 10 CFU of UW551, were subjected to qPCR detection by the RsSA2 set with StepOnePlus thermocycler, resulting in average Ct values of 22.05, 24.62, 27.41, 31.01, and 34.51 respectively (Figure 1). When a 1 ml gDNA sample, equivalent to 1 CFU UW551, was added to the qPCR reaction, it was not amplified in the StepOnePlus thermos-cycler (Figure 1).
Figure 1: qPCR detection of Rs UW551 by RsSA2 TaqMan probe with the portable POCKIT™ system. The average Ct values generated by the RsSA2 probe with the StepOnePlus qPCR system for the same amount of UW551 cells are displayed.
When 1 ml gDNA samples, equivalent to 2.2 × 104, 1 × 104, 1 × 103, 1 × 102, and 10 CFU of UW551 bacterial cells, and 1 ng gDNA isolated from pure UW551 bacterial cells, were added to the qPCR reactions with the RsSA2 set in the POCKIT™ instrument, the “+” detection signals were generated (Figure 1). When 1 ml gDNA sample, equivalent to 1 CFU UW551, was added to the POCKIT™ instrument, the RsSA2 set did not detect the presence of UW551 gDNA and generated a “-” signal (Figure 1). These data indicate that the r3b2-specific RsSA2 TaqMan probe could detect Rs r3b2 with the POCKIT™ system as sensitively as the StepOnePlus thermos-cycler. Furthermore, the POCKIT™ system could detect Rs much more rapidly (32 minutes) than the StepOnePlus (2 hours), and is small enough to be hand-held.
The RsSA2 TaqMan probe can specifically detect Rs r3b2 with the POCKIT™ system
In order to investigate whether the RsSA2 TaqMan probe could specifically detect Rs r3b2, several different races, biovars and sequevars of Rs were tested with the POCKIT™ system. These different Rs strains were spiked into healthy geranium stem sections, and the gDNA was isolated using extraction buffer and applied to the POCKIT™ system (Figure 2A). As shown in Figure 2A, both extracted gDNA from 3 × 104 CFU of UW551 and 1 ng gDNA from pure UW551 cells generated “+” detection signals, while F26, a r1b1 strain, and the water (H2O)-spiked sample generated the same “-” signal. The POCKIT system also produced the “+” signal for 3 × 104 CFU PD1958, a r3b2T strain in Figure 2B, confirming that PD1958 is a phylotype IIB sequevar 2 strain of Rs . The POCKIT, however, produced “-” signals for both JJRs-2, a r1b1 strain, and Rs08-01, a strain that was identified as a biovar 3 with an unknown race (Figure 2B). Figure 2C indicates that P673 (biovar 1; phylotype IIB-sequevar 4), Pss51 (biovar 4; phylotype I-sequevar 15), and Molk2 (biovar 1; phylotype IIB-sequevar 3)  all produced “-” signals. Additionally, Fig. 2D shows that the RsSA2 TaqMan probe did not detect UW349 (biovar 2T; phylotype IIB-sequevar 27) or GMI1000 (biovar 3; phylotype I-sequevar 18)  with the POCKIT™ system. The “-” signals for P673, Pss51, Molk2, UW349 and GMI1000 by POCKIT™ validated the negative detection results by Stulberg and Huang  with the BioRad CFX96 thermo-cycler. These results indicate that the RsSA2 TaqMan probe can specifically detect Rs strains that belong to biovar 2 phylotype IIB-sequevar 1 or 2 when used in the POCKIT™ system.
Figure 2: Detection of Rs strains by the RsSA2 TaqMan probe with the POCKIT™ system.
The RsSA2 TaqMan probe can specifically detect Rs r3b2 in inoculated plants with the POCKIT™ system
We next tested the application of the RsSA2 TaqMan probe/POCKIT™ system to detect Rs in inoculated geranium plants. This work was conducted in the USDA APHIS FSAP-approved select agent containment facility at the USDA-ARS laboratory in Beltsville, MD. Five sets of four Qiagen-extracted DNA samples from Rs-inoculated geranium plants were tested, totaling twenty plants. Each set contained DNA samples from one water-inoculated healthy plant, one UW551-inoculated symptomatic plant, one UW551-inoculated asymptomatic plant, and one K60 (non-r3b2)-inoculated symptomatic plant (Table 1). The RsSA2 probe consistently produced “-” signals for all the water-inoculated healthy samples CK1-CK5 with POCKIT™ (Table 1). The “-” signal of CK5 was verified by the qPCR “NA” (no amplification) reaction with the BioRad CFX96 thermo-cycler. Four of the five UW551-inoculated symptomatic plants S1, S2, S3 and S5 were tested “+”. The false negative result of S4 in the 4th sample set was later proven to be positive upon retesting in the 6th and 7th sample sets. It was also shown to produce a Cq value of 16.3 by qPCR analysis with the BioRad CFX96 thermo-cycler. Based on this, the initial result may have resulted from operation error.
Table 1: Detection of Ralstonia solanacearum in inoculated geranium plants by RsSA2 TaqMan probe and POCKIT™
Three out of the five UW551-inoculated asymptomatic plants tested “+” by the RsSA2 probe with POCKIT™ (Table 1). When two of the three POCKIT™-positive samples AS2 and AS3 were also tested by qPCR for verification, they produced Cq values of 23.23 and 20.14 by the CFX thermo-cycler (Table 1). This indicates that even though the UW551-inoculated plants had not shown typical symptoms, the presence of UW551 could be detected by RsSA2 probe with POCKIT™ as sensitively as with the BioRad CFX thermo-cycler. The AS4 and AS5 UW551-inoculated asymptomatic plants tested negative initially and also in the re-tests. They were confirmed negative through testing with the BioRad CFX thermo-cycler, which produced a Cq value of 38.31 for AS4 and no signal (NA) for AS5 (Table 1).
Three out of five Rs non-r3b2 strain K60-inoculated symptomatic plants N3-N5 tested negative by RsSA2 with POCKIT™ (Table 1). The N1 plant initially gave a false positive reaction with POCKIT™ but was later shown to be a negative plant in two retests (sample sets 6 and 7). When a questionable signal is produced with POCKIT, normally a “?” will be displayed, as in the case of N2. This sample was later shown to be negative in the sixth sample set test and produced a Cq value of 39.9 by the CFX thermo-cycler.
The portable BLItz® system is reactive to Rs strains using the Forsite Rs MAb-coupled sensor
The BLI-based BLItz® system by Pall ForteBio was chosen to develop the antibody-mediated detection of Rs as a second, independent, non-PCR method. The protocol included binding the Forsite Diagnostics Rs MAb (IgM) to the ProL sensor, loading pathogen sample and associating the same MAb to amplify the signal. As shown in Figure 3, each curve represents the coupling of antibody, pathogen sample and amplifying antibody on a single ProL sensor. After dipping the ProL sensor into the “Drop” containing 4 ml of 5.5 mg/ml Forsite Rs MAb, the binding curve rose as the IgM MAb interacted with the embedded Protein L at the sensor tip; 80 seconds later, the sensor was typically saturated with the antibody. The sensor was then dipped in PBS to wash off the unbound antibody for 30 seconds. The antibody-coupled sensor was dipped into the “Drop” containing 4 ml of Rs sample for 120 seconds to allow Rs to be bound by the MAb. After washing the sensor in PBS for 20 seconds, it was dipped into the “Drop” containing 4 ml Forsite Rs MAb for 90 seconds, the binding curve rose again indicating a specific interaction between the antibody and the Rs sample. In this experiment, all Rs strains were bacterial cells grown from pure cultures, heat-killed, centrifuged, and resuspended in PBS buffer. Figure 3 shows that at the same 0.3 mg/ml concentration, the r3b2 UW551 strain produced the highest signal compared to r3b2T strain PD1958 and r1b1 strain F26 and the r1b1 positive control from Agdia. As mentioned above, 0.3 mg/ml Rs is equivalent to approximately 1 × 107 CFU/ml. Only 4 ml of bacterial cells was used, showing 4 × 104 CFU cells could be detected by the BLItz® system. Notably, both PBS buffer and the negative control of geranium extract from Agdia produced similarly low signals. The total run time of one detection was approximately 6 minutes.
Figure 3: Detection of different Rs strains with the Forsite Rs MAb-coupled sensor using the BLItz® system. All Rs strains and the geranium negative control (- Ctr) from Agdia were at a concentration of 0.3 mg/ml protein.
The BLItz® system can detect r3b2 Rs bacterial cells in spiked plant extracts with the Forsite Rs MAb-coupled sensor
Figure 4 shows that 4 × 104 CFU W551 cells in PBS buffer could be detected by the Forsite MAb-coupled ProL sensor. When 4 ml of 0.3 mg/ml UW551 cells (equivalent to 4 × 104 CFU) were spiked into 0.3 mg/ml geranium and potato negative control extracts (Agdia), the bacterial cells were detected by the Forsite Rs MAb-coupled sensor almost as efficiently as the pure culture UW551 cells in PBS with the BLItz® system (Figure 4A). The healthy geranium and potato extracts produced similarly low signals as the PBS buffer (Figure 4A). These data indicate that the Forsite Rs MAb could specifically detect, without inhibition, UW551 in plant extracts.
Figure 4: (A) Detection of 4 × 104 CFU r3b2 UW551 in spiked geranium and potato extracts by BLItz® Forsite Rs MAb-sensor. Both bacterial cells and plant extracts were tested at 0.3 mg/ml concentration. (B) Detection limit of UW551 spiked in geranium extract using the Forsite Rs MAb-sensor. (C) Detection of Rs r3b2 UW551, r1b1 F26, and r1b1 positive control (+ Ctr) by Agdia Rs ImmunoStrip® at different concentrations. The Rs positive control (+ Ctr) and negative control (- Ctr) were both purchased from Agdia.
The detection limit of UW551 using the Forsite Rs MAb-sensor with the BLItz® system was tested next. The ProL sensor coupled with 5.5 mg/ml Forsite Rs MAb had an upper detection limit of 2 × 107 CFU/ml (equivalent to 0.6 mg/ml by protein concentration) with UW551 spiked into 0.3 mg/ml geranium extract. When the sensor was dipped into 4 ml of UW551 at 2 × 107 CFU/ml, representing 8 × 104 CFU, the detection signal of 2.06 nm was similar to that (1.93 nm) from 4 × 104 CFU (1 × 107 CFU/ml, or 0.3 mg/ml) (Figure 4B). The 2 × 103 CFU UW551 sample had a significant decrease in the detection signal (1.57 nm) at the end of 90 s dipping in the amplifying antibody, compared to the 2 × 104 CFU sample (1.82 nm). The detection signal from the 1 × 103 CFU UW551 sample was further reduced to 1.28 nm, which was only slightly higher than the 1.11 nm signal from the healthy geranium extract (Figure 4B). These results indicate that the Forsite Rs MAb-sensor could detect UW551 as low as 1 × 103 CFU (at the spike concentration of 2.5 × 105 CFU/ml) when spiked in geranium extract.
The Rs ImmunoStrip® from Agdia, Inc. was tested and compared to the efficiency of Rs detection using BLItz® and Forsite Rs MAb-sensor. Figure 4C shows that the Rs ImmunoStrip® could detect r3b2 UW551, r1b1 F26 and the r1b1 (+) control samples at 2.5 × 106 CFU/ml, with r1b1 strains showing slightly stronger detection signals. The Rs ImmunoStrip® could detect r1b1 strains at 2.5 × 105 CFU/ml concentration, although the signal from r3b2 UW551 was barely visible. At 2.5 × 103 CFU/ml, the Rs ImmunoStrip® could not detect r1b1 strains compared to the results obtained by BLItz® (Figure 4B) where 2.5 × 105 CFU/ml UW551 produced a distinct detection signal. Additionally, only 4 ml of 2.5 × 105 CFU/ml UW551 was used in the BLItz® system, equivalent to 1 × 103 CFU bacterial cells; in contrast, significantly more than 4 ml of 2.5 × 105 CFU/ml UW551 had to be used for the Rs ImmunoStrip® to produce the barely visible detection signal in Figure 4C. This indicates that the BLItz® system with the Forsite Rs MAb was more sensitive than the Rs ImmunoStrip®, and both methods required a similar amount of time (5-6 minutes).
Figure 5: (A) Detection of Rs strains using Forsite Rs MAb-sensor in the BLItz® system. All strains were tested at a 0.3 mg/ml concentration (equivalent to 1 × 107 CFU/ml; 4 μl, 4 × 104 CFU). (B) Forsite Rs MAb is more reactive to r3b2 UW551 strain than to non-r3b2 Rs strains and other bacteria. All samples were tested at a 0.3 mg/ml concentration.
Forsite Rs MAb-sensor detects Rs strains with varying efficiencies using BLItz®
The Forsite Rs MAb-sensor was tested for its ability to detect other Rs strains (at 0.3 mg/ml concentration, or 1 × 107 CFU/ml; 4 ml, 4 × 104 CFU) with the BLItz® system. Figure 5A shows that the Forsite Rs MAb-sensor could detect UW349, another biovar 2 (2T) (phylotype IIB-sequevar 27), as efficiently as UW551. The detection signal from GMI1000, a biovar 3 (phylotype I-sequevar 18), was similar to that from UW551. Detection signals from Pss51, a biovar 4 (phylotype I-sequevar 15), and Molk2, a biovar 1 (phylotype IIB-sequevar 3), were much lower than UW551. Strain P673, a biovar 1 (phylotype IIB-sequevar 4), produced an even lower signal than the healthy geranium extract. These results demonstrate that the while the Forsite Rs MAb-sensor was more reactive with Rs biovar 2 and 2T strains tested, it produced good signal for most other biovars tested and demonstrate the Forsite Rs MAb-sensor is a good screening tool.
To evaluate the reactivity of the Forsite Rs MAb to Rs r3b2 strains, two non-r3b2 Rs strains, Rs C and Rs J, r1b1 strain F26, Escherichia coli (E. coli) (DH5a strain) and Pseudomonas syringae (Ps Avr strain) were tested. As shown in Figure 5B, F26, Rs C, Rs J and E. coli produced signals as low as the healthy geranium extract, and the Ps Avr’s signal was even lower. In this test the Forsite Rs MAb is more reactive to Rs r3b2 than too other Rs strains.
Forsite Rs MAb-sensor can be stored at room temperature for at least 3 months
The ProL sensors from Pall ForteBio use thin glass rods with tips embedded with protein L and coated with 40% sucrose. The sensors can be stored dry at room temperature without losing the binding capacity of protein L to IgM. We tested the detection capability of the Forsite Rs MAb-coupled sensors stored at room temperature over a period of storage times. The same amount of Forsite Rs MAb was coupled onto several dozen ProL sensors. The antibody-bound sensors were then dipped into a 40% sucrose solution, air-dried and stored at room temperature. The stored antibody-coupled sensors were tested for their detection of UW551 after 1 day, 2 weeks, 3 months, 8 months and 12 months of room temperature storage. At initial coating prior to storage, the binding signal was 1.95 nm for UW551 at 0.3 mg/ml (1 × 107 CFU/ml) (Figure 6A). After 1 day of storage, the signal dropped slightly to 1.70 nm and decreased to 1.55 nm 2 weeks after storage. Three months after storage, the detection signal for 0.3 mg/ml UW551 was 1.40 nm, which was still higher compared to the signals for PBS buffer (1 nm) and healthy geranium extract (0.81 nm). Freshly coupled Forsite Rs MAb sensors tested with PBS buffer and healthy geranium extract consistently measured 1.0 nm. This indicates that the Rs detection efficiency of Forsite Rs MAb-sensor was reduced by approximately 28% after 3 months of storage. After 8 months of storage, however, the detection efficiency of Forsite Rs MAb-sensor was reduced to 0.62 nm for 0.3 mg/ml UW551 (Figure 6B) and lower than the value for PBS and healthy geranium and therefore determined not stable for use after 8 months of storage. Additionally, after 12 months of storage the Rs MAb sensor was not able to generate any signal for UW551.
Figure 6: Detection of UW551 with Forsite Rs MAb-coupled sensors following storage at room temperature for 1 day, 2 weeks and 3 months (A) and 8 months and 12 months (B). All samples were tested at a 0.3 mg/ml concentration.
We have adapted and evaluated two portable platforms for the detection of Rs r3b2. As a quarantine plant pathogen and also a USDA-APHIS select agent, it is necessary to quickly and accurately determine if the Rs strain detected is r3b2. The POCKIT™ Nucleic Acid Analyzer system was the first platform we tested for the feasibility of using the recently published TaqMan-based qPCR primers and probe set RsSA2 assay for fast, accurate and sensitive detection of Rs r3b2 in a field deployable system. Our data showed that when used in POCKIT™, the r3b2-specific RsSA2 TaqMan assay set could detect as low as an equivalent of 10 CFU of the r3b2 strain UW551 when the heat-killed bacterial cells were spiked into the healthy geranium extract (Figure 1). Additionally, the RsSA2 assay used with the POCKIT™ system could detect other Rs biovar 2 strains (Figure 2) in the spiked geranium extract. None of the non-r3b2 (non-IIB, seq1, 2) strains tested was detected by RsSA2/POCKIT™ demonstrating the specificity of the assay. The time required for POCKIT™ detection included 10 minutes for extraction plus 32 minutes for the qPCR reaction, which is shorter than the standard 2 hours needed for a typical extraction and qPCR reaction. Most importantly, the small footprint, low cost and ease of use of the POCKIT™ instrument makes it an ideal platform for rapid, sensitive and specific detection of target organisms at ports of entry and in field settings.
We have also shown that the RsSA2 TaqMan set used in the POCKIT™ system could detect Rs r3b2 UW551 in live inoculated geranium plants (Table 1). UW551 could even be detected in inoculated but asymptomatic plants as sensitively as the BioRad CFX thermo-cycler. Our data showed that false positive and false negative rates were very low, 1 out of 20 for each incidence for the RsSA2 probe/POCKIT™ system, and the issue can be resolved by testing each sample in duplicate and verifying the result by qPCR if contradicting results are obtained by POCKIT™.
Using companion diagnostic protocols in a multi-platform diagnostic system adds strength and confidence to test results. This can be achieved by combining serological and DNA-based detection protocols. For detection of Rs and then identification of r3b2, we combined the RsSA2 qPCR using the POCKIT™ system and evaluated a second platform, the BLItz® system by Pall ForteBio. This system uses inexpensive sensors that were coupled with the Rs MAb from Forsite Diagnostics Ltd. and could distinguish r3b2 UW551 from other non-r3b2 strains tested at the same concentration (Figure 3). Heat-killed Rs UW551 at a concentration of 1 × 103 CFU spiked geranium extract could be detected by Forsite Rs MAb-sensor with BLItz® (Figure 4B). Compared to the ImmunoStrip® (Agdia), the BLItz® system appeared to be more sensitive (Figure 4C). The total run time required for Forsite Rs MAb-sensor/ BLItz® system to detect Rs was approximately 6 minutes. Lastly, the Forsite Rs MoAb-coupled sensors could be stored at room temperature for at least 3 months, which greatly facilitates field detection and on-site operations. Also, quantification using the Forsite Rs MAb is complicated by the fact that it was generally more reactive with Rs r3b2 strains, meaning low concentrations of r3b2 may produce a signal similar to non-r3b2 strain samples at high concentrations. To specifically identify Rs r3b2 in samples testing positive for Rs using BLItz® a companion diagnostic assay such as the RsSA2 TaqMan probe/POCKIT™ system should be used.
We have shown that two portable systems, the TaqMan probe/qPCR-based POCKIT™ and the BLI-based BLItz® using MAb-coupled sensors, could be used to detect low levels of Rs r3b2 with speed, accuracy and sensitivity. The small footprints of these two systems make on-site pathogen detection feasible.
We gratefully acknowledge the support to R. Di (Rutgers University) from USDA Farm Bill Section 10007 funding through the USDA-APHIS-PPQ-CPHST Cooperative Agreements (# USDA-APHIS-12-8130-1511-CA and # USDA-APHIS-13-8130-1511-CA). We also acknowledge the APHIS Science Fellows Program financial support of Liming Zhao. We sincerely thank Elizabeth Twieg and Zhaowei Liu from the USDA-APHIS-PPQ-CPHST Beltsville Laboratory for the preparation of some of the heat-killed Rs cultures provided for use in this study. We are also thankful to John Rascoe from USDA-APHIS-PPQ-CPHST Beltsville Laboratory for the critical review of the manuscript.
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