Rapid PCR Amplification Research Articles

Development of rapid DNA extraction and PCR amplification methods for fungi and parasitic plants

Development of rapid DNA extraction and PCR amplification methods for fungi and parasitic plants

A Disposable Electromagnetic Bi-Directional Micropump Utilizing a Rotating Multi-Pole Ring Magnetic Coupling.

Electromagnetic bi-directional micropumps (EMBM) are indispensable for the development of portable devices which enable fluid transportation in forward and reverse directions. However, the high disposal cost of rare-earth magnets attached to the fluidic part and the low pump density due to multiple motors limit their practical application in disposable multi-channel microfluidic applications such as droplet-based oscillatory-flow (DBOF) rapid PCR amplification. Therefore, this paper presented a low-cost, disposable, high-pump-density EMBM. To reduce the disposal cost, we separated the magnets from the disposable fluidic part and used cylindrical holes to store and guide the magnet, which resulted in the ability to reuse all the magnets. To increase the pump density, we used the combination of one motor and one multi-pole ring magnet to drive several channels simultaneously. A proof-of-concept prototype with a pump density of 0.28 cm−2 was fabricated and experimentally evaluated. The fabricated micropump exhibited a maximum flow rate of 0.86 mL/min and a maximum backpressure of 0.5 kPa at a resonant frequency around 50 Hz. The developed multi-channel micropump with reusable magnets is highly beneficial to the development of low-cost and high-throughput rapid PCR amplification microchips and therefore can have a significant impact on timely infectious disease recognition and intervention.

The development of miniSTRs as a method for high-speed direct PCR.

There are situations in which it would be very valuable to have a DNA profile within a short time; for example, in mass disasters or airport security. In previous work, we have promoted reduced size STR amplicons for the analysis of degraded DNA. We also noticed that shorter amplicons are more robust during amplification, making them inhibition resistant, and potentially applicable to high-speed direct PCR. Here, we describe a set of miniSTRs capable of rapid direct PCR amplification. The selected markers are a subset of the Combined DNA Index System (CODIS) loci modified to permit high-speed amplification. Using the proposed protocol, the amplification of eight loci plus amelogenin directly from a saliva sample can be completed in 7 min and 38 s using a two-step PCR with 30 cycles of 98°C for 2s and 62°C for 7s on a Streck Philisa thermocycler. Selection of DNA polymerase, optimization of the two-step PCR cycling conditions, the primer concentrations, and the dilution of saliva is described. This method shows great potential as a quick screening method to obtain a presumptive DNA profile when time is limited, particularly when combined with high-speed separation and detection methods.

Ultrafast visual nucleic acid detection with CRISPR/Cas12a and rapid PCR in single capillary

Rapid and sensitive nucleic acid detection is critical for point-of-care testing (POCT). Here, we proposed an ultrasensitive visual nucleic acid detection method that could be accomplished in <10 min. By integrating rapid PCR amplification and Cas12a cleavage as one-pot reaction in capillary, the operation was greatly simplified and amplicon contamination was totally avoided. Rapid PCR amplification was performed in portable thermo cups and Cas12a cleavage reaction was triggered with body-heat. Fluorescent results could be easily identified by naked eye with the help of a mini UV-touch. Results indicated that the proposed method possesses detection sensitivity at single molecule level, with high robustness, specificity and reliability. Thus, we have created a field-deployable nucleic acid detection platform with high performance and minimal equipment. It has broad application prospects for POCT in fields ranging from food safety determination to clinical diagnosis.

End-point dual specific detection of nucleic acids using CRISPR/Cas12a based portable biosensor

A CRISPR/Cas12a based portable biosensor (Cas12a-PB) was developed to simultaneously visually detect CaMV35S promoter and Lectin gene from genetically modified (GM) soybean powders (Roundup Ready@). The Cas12a-PB, mainly made of polymethylmethacrylate (PMMA) and PMMA tape, has a connection structure, three channels and three detection chambers. The CRISPR/Cas12a detection reagents were preloaded in detection chambers and the reaction tube was connected to the connection structure by screw threads. After amplification, the amplicons were gone into three detection chambers by swinging the Cas12a-PB to conduct dual detection. Positive samples would produce green fluorescence while negative samples were black under the irradiation of 490 nm LED light. In this study, the Cas12a-PB successively combined with ordinary PCR, rapid PCR and loop-mediated isothermal amplification (LAMP) to achieve dual detection, which made detection process more convenient and portable. As low as 0.1% transgenic ingredients in soybean powders could be detected and the specificity of Cas12a-PB was confirmed with GM maize powders (MON810, GA21), GM soybean powders (DP305423), non-GM peanut and rice as targets. In the end, an amplification chamber combining with Cas12a-PB on a PMMA chip was further designed to eliminate the use of reaction tube and mineral oil, which made operation simpler. The established Cas12a-PB would provide a new reliable solution for multiple targets detection in clinic diagnostics, food safety, etc.

A fast and visual method for duplex shrimp pathogens detection with high specificity using rapid PCR and molecular beacon

Molecular diagnosis of genome is one of the major methods for pathogens detection. The commonly used PCR method can realize an exponential amplification of the target gene but is time-consuming. In this work, we proposed a duplex and visual method using rapid PCR combined with molecular beacons to specifically detect two kinds of shrimp pathogens in one reaction tube. We only need to observe the fluorescence change of the reaction tube with naked eye to determine the result. A home-made automatic transfer equipment allows reaction tubes shuttling quickly between two water baths to achieve rapid PCR amplification. A simple device was also designed to present the detection results easily determined with naked eye. This duplex and visual detection method is fast, low-cost and of high specificity. From DNA extraction to results judgment, only 15 min was enough. Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) and Vibrio parahaemolyticus (VP) are two common shrimp pathogens which were chosen as our detection objects. This method may give a possibility to conduct end-point visual duplex detection, which may make a positive influence on the pathogen prevention.

Rapid ABO genotyping by high-speed droplet allele-specific PCR using crude samples.

ABO genotyping has common tools for personal identification of forensic and transplantation field. We developed a new method based on a droplet allele-specific PCR (droplet-AS-PCR) that enabled rapid PCR amplification. We attempted rapid ABO genotyping using crude DNA isolated from dried blood and buccal cells. We designed allele-specific primers for three SNPs (at nucleotides 261, 526, and 803) in exons 6 and 7 of the ABO gene. We pretreated dried blood and buccal cells with proteinase K, and obtained crude DNAs without DNA purification. Droplet-AS-PCR allowed specific amplification of the SNPs at the three loci using crude DNA, with results similar to those for DNA extracted from fresh peripheral blood. The sensitivity of the methods was 5%-10%. The genotyping of extracted DNA and crude DNA were completed within 8 and 9minutes, respectively. The genotypes determined by the droplet-AS-PCR method were always consistent with those obtained by direct sequencing. The droplet-AS-PCR method enabled rapid and specific amplification of three SNPs of the ABO gene from crude DNA treated with proteinase K. ABO genotyping by the droplet-AS-PCR has the potential to be applied to various fields including a forensic medicine and transplantation medical care.

Fast and Efficient Gene Editing in Human Hematopoietic Cells

Although CRISPR/Cas9 is now accessible to a wide variety of cell-types and model systems, efficient editing of hematopoietic cells remains challenging. We have designed and optimized a protocol for rapid and efficient delivery of CRISPR/Cas9 to hematopoietic cell lines and primary cells. Combining electroporation's high transfection efficiency and the reduced cellular toxicity of Cas9 protein versus plasmid in suspension cells, we are able to produce highly efficient gene disruption and knock-in in a variety of human cell types, including acute myeloid leukemia (AML) cell lines, B-acute lymphoid leukemia (ALL) cell lines, primary T-lymphocytes and primary hematopoietic stem/progenitor cells (HSPCs). Our protocol involves rapid sgRNA template design and PCR amplification, followed by overnight in-vitro transcription, sgRNA purification and sgRNA-Cas9 ribonucleoprotein (RNP) formation. We began by testing the protocol on three AML cell lines, in which we observed up to 98% knock-out (KO) of the ubiquitous hematopoietic marker CD45 (%CD45neg cells by flow cytometry: HL-60 - 98%, OCI-AML2 - 92%, Kasumi - 87%). Using multiple guides, we also induced KO of two B-cell markers (CD19 and CD22) in three B-cell cancer cell lines (BV173, Daudi and Nalm-6). In these three cell lines, up to 70% of cells displayed combined loss of both cell surface receptors, indicating disruption of all four alleles (%CD19negCD22neg cells by flow cytometry: BV-173 - 58%, Nalm-6 - 70%, Daudi - 18%).We then optimized our editing strategy in human primary cells. We observed highly efficient CD45 loss (86±2%; n=3) in activated T-cells by flow cytometry and confirmed this KO frequency using high-throughput sequencing. We next measured CD45 gene disruption in CD34+ HSPC cells isolated from cord blood and found that our system had 75±10% editing efficiency (n=4). Importantly, a 48-hour period of cytokine stimulation with SCF/TPO/FLT3L prior to electroporation was required for efficient gene knockout (0hr: 8±4%, 24hr: 41±12%, 48hr: 73±16%; p0vs24=0.0002, p24vs48=0.003; n=8). Our protocol induced efficient gene disruption of several relevant targets in CD34+ cells including DNMT3A ex7 (69±4%; n=5), DNMT3A ex10 (86±14%; n=10) and NR3C1 (75±6%; n=5), and near complete loss of protein by western blot. To verify that the edited CD34+ HSPCs cells maintained engraftment and multilineage differentiation capacity, we transplanted Cas9 only (n=8) and Cas9/hCD45-sg1 RNP edited cells (n=8) into sub-lethally irradiated NOD scid gamma (NSG) mice. To avoid possible donor-dependent bias, each experimental pair (i.e. one Cas9 only replicate and one Cas9/hCD45-sg1 RNP treated replicate) was performed on cells derived from a single cord blood. Human cells successfully engrafted in the bone marrow of 16/16 recipients and spleens of 13/16 recipients. Importantly, we observed significant levels of engraftment by hCD45neg cells in the bone marrow of 7/8 mice and in the spleen of 5/8 mice transplanted with Cas9/hCD45-sg1 RNP edited cells (Figure 1A; Figure 1B shows one representative pair). High-throughput sequencing confirmed that engrafted human cells in BM displayed hCD45indel frequencies consistent with the flow cytometry data.Finally, we considered whether these editing strategies could be used to introduce specific point mutations into primary human HSPCs using Cas9-mediated homology directed repair (HDR). Single-stranded oligonucleotide HDR templates (ssODNs) with 90bp homology arms to the human CD45 locus were designed to introduce three basepair changes, two of which result in the generation of a BsiWI site near the CD45-sg1 cut site. High-throughput sequencing of treated human HSPC samples revealed efficient precise knock-in (22±4%; n=4) of the mutant allele.In conclusion, we describe a fast and efficient protocol for both gene disruption and targeted gene editing of human hematopoietic cells, including HSPCs, using the CRISPR/Cas9 system. The ability to quickly and efficiently edit primary human HSPCs using HDR makes it possible to introduce or repair genetic variants identified in association with hematologic diseases such as leukemia or bone marrow failure. Moreover, the high efficiency of this system offers the possibility to perform large-scale combinatorial gene editing in HSPCs to model complex mutational landscapes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Toxicity of the Lessepsian pufferfish Lagocephalus sceleratus from eastern Mediterranean coasts of Turkey and species identification by rapid PCR amplification

Pufferfish, Lagocephalus sceleratus, L. suezensis and L. spadiceus, are widely distributed Lessepsian species along the Turkish and other eastern Mediterranean coasts. L. sceleratus is considered one of the worst invasive species in the Mediterranean Sea and recently caused human intoxication by accidental consumption. Therefore, methods to assess pufferfish toxicity and accurately identify toxic pufferfish are important for food safety. Firstly, 20 specimens of L. sceleratus were collected from the eastern Mediterranean coasts of Turkey. Tetrodotoxin (TTX) content was determined among different tissues (muscle, liver, gonad, kidney, intestine and skin) by liquid chromatography-tandem mass spectrometry. All of the specimens contained TTX. The highest TTX concentration was detected in the ovary (80.0 µg TTX/g), followed by the intestine (48.8 µg TTX/g), kidney (34.0 µg TTX/g) and liver (25.4 µg TTX/g). The flesh and testis contained detectable amounts of TTX (3.4 µg TTX/g and 2.6 µg TTX/g, respectively). TTX analogs were also detected in the tissues, with 4,9-anhydro-5,6,11-trideoxyTTX and 5,6,11-trideoxyTTX being the major analog. A species-specific PCR method was developed to identify three species of genus Lagocephalus based on the nicotinamide adenine dinucleotide dehydrogenase subunit 2 or cytochrome oxidase subunit I regions of the mitochondrial DNA of L. sceleratus, L. suezensis, and L. spadiceus. The designed primers amplified only DNA of target species and not those of other fish and shellfish species. The species-specific PCR method successfully identified these pufferfish species and could be applied to thermally treated products of the pufferfish with only 1 ng of extracted DNA.

Rapid real-time PCR and high resolution melt analysis in a self-filling thermoplastic chip.

A microfluidic platform designed for point-of-care PCR-based nucleic acid diagnostics is described. Compared to established microfluidic PCR technologies, the system is unique in its ability to achieve exceptionally rapid PCR amplification in a low cost thermoplastic format, together with high temperature accuracy enabling effective validation of reaction product by high resolution melt analysis performed in the same chamber as PCR. In addition, the system employs capillary pumping for automated loading of sample into the reaction chamber, combined with an integrated hydrophilic valve for precise self-metering of sample volumes into the device. Using the microfluidic system to target a mutation in the G6PC gene, efficient PCR from human genomic DNA template is achieved with cycle times as low as 14 s, full amplification in 8.5 min, and final melt analysis accurately identifying the desired amplicon.

Cathepsin L is an immune-related protein in Pacific abalone (Haliotis discus hannai)--Purification and characterization.

Cathepsin L, an immune-related protein, was purified from the hepatopancreas of Pacific abalone (Haliotis discus hannai) by ammonium sulfate precipitation and column chromatographies of SP-Sepharose and Sephacryl S-200 HR. Purified cathepsin L appeared as two bands with molecular masses of 28.0 and 28.5 kDa (namely cathepsin La and Lb) on SDS-PAGE under reducing conditions, suggesting that it is a glycoprotein. Peptide mass fingerprinting (PMF) analysis revealed that peptide fragments of 95 amino acid residues was high similarity to cathepsin L of pearl oyster (Pinctada fucata). The optimal temperature and pH of cathepsin L were 35 °C and pH 5.5. Cathepsin L was particularly inhibited by cysteine proteinase inhibitors of E-64 and leupeptin, while it was activated by metalloproteinase inhibitors EDTA and EGTA. The full-length cathepsin L cDNA was further cloned from the hepatopancreas by rapid PCR amplification of cDNA ends (RACE). The open reading frame of the enzyme was 981 bp, encoding 327 amino acid residues, with a conserved catalytic triad (Cys134, His273 and Asn293), a potential N-glycosylation site and conserved ERFNIN, GNYD, and GCGG motifs, which are characteristics of cathepsin L. Western blot and proteinase activity analysis revealed that the expression and enzyme activity of cathepsin L were significantly up-regulated in hepatopancreas at 8 h following Vibrio parahaemolyticus infection, demonstrating that cathepsin L is involved in the innate immune system of abalone. Our present study for the first time reported the purification, characterization, molecular cloning, and tissue expression of cathepsin L in abalone.

Rapid PCR protocols for forensic DNA typing on six thermal cycling platforms.

Rapid PCR protocols for the amplification of typing STR multiplexes were evaluated on six different thermal cyclers. Through the use of a faster DNA polymerase coupled with the use of rapid thermal cyclers the amplification cycling times were reduced down to as little as 14 min using PCR primers from the commercially available multiplex STR typing kit Identifiler. Previously described two-step and three-step thermal cycling protocols were evaluated for the six thermal cyclers on 95 unique single-source DNA extracts. CE characterization of the PCR products indicates good peak balance between loci (median values greater than 0.84), and N minus four stutter ratios on averages were 30 to 40% higher than for standard Identifiler PCR conditions. Nonspecific amplification artifacts were observed, but were not observed to migrate within the allele calling bins. With the exception of one locus (D18S51) in a single sample, genotyping results were concordant with manufacturer's recommended amplification conditions utilizing standard thermal cycling procedures. Assay conditions were robust enough to routinely amplify 250 to 500 pg of template DNA. This work describes the protocols for the rapid PCR amplification of STR multiplexes on various PCR thermal cyclers with the future intent to support validation for typing single-source samples in a database laboratory.

Rapid PCR amplification protocols decrease the turn-around time for detection of antibiotic resistance genes in Gram-negative pathogens

A previously designed end-point multiplex PCR assay and singleplex assays used to detect β-lactamase genes were evaluated using rapid PCR amplification methodology. Amplification times were 16–18 minutes with an overall detection time of 1.5 hours. Rapid PCR amplifications could decrease the time required to identify resistance mechanisms in Gram-negative organisms.

A New Rapid Method for Clostridium difficile DNA Extraction and Detection in Stool: Toward Point-of-Care Diagnostic Testing

We describe a new method for the rapid diagnosis of Clostridium difficile infection, with stool sample preparation and DNA extraction by heat and physical disruption in a single-use lysis microreactor (LMR), followed by a rapid PCR amplification step. All steps can be accomplished in <20 minutes overall. Gel electrophoresis is currently used to detect the amplification product, pending real-time availability with an ultra-rapid thermocycler. Compared with the dual enzyme immunoassay (EIA) screening test (C. diff Quik Chek Complete; Techlab, Blacksburg, VA), the novel LMR/PCR assay showed complete concordance with all glutamate dehydrogenase (GDH) results (GDH(+)/toxin(+), n = 48; GDH(-)/toxin(-), n = 81). All 69 stool samples with discordant EIA results (GDH(+)/toxin(-)) were tested by both the LMR/PCR assay and the loop-mediated isothermal amplification test (LAMP) (Illumigene C. difficile; Meridian Bioscience, Cincinnati, OH). In 64/69 EIA-discordant samples, LAMP and LMR/PCR results matched (both positive in 29 sample and both negative in 35 samples); in the remaining 5 samples, results were discrepant between the LAMP assay (all five negative) and the LMR/PCR assay (all 5 positive). Overall, LMR/PCR testing matched the current algorithm of EIA and/or LAMP reflex testing in 193/198 (97.5%) samples. The present proof-of-concept study suggests that the novel LMR/PCR technique described here may be developed as an inexpensive, rapid, and reliable point-of-care diagnostic test for C. difficile infection and other infectious diseases.

Centrifugal microfluidic platform for rapid PCR amplification using integrated thermoelectric heating and ice-valving

We present a novel centrifugal microfluidic system for the rapid amplification of a Bacillus anthracis gene, with integrated pumping and thermoelectric-based heating and valving. Polymerase chain reaction is a powerful method for rapid and accurate diagnosis of infectious diseases and bio-warfare agents. The fully integrated system and disposable disc developed here automates the fluidic pumping, valving, and rapid thermocycling required for polymerase chain reaction. We demonstrate the first utilization of ice-valving in an integrated centrifugal microfluidic system as a method for sealing the thermocycling chamber to reduce fluid loss due to evaporation. The automated centrifugal system is validated via amplification of a B. anthracis gene. Fluidic thermal profiles are monitored and optimized to minimize non-specific amplification, reducing the total amplification time from 110 min down to 53 min.

Direct and rapid PCR amplification using digested tissues for the diagnosis of drowning

We developed a direct and rapid method for the diagnosis of death by drowning by PCR amplification of phytoplankton DNA using human tissues. The primers were designed based on the DNA sequence of the 16S ribosomal RNA gene (16S rDNA) of Cyanobacterium. Samples of lung, liver and kidney tissues were collected from 53 autopsied individuals diagnosed as death by drowning. Without DNA extraction, the tissue fragments were incubated directly in a digest buffer developed in this study, for 20 min. Using 1 microL of the tissue digest solution in PCR, the 16S rDNA was successfully amplified. The specific 16S rDNA fragment was identified from the standard picoplankton Euglena gracilis, the tissues of bodies died from drowning and water samples from the drowning scenes. On the other hand, no PCR products were found in the tissues of individuals who died from causes other than drowning. Various quantities of tissue weighing 1, 5, 10, 20 and 30 mg were tested, and the PCR amplification detected the specific 16S rDNA fragment from all the quantities of tissue tested. This method was found to be more reliable, sensitive, specific and rapid when compared to the conventional diagnosis of death by drowning using the diatom test by acid digestion method.

Application of Bio-Cell Chip Technology to Rapid Genomic DNA Preparation and PCR Amplification.

Abstract 4197 BackgroundThough primary cells obtained from patients are necessary to scientists, it has been problems that their amount is not sufficient to perform various assays and that the cell specimens are often impaired, or their constituents or products are destroyed during the transport to the scientists. To address these problems, we developed a bio-cell chip, a chip having hundreds kind of cells arrayed and immobilized on a small slide. We arrayed the cells in a tiny space by spotting them onto a supporting matrix, so that hundreds of assays can be conducted with only a small amount of sample and several hundreds kinds of samples can be assayed under the same condition. We developed the direct extraction of DNA from bio-cell chip and successfully amplified the extracted DNA. Here, we introduce simple and convenient method of DNA extraction utilizing bio-cell chip, which can handle hundreds of specimen at one time. MethodsPeripheral blood anticoagulated with EDTA or sodium citrate were centrifuged at 2000 rpm for 20 minutes, and leukocyte layers were transferred to 1.5 ml tube by careful pipetting. Buffy coats are adjusted to the cell count of 1,000 cells/ μl to 10,000 cells/ μl with Phosphate Buffered Saline. One microliter of cell suspension are loaded onto each of 92 well bio cell-chip and air-dried. To investigate the duration of DNA stability of cells loaded on bio cell-chip, loaded bio cell-chip were stored at room temperature for 5day, 15day, and 1 month before DNA extraction. Cell-chip preparation was done as previously published. Briefly, the substrate for the bio cell-chip was a slide glass on which a 1 mm thick perforated poly- dimethylsiloxane (PDMS) layer was bonded to form 16×6 wells array for subsequent cell seeding. Each cylindrical shape with 1.5 mm in diameter and positioned within a lattice pattern with its own indexing number printed on the slide glass. Maximum volume that can be loaded in each cylinder was 10ul. For DNA extraction, 2 ul of BR-A buffer (GenScript BloodReadyTMMultiplex PCR System) was dropped onto each of the wells of bio-cell chip and dried cells in the bottom of wells were mixed with BR-A buffer solution thoroughly by pipetting up and down for 10-15 times. Then, BR-A buffer additionally dropped into each well up to 20ul volume finally and mixed well. One or multiple gene targets can be amplified using a pair of primers or multiple pairs of primers from the mixture of buffer with cells without DNA purification. Then, PCR Amplification was carried out using the MJ Research DNA thermal cycler programmed to denature DNA at 95°C for 30s, and 40cycles of annealing at 62°C and extension at 72°C for 30sec in a 20μl mixture containing 1U BioTherm‘DNA Polymerase (Genecraft, GERMANY), 1μl(10pM/ul) of each oligonucleotide primer, 2.5mM dNTP, and genomic DNA. Results and conclusionThe PCR amplification results were successful using BioTherm‘DNA Polymerase. When we carried out PCR amplification using the PCR Premix (BloodReadyTM PCR System), we had no PCR products. The target PCR products were observed from genomic DNA sample with above 100 cells. Also, PCR amplification for genomic DNA from stored cell for 5day, 15 day, and 1 month was successful. With this extraction method, PCR was successful and not only minimize the simplification of experimental procedure but also save the time. Disclosures:No relevant conflicts of interest to declare.

Rapid ID Technology (RIDT) in Plants: High-Speed DNA Fingerprinting in Grain Seeds for the Identification, Segregation, Purity, and Traceability of Varieties Using Labautomation Robotics

A rapid and inexpensive DNA diagnostic platform for fingerprinting Cdn. registered, wheat varieties has been developed. Two current, real-time applications being used in Canada include the determination of purity (% contamination) of grain shipments in rail cars and the monitoring of field plots that represent a midge varietal blend. The quantification of a sample is accomplished by fingerprinting single seeds and a sample having a mixture of varieties can be assayed. The Rapid ID Technology (RIDT) platform enables high-speed, high-throughput, robotic labautomation and low-cost single nucleotide polymorphism (SNP)-DNA fingerprinting in wheat. The inexpensive seed DNA extraction method, rapid PCR amplification, and miniaturization of the Invader assay for SNP scoring are all paramount for fingerprinting millions of single seeds per year in numerous rail cars/field plots at a low cost. The cost factor is the dominant concern in any fingerprinting program and the total cost for consumables for a single marker tested is estimated at less than $0.60 per seed. The RIDT platform can also segregate closely related cultivars, an important characteristic since many varieties have a common genetic background. The results “from seed to fingerprint data” at a central lab can then be transferred electronically to any location using a laboratory information management system.

High-Throughput Quantitative Polymerase Chain Reaction in Picoliter Droplets

Limiting dilution PCR has become an increasingly useful technique for the detection and quantification of rare species in a population, but the limit of detection and accuracy of quantification are largely determined by the number of reactions that can be analyzed. Increased throughput may be achieved by reducing the reaction volume and increasing processivity. We have designed a high-throughput microfluidic chip that encapsulates PCR reagents in millions of picoliter droplets in a continuous oil flow. The oil stream conducts the droplets through alternating denaturation and annealing zones, resulting in rapid (55-s cycles) and efficient PCR amplification. Inclusion of fluorescent probes in the PCR reaction mix permits the amplification process to be monitored within individual droplets at specific locations within the microfluidic chip. We show that amplification of a 245-bp adenovirus product can be detected and quantified in 35 min at starting template concentrations as low as 1 template molecule/167 droplets (0.003 pg/microL). The frequencies of positive reactions over a range of template concentrations agree closely with the frequencies predicted by Poisson statistics, demonstrating both the accuracy and sensitivity of this platform for limiting dilution and digital PCR applications.

Asian Soybean Rust Caused by Phakopsora pachyrhizi on Soybean and Kudzu in Florida

Asian soybean rust caused by Phakopsora pachyrhizi was found on soybean and kudzu in Florida in November of 2004. The initial diagnosis of soybean rust was based on observations of symptoms and urediniospores. The two species of Phakopsora that cause rust diseases on soybean, P. pachyrhizi and P. meibomiae, cannot be differentiated with light microscopy. A rapid DNA extraction and PCR amplification protocol discriminated between the two species. The sequence of the amplified DNA product confirmed this first report of P. pachyrhizi in Florida. Accepted for publication 9 May 2005. Published 13 June 2005.