Pg μL Research Articles

Phase-Regulated Sensing Mechanism of MoS2 Based Nanohybrids toward Point-of-Care Prostate Cancer Diagnosis.

Alpha-methylacyl-CoA racemase (AMACR) has been proven to be consistently overexpressed in prostate cancer epitheliums, and is expected to act as a positive biomarker for the diagnosis of prostate carcinoma in clinical practice. Here, a strategy for specific determination of AMACR in real human serum by using an electrochemical microsensor system is presented. In order to implement the protocol, a self-organized nanohybrid consisting of metal nanopillars in a 2D MoS2 matrix is developed as material for the sensing interface. The testing signal outputs are strongly enhanced with the presence of the nanohybrids owing to that the metal pillars provide an efficient mass difussion and electron transfer path to the MoS2 film surface. Furthermore, the phase-regulated sensing mechanism over MoS2 is noticed and demonstrated by density functional theory calculation and experiments. The explored MoS2 based nanohybrids are employed for the fabrication of an electrochemical microsensor, presenting good linear relationship in both ng µL-1 and pg µL-1 ranges for AMACR quantification. The sampling analysis of human serum indicates that this microsensor has good diagnostic specificity and sensitivity toward AMACR. The proposed electrochemical microsensor system also demonstrates the advantages of convenience, cost-effectiveness, and disposability, resulting in a potential integrated microsystem for point-of-care prostate cancer diagnosis.

Metal-polymer hybrid nanomaterial for impedimetric detection of human papillomavirus in cervical specimens

The human papillomavirus (HPV) is one of the main sexually transmitted pathogens that infect the anogenital epithelium and mucous membranes. HPV genotypes can be classified as high and low oncogenic risk, with infection by the former resulting in cervical cancer in approximately 100 % of the cases. In this work, we developed an ultrasensitive electrochemical biosensor for the detection and identification of different HPV genotypes. A nanostructured platform based on a matrix of polyaniline (PANI) containing gold nanoparticles (AuNps) was designed for the chemical immobilization of a DNA probe capable of recognizing different HPV types. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and atomic force microscopy (AFM) were used to characterize the genosensor. The impedimetric responses indicate that the proposed sensor was able to detect HPV (types 6, 11, 16, 31, 33, 45, and 58) in cervical specimens (cDNA samples). We obtained different profiles of electrochemical responses for the high and low-risk HPV genotypes. By adopting a three-dimensional quantitative analysis of impedance response variables, it was possible to identify the existence of a pattern of association for samples of high oncogenic risk, which may lead to the differential diagnosis of HPV. The biosensor demonstrated an excellent analytical performance for the detection of HPV genotypes with high sensibility and selectivity. The genosensor exhibited a linear range of response in the 1 pg μL−1 to 100 pg μL−1 range. Besides, a limit of detection (LOD) of 2.74 pg μL−1 and 7.43 pg μL−1 was obtained for HPV11 and HPV16, respectively, with regression coefficients of 99.88 % and 99.47 %. Thus, the proposed sensor may serve as a good prognostic indicator for patients infected with papillomavirus.

Real-time PCR assay for detection of Dickeya fangzhongdai causing bleeding canker of pear disease in China

Bleeding canker, caused by Dickeya fangzhongdai, is a devastating disease of pear in China. The bacterium causes cankers, branch die-back, and eventually kills pear trees. The typical sign of bleeding canker infection is a rusty-brown bacterial ooze that exudes down from cankers onto branches or trunks. However, early symptoms and signs are inconspicuous, which makes effective disease management difficult. Detection and identification of D. fangzhongdai are time-consuming and difficult because no rapid method exists to date. In this study, a TaqMan real-time PCR assay was developed for D. fangzhongdai based on an elongation factor G (fusA) gene. The real-time PCR assay detected 0.2 pg μL−1 DNA and 1×103 CFU mL−1 of D. fangzhongdai. Based on this assay, bleeding canker on asymptomatic pear trees can be diagnosed as early as 5 days after infection. The real-time PCR assay can facilitate disease management by providing early and accurate diagnosis of the bleeding canker disease of pear.

Development of loop-mediated isothermal amplification (LAMP) assay for rapid detection of Fusarium proliferatum causing ear and kernel rot on maize

Fusarium proliferatum, one of the predominant causal agents associated with ear and kernel rot in China, reduces maize yield and quality, and produces various mycotoxins that are harmful to animals and humans. A key challenge for the integrated management strategy of this disease is rapid and accurate detection of F. proliferatum on maize. Therefore, a new loop-mediated isothermal amplification (LAMP) assay was developed for the detection of F. proliferatum from the diseased maize. The primers were selected and designed using the translation elongation factor-1α (TEF-1α) gene region as target DNA sequence. The amplicons were successfully inspected by visual assessment using SYBR Green I as well as electrophoresis on agarose gel. The target gene was efficiently amplified at 62 °C for 30 min. The minimum detection concentration was 10 pg μL−1. The LAMP assay was found to be highly specific for F. proliferatum from the other fungal species. In field trial, F. proliferatum could be detected from suspected diseased maize samples. The results showed that the new and readily available LAMP assay has potential to apply successfully for the detection of F. proliferatum causing ear and kernel rot on maize in the field.

Reliability of gastrointestinal barrier integrity and microbial translocation biomarkers at rest and following exertional heat stress.

PurposeExertional heat stress adversely distrupts (GI) barrier integrity and, through subsequent microbial translocation (MT), negativly impacts health. Despite widespread application, the temporal reliability of popular GI barrier integity and MT biomarkers is poorly characterised.MethodFourteen males completed two 80‐min exertional heat stress tests (EHST) separated by 7–14 days. Venous blood was drawn pre, immediately‐ and 1‐hr post both EHSTs. GI barrier integrity was assessed using the serum Dual‐Sugar Absorption Test (DSAT), Intestinal Fatty‐Acid‐Binding Protein (I‐FABP) and Claudin‐3 (CLDN‐3). MT was assessed using plasma Lipopolysaccharide Binding Protein (LBP), total 16S bacterial DNA and Bacteroides DNA.ResultsNo GI barrier integrity or MT biomarker, except absolute Bacteroides DNA, displayed systematic trial order bias (p ≥ .05). I‐FABP (trial 1 = Δ 0.834 ± 0.445 ng ml−1; trial 2 = Δ 0.776 ± 0.489 ng ml−1) and CLDN‐3 (trial 1 = Δ 0.317 ± 0.586 ng ml−1; trial 2 = Δ 0.371 ± 0.508 ng ml−1) were increased post‐EHST (p ≤ .01). All MT biomarkers were unchanged post‐EHST. Coefficient of variation and typical error of measurement post‐EHST were: 11.5% and 0.004 (ratio) for the DSAT 90‐min postprobe ingestion; 12.2% and 0.004 (ratio) at 150‐min postprobe ingestion; 12.1% and 0.376 ng ml−1 for I‐FABP; 4.9% and 0.342 ng ml−1 for CLDN‐3; 9.2% and 0.420 µg ml−1 for LBP; 9.5% and 0.15 pg µl−1 for total 16S DNA; and 54.7% and 0.032 for Bacteroides/total 16S DNA ratio.ConclusionEach GI barrier integrity and MT translocation biomarker, except Bacteroides/total 16S ratio, had acceptable reliability at rest and postexertional heat stress.

Detection of cardiovascular disease associated miR-29a using paper-based microfluidics and surface enhanced Raman scattering.

The development of viable point-of-care diagnostic formats is integral to achieving better patient care and improved outcomes. The need for robust and low-cost tests is especially important in under-resourced and rural settings. Perhaps the greatest challenge is ensuring that an untrained individual is capable of operating and interpreting the test, out with a care facility. Here we present a paper-based diagnostic device capable of sensing miR-29a using both colorimetric and surface enhanced Raman scattering (SERS) analysis. Rather, than carry out the two types of analyses in tandem, we envisage that the colorimetric output is easy enough to be interpreted by the untrained-individual administering the test to provide them with qualitative feedback. If deemed positive, the test can be further validated at a centralized care facility using a handheld-Raman spectrometer to provide a semi-quantitative result. Detection of miR-29a, a microRNA associated with myocardial infarction, was achieved at a level of pg μL-1 through the combination of three-dimensional paper-based microfluidics, colorimetric detection, and surface enhanced Raman scattering (SERS) analysis. RGB analysis of the colorimetric output generated from samples containing miR-29a at different concentrations (18-360 pg μL-1) showed differentiation from the control sample, however significant repeat variability indicated that it could not be used for quantifying miR-29a levels. However, the SERS analysis exhibited greater reproducibility at varying concentrations, achieving an LoD of 47 pg μL-1. The union of the paper-based device and the two analysis methods resulted in the production of a sensitive, reproducible and facile, point of care test (POCT), which paves the way for future implementation in the diagnosis of a range of diseases.

Highly sensitive detection of Staphylococcus aureus by a THz metamaterial biosensor based on gold nanoparticles and rolling circle amplification.

A highly sensitive method for detecting Staphylococcus aureus (S. aureus) is urgently needed to reduce the impact and spread of hospital-acquired infections and food-borne illness. For this purpose, this paper presents a THz metamaterial biosensor based on gold nanoparticles (AuNPs) and rolling circle amplification (RCA). The RCA process amplified the S. aureus DNA fragments and generated copious yields of long single-strand DNA molecules. These molecules were then conjugated with the AuNPs to form complexes that delivered exceptional increases in the refractive indices of the samples, and resulted in corresponding improvements in the THz response of the metamaterial. Under optimal conditions, the shifts in the metamaterial's resonance frequency displayed a linear relationship with concentrations of synthetic S. aureus DNA in the range from 10 fM to 10 pM, with a limit of detection of 2.77 fM. We also tested the practical application of this biosensor in measurements of genomic DNA in clinical bacterial strains, where the sensor showed a detection limit of 0.08 pg μL−1 and a linear range from 0.1 to 5 pg μL−1. It also exhibited reasonable specificity, resisting interference from three other pathogenic bacteria. These findings indicate that the proposed approach offers a cost-effective THz biosensing strategy that can be easily fabricated and conveniently operated to aid the diagnosis of infectious disease and food safety control.

A loop‐mediated isothermal amplification (LAMP) assay based on unique markers derived from genotyping by sequencing data for rapid in planta diagnosis of Panama disease caused by Tropical Race 4 in banana

The socio‐economic impact of Fusarium odoratissimum, which is colloquially called tropical race 4 (TR4), is escalating as this fungal pathogen spreads to new banana‐growing areas. Hence, the development of simple, reliable and rapid detection technologies is indispensable for implementing quarantine measures. Here, a versatile loop‐mediated isothermal amplification (LAMP) assay has been developed that is applicable under field and laboratory conditions. DNA markers unique to TR4 isolates were obtained by diversity arrays technology sequencing (DArTseq), a genotyping by sequencing technology that was conducted on 27 genotypes, comprising 24 previously reported vegetative compatibility groups (VCGs) and three TR4 isolates. The developed LAMP TR4 assay was successfully tested using 22 TR4 isolates and 45 non‐target fungal and bacterial isolates, as well as on infected plants under greenhouse and field conditions. The detection limit was 1 pg µL−1 pure TR4 DNA or 102 copies plasmid‐localized TR4 unique sequence (SeqA) per reaction, which was not affected by background DNA in complex samples. The LAMP TR4 assay offers a powerful tool for the routine and unambiguous identification of banana plants infected with TR4, contributing to advanced diagnosis in field situations and monitoring of fusarium wilt.

QPCR and loop mediated isothermal amplification for rapid detection of Ustilago tritici.

Loose smut of wheat caused by the basidiomycete fungus Ustilago tritici, a seed-borne disease, is difficult to control because of the expanse of wheat planting area and difficulty in pathogen detection. In this study, real-time fluorescence quantitative PCR (qPCR) and loop-mediated isothermal amplification (LAMP) assays are used to rapidly amplify the DNA of U. tritici. Five pairs of primers for qPCR and two series primers for LAMP were designed. Primarily, the specificity of the primer was assessed by using genomic DNA of U. tritici, Fusarium graminearum, Blumeria graminis, Rhizoctonia cerealis, Puccinia striiformis, Bipolaris sorokiniana, and Alternaria solani as templates. Further, the amplification systems were optimized. Finally, the sensitivity of qPCR and LAMP assays were evaluated. The results showed that the primer Y-430 F/R, Y-307 F/R, Y-755 F/R, and Y-139 F/R for qPCR and primers L-139 and L-988 for LAMP could be used for U. tritici detection. In the sensitivity test, the detection limit of qPCR assay was identified as 10 pg μL−1 of genomic DNA, the detection limit for LAMP assay was 100 fg μL−1. We successfully performed qPCR and LAMP assays on wheat loose smut wheat samples. This paper establishes two methods for U. tritici detection, which can be used for diagnosis of wheat loose smut in the laboratory and in the field.

Matrix solid-phase dispersion (MSPD) as simple and useful sample preparation technique for determination of polybrominated diphenyl ethers (PBDEs) in dust

In the present study a novel analytical procedure for the determination of polybrominated diphenyl ethers in dust samples was developed.The main aim of the research was the selection of the optimum conditions of the matrix solid-phase dispersion before the final determination of polybrominated diphenyl ethers in dust samples. In order to assess the best usefulness of this technique, a favourable ratio of sample amount to the mass of dispersing sorbent, as well as the type of this sorbent used has been tested. The type of sorbent responsible for additional purification (clean-up sorbent) of the extract during matrix solid-phase dispersion was also selected. Gas chromatography coupled with mass spectrometry will be used at the final determination step.Preliminary results indicate that the use of matrix solid-phase dispersion can be a promising alternative to other time-consuming and multi-stage analytical procedures. The proposed method provided satisfactory recoveries (76–119%) and limits of detection: 2.1–4.4 pg μL−1 for tri-heptaBDE in linear range of 5–100 pg μL−1; 480 pg μL−1 for decaBDE in linear range of 500–2000 pg μL−1 from only 0.05 g of a dust sample. Finally, the method was applied to study the content of selected polybrominated diphenyl ethers in real dust samples. Some polybrominated diphenyl ether congeners reached up to (16.3 ± 3.0)·102 ng g−1.

Label-free nanostructured biosensor for Schistosoma mansoni detection in complex biological fluids

Schistosomiasis is a neglected tropical disease with a worldwide prevalence. Neuroschistosomiasis is the most severe presentation of the disease and affects the central nervous system. In this work, Schistosoma mansoni detection was based on self-assembled layers of 3-mercaptopropyltrimethoxysilane (MPTS) and electrosynthesized gold nanoparticles (AuNPs). The DNA probe was chemisorbed onto AuNPs. The biosensor was characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The impedimetric response of the MPTS-AuNPs-DNAprobe system indicates an effective modification of the electrode surface. Topographical atomic force microscopy images were used to characterize the self-assembled layers on the gold electrode surface. The proposed biosystem was able to recognize the S. mansoni genome sequence at different concentrations in samples of urine, cerebrospinal fluid, and serum. Several concentration ranges were evaluated: urine (27–50 pg μL−1), cerebrospinal fluid (25–60 pg μL−1), and serum (27–42 pg μL−1). The limit detection (LOD) of the biosensor was 0.6 pg μL−1. The developed label-free genosensor was able to detect small concentrations of S. mansoni DNA in complex biological fluids.

Species-specific COI primers for rapid identification of a globally significant invasive pest, the cassava mealybug Phenacoccus manihoti Matile-Ferrero

The globally invasive cassava mealybug Phenacoccus manihoti Matile-Ferrero is a pernicious pest of cassava, and its recent introduction into Asia has raised considerable alarm. To slow or prevent further invasion, an accurate, simple, and developmental-stage-independent detection method for P. manihoti is required. In the present study, a PCR method based on a species-specific mitochondrial DNA cytochrome oxidase I (SS-COI) marker was developed for rapid identification of P. manihoti. One pair of SS-COI primers (PMSSZW-1F and PMSSZW-1R) was designed based on sequence variations in the COI gene among P. manihoti and related mealybug species. Specificity of the primer pair was validated on 21 closely related species. Sensitivity tests were performed on four immature developmental stages and female adults. Efficacy tests demonstrated that at the relatively low concentration of (135.2±14.7) pg μL−1 resuspended DNA, the specific fragment was detected in all replicates. Furthermore, the SS-COI primer pair was assayed on three populations of P. manihoti from major exporting countries of cassava. The PCR assay was proved to be a rapid, simple, and reliable molecular measure for the identification of P. manihoti. This tool will be useful for quarantine, monitoring, and management of this invasive pest.

Establishment of the molecular beacon-loop-mediated isothermal amplification method for the rapid detection of Porphyromonas gingivalis

Porphyromonas gingivalis, a clinically important oral pathogen causing periodontal disease, is difficult to culture in routine conditions. Hence, it is necessary to establish a reliable technique to detect this pathogen. Previously, our laboratory developed a new isothermal detection method, called MB-LAMP (molecular beacon-Loop-mediated isothermal amplification), which combines the advantages of LAMP and qPCR through the accurate and quantitative detection of LAMP products. This approach offers significant potential for the point-of-care detection of P. gingivalis. Here, MB-LAMP was used to detect P. gingivalis targeting a specific fragment, and the sensitivity was as high as 1.4 × 10−1 pg μL–1. The method showed no cross-reaction with 14 other bacterial pathogens. For clinical samples, this assay showed a high diagnostic sensitivity (100%) and specificity (100%), equivalent to that of real-time quantitative polymerase chain reaction (real-time qPCR). Moreover, detection with MB-LAMP was significantly faster than that with real-time qPCR, reducing the time required for clinical diagnosis. Finally, we established an absolute quantification method with MB-LAMP for P. gingivalis using pilot samples. Thus, the highly specific, sensitive, and rapid assay developed in this study makes it feasible to diagnose P. gingivalis.

A new biosensor for osteoporosis detection

Osteoporosis is a disease that is characterized by deterioration of bone tissue and increased risk of fracture as it leads to a decrease in bone mineral density, which is an important public health problem. Today, bone mineral density is measured by radiological techniques. Alternative techniques are needed because of the disadvantages such as excessive radiation intake, the cost of radiological techniques, and the necessity for specialist personnel for the devices. The quantitative determination of biochemical markers that play a role in bone mineralization may be a good alternative for the osteoporosis diagnosis and especially in the follow-up of treatment.In this study, a specific and sensitive immunological biosensor, which quantitatively determines the osteocalcin molecule, has been developed to be used in the early osteoporosis diagnosis and to evaluate the response to the drug treatment. Anti-osteocalcin antibody was immobilized onto gold electrode surface via covalent immobilization method by using 6-mercaptohexanol, 1,4-butanedioldiglycidyl ether, ethanolamine, and glutaraldehyde. Immobilization steps and biosensor characterization were specified by cyclic voltammetry and electrochemical impedance spectroscopy. The detection time and range of Ocn biosensor were determined as 45 min and 10–60 pg µL−1 Ocn concentration, respectively. The Ocn biosensor was successfully applied in artificial serum samples spiked with Ocn.

New strategy for the gene mutation identification using surface enhanced Raman spectroscopy (SERS)

An early and accurate diagnosis of a specific DNA mutations has a decisive role for effective treatment. Especially, when an immediate decision on treatment most needs to be made, the rapid and precise confirmation of clinical findings is vital. Herein, we show a new strategy for the gene mutation (BRAF c.1799T>A; p. V600E) identification using highly SERS-active and reproducible SERS substrate (photo-etched GaN covered with a thin layer of sputtered gold) and surface enhanced Raman scattering (SERS) spectroscopy. The detection is based on the conformation change (gauche → trans) of the alkanethiol linker modifying the capture DNA during the hybridization process. The value of the intensity ratio of the ν(C–S) bands of the trans and gauche conformer higher than 1.0 indicated the presence of mutation. The demonstrated new DNA SERS (bio)sensor is characterized by the low detection limit at the level of pg μL–1, wide analytical range from 6.75 pg μL–1 to 67.5 ng μL–1 and high selectivity. The proposed bioactive platforms, based on nanostructured GaN substrates modified with thiolated ssDNA (single stranded DNA) can be successfully used in the analysis of clinical samples.

Quantitative polymerase chain reaction (Q-PCR) and fluorescent in situ hybridization (FISH) detection of soilborne pathogen Sclerotium rolfsii

Early detection of soilborne disease is essential for prediction of disease development and successful disease management. Traditional methods of monitoring soilborne diseases based on plant growth and symptoms did not take into account the presence and inoculum potential of pathogens in the soil, which generally delay the prevention of diseases. To detect and quantify soil populations of Sclerotium rolfsii, the causal agent of southern blight of tomatoes, quantitative polymerase chain reaction (Q-PCR) was used to measure the amount of soil general fungal and specific S. rolfsii DNA. Significantly higher amounts of total fungal and S. rolfsii DNA were detected in inoculated than in uninoculated soil in a greenhouse inoculation experiment. Fluorescent in situ hybridization (FISH) was successfully developed to detect the abundance of S. rolfsii through whole cell hybridization and was further applied for visual detection of the abundance of S. rolfsii in the soil samples of greenhouse inoculation experiment. Natural abundance of S. rolfsii in soil with a DNA amount of 0.06 pg µl−1 DNA extraction, which was equivalent to 8 pg g−1 soil, was successfully detected. The hybridization signal detected in soil for S. rolfsii had a great correlation with the amount of infested DNA levels of S. rolfsii. Molecular detection and quantification of soilborne pathogens in plant roots and in the soil could provide valuable information to predict disease development and apply management practices for soilborne diseases.

A DNA-modified hydrogel for simultaneous purification, concentration and detection of targeted cfDNA in human serum.

Cell-free DNA (cfDNA) in blood, which stems from the fetus of pregnant women and tumor in cancer patients, has gained attention in molecular diagnosis. However, cfDNA is less stable, and its amount in the serum is extremely low; these are critical barriers for the utilization of this resource. In this study, a DNA-modified polyacrylamide hydrogel (DNA-Gel) was prepared, and a specialized device was designed to simultaneously catch, purify, concentrate, and detect targeted cfDNA by electrophoresis. We demonstrated that 20–1000 bp ssDNA and dsDNA could be caught and released by the DNA-Gel-based device with high specificity and sensitivity. Upon increasing the number of cycles and electrophoresis time, higher DNA purity and density were achieved, and the separation of serum proteins, untargeted cfDNA, and other charged molecules was promoted. As low as 10 pg μL−1 of DNA could be detected using the DNA-Gel after four cycles of concentration. We also detected 1 fg μL−1 of DNA in the serum with 16 cycles of concentration, followed by 25 PCR cycles. We also designed a device to obtain DNA from the DNA-Gel. We found that the DNA loss rate was around 50%, and A260/A280 was close to 1.7. Thus, we have designed a cost-effective and highly economical device to purify DNA at low concentrations with high specificity and selectivity.

Detection and quantification of black foot and crown and root rot pathogens in grapevine nursery soils in the Western Cape of South Africa

Black foot disease (BFD) and crown and root rot (CRR) are important soilborne diseases that affect young grapevines in nurseries and vineyards. A 3-year survey (2013–2015) of five open-field grapevine nurseries was conducted in the Western Cape Province of South Africa. The survey involved the isolation of BFD and CRR pathogens from grafted rootstocks (ten plants per nursery, per year) that were rooted in soil for 1 year. In 2013 and 2015, grapevines were sampled, while in 2014, sampling was focused on rotation crops and weeds (ten plants each). The rotation crops included white mustard, lupins, canola, triticale and forage radish. The weed species sampled included Johnson grass, ryegrass, winter grass, Cape marigold and corn spurry. Soil samples from ten sites per nursery were also collected in close proximity to the sampled plants, at depths of 0–30 cm and 30–60 cm (ten samples per depth). Isolations were made from the grapevines, rotation crops and weeds. Pathogen detection and quantification in the soil were determined using quantitative real-time polymerase chain reaction technology. The predominant BFD pathogens isolated from grapevines were Campylocarpon fasciculare , Ca. pseudofasciculare and Dactylonectria macrodidyma . The predominant CRR pathogens were Pythium irregulare and Phytopythium vexans. Dactylonectria macrodidyma , D. novozelandica , D. pauciseptata , Py. irregulare , Py. ultimum var. ultimum and Py. heterothallicum were isolated from triticale roots. Dactylonectria spp. were also isolated from corn spurry, while Py. irregulare and Py. ultimum var. ultimum were isolated from numerous weeds and rotation crops. Mean soil DNA concentrations of Ilyonectria and Dactylonectria were from 0.04 to 37.14 pg μL -1 , and for Py. irregulare were between 0.01 and 3.77 pg μL -1 . The Phytophthora mean soil DNA concentrations ranged from 0.01 to 33.48 pg μL -1 . The qPCR protocols successfully detected and quantified BFD and CRR pathogens in grapevine nursery soil. This is the first report of D. pauciseptata and D. alcacerensis in South African grapevine nurseries.

A Set of Conventional and Multiplex Real-Time PCR Assays for Direct Detection of Elsinoë fawcettii, E. australis, and Pseudocercospora angolensis in Citrus Fruits.

Elsinoë fawcettii, E. australis, and Pseudocercospora angolensis are causal agents of citrus scab and spot diseases. The three pathogens are listed as quarantine pests in many countries and are subject to phytosanitary measures to prevent their entry. Diagnosis of these diseases based on visual symptoms is problematic, as they could be confused with other citrus diseases. Isolation of E. fawcettii, E. australis, and P. angolensis from infected tissues is challenging because they grow slowly on culture media. This study developed rapid and specific detection tools for the in planta detection of these pathogens, using either conventional PCR or one-tube multiplex real-time PCR. Primers and hybridization probes were designed to target the single-copy protein-coding gene MS204 for E. fawcettii and E. australis and the translation elongation factor (Tef-1α) gene for P. angolensis. The specificity of the assays was evaluated by testing against DNA extracted from a large number of isolates (102) collected from different citrus-growing areas in the world and from other hosts. The newly described species E. citricola was not included in the specificity test due to its unavailability from the CBS collection. The detection limits of conventional PCR for the three pathogens were 100, 100, and 10 pg μl-1 gDNA per reaction for E. fawcettii, E. australis, and P. angolensis, respectively. The quadruplex qPCR was fully validated assessing the following performance criteria: sensitivity, specificity, repeatability, reproducibility, and robustness. The quadruplex real-time PCR proved to be highly sensitive, detecting as low as 243, 241, and 242 plasmidic copies (pc) μl-1 of E. fawcettii, E. australis, and P. angolensis, respectively. Sensitivity and specificity of this quadruplex assay were further confirmed using 176 naturally infected citrus samples collected from Ethiopia, Cameroon, the United States, and Australia. The quadruplex assay developed in this study is robust, cost-effective, and capable of high-throughput detection of the three targets directly from citrus samples. This new detection tool will substantially reduce the turnaround time for reliable species identification and allow rapid response and appropriate action.

Phenacoccus madeirensis Green (Hemiptera: Pseudococcidae): New geographic records and rapid identification using a species-specific PCR assay

The highly invasive Madeira mealybug Phenacoccus madeirensis Green (Hemiptera: Pseudococcidae) is a polyphagous pest which seriously harms crops of staple foods, fruits, vegetables, and ornamental plants. A method for rapid and accurate identification is needed to slow further spread of this pernicious pest. DNA barcoding and taxonomic keys were used to identify specimens of P. madeirensis collected in Rwanda and China. We designed a species-specific PCR (SS-PCR) primer pair for P. madeirensis based on variable sequences of the mitochondrial cytochrome c oxidase subunit I gene among P. madeirensis and 29 related species. This SS-PCR assay allows for rapid and precise identification. With this assay, no cross-reaction was detected from the 21 closely related mealybug species. All specimens from all ontogenic stages, adult debris, and individuals from 11 populations of P. madeirensis from different geographical areas and/or host plants were detected. The detection limit of this method was 192.58 ± 16.48 pg μL−1 of P. madeirensis DNA. These demonstrated the high sensitivity and effectiveness of this method. Using this method, we found the first described occurrence of P. madeirensis in Southern Province, Rwanda, indicating ongoing spread, and an entirely new invasion in Fujian, China. Invaded regions of China were delineated by a nationwide field study. The SS-PCR assay developed in this study is a rapid, straightforward, and reliable method for the identification of P. madeirensis, and will prove practical in plant quarantine, thus slowing or preventing further invasion and spread, and allowing for sustainable and effective management of this cosmopolitan invader.