Evaluation of an enzyme-linked immunosorbent assay for detection of Mycosphaerella pinodes in pea seeds

A polyclonal antiserum was raised against soluble mycelial extracts of Mycosphaerella pinodes aiming at pathogen detection in infected pea seeds by ELISA. When tested against the homologous antigen, it allowed the detection of 5 ng fungal soluble protein ml^-1 buffer, by double-antibody sandwich ELISA (DAS-ELISA). Positive reactions were obtained with isolates of M. pinodes of wide geographical origins but also with all tested isolates of Ascochyta pisi and Phoma medicaginis var. pinodella, two closely related pathogens forming with the target organism the Ascochyta complex. Out of the 11 other genera of pea seed-borne fungi tested, only two (Alternaria sp. and Stemphylium sp.) cross-reacted strongly by both antigen-coated plate (ACP-ELISA) and DAS-ELISA. Cross-absorption of the crude antiserum could not lead to a species-specific antiserum; however, a combination of P. medicaginis var. pinodella and Stemphylium sp. antigens resulted in an antiserum preferentially recognising A. pisi and M. pinodes. The cross-absorbed antiserum detected 50 and 500 ng of fungal protein ml^-1 buffer and healthy seed extracts respectively. DAS-ELISA proved suitable for the detection and quantification of M. pinodes in infected pea seeds tested singly.     

Detection of Double-Stranded RNA (DSRNA) in Crude Extracts of Virus-Infected Plants by Indirect ELISA

An antiserum against polyinosinic-polycytidylic acid (In-Cn) was used to detect double-stranded RNA (dsRNA) by indirect ELISA (ELISA-I). DsRNA from cucumber mosaic virus (CMV) and plum pox virus (PPV)-infected plants was detected using different types of extracts. The pH of the extraction buffer was very important in dsRNA detection, the highest optical density values being obtained at pH 6 or in aqueous extracts. Extracts heated at 80°C for 2 min showed increased optical density values compared with unheated extracts. DsRNA from Nicotiana benthamiana plants infected with each of six PPV isolates was readily detected by ELISA-I 50 days after inoculation. ELISA values then obtained with the In-Cn antiserum were generally higher than those obtained by double antibody sandwich ELISA using an antiserum to virus coat protein. Purified dsRNA from the same infected plants showed no visible band, but it produced a fluorescent background when analysed by polyacrylamide gel electrophoresis.     

Microbial enrichment and multiplexed microfiltration for accelerated detection of Salmonella in spinach

To attain Salmonella detection thresholds in spinach suspensions using enrichment media requires at least 24 hr. Separation and concentration of selected microorganisms via microfiltration and microfugation reduce time for sample preparation, especially when working with large volumes of vegetable suspensions. This facilitates accelerated detection of Salmonella in spinach suspensions, and may contribute to effectively monitoring this pathogen before it reaches the consumer. We report a microfiltration-based protocol for accelerated sample preparation to concentrate and recover ≤1 colony forming unit (CFU) Salmonella/g pathogen-free spinach. Store-bought samples of spinach and a spinach plant subjected to two environmental conditions (temperature and light exposure) during its production were tested. The overall procedure involves extraction with buffer, a short enrichment step, prefiltration using a nylon filter, crossflow hollow fiber microfiltration, and retentate centrifugation to bring microbial cells to detection levels. Based on 1 CFU Salmonella/g frozen spinach, and a Poisson distribution statistical analyses with 99% probability, we calculated that 3 hr of incubation, when followed by microfiltration, is sufficient to reach the 2 log concentration required for Salmonella detection within 7 hr. Longer enrichment times (5 hr or more) is needed for concentrations lower than 1 CFU Salmonella/g of ready to eat spinach. The recovered microbial cells were identified and confirmed as Salmonella using both polymerase chain reaction (PCR) and plating methods. Different environmental conditions tested during production did not affect Salmonella viability; this demonstrated the broad adaptability of Salmonella and emphasized the need for methods that enable efficient monitoring of production for the presence of this pathogen.     

A “Nonsterile” Method for Selecting and Growing <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Transformants (T<Subscript>2</Subscript> Transgenic Lines) Resistant to Kanamycin

A breakthrough in transgenic Arabidopsis thaliana research was the development of the floral dip transformation protocol, a simple and reliable method of obtaining transformants, T₁ transgenic lines, at high efficiency while avoiding the use of tissue culture. However, the traditional protocol (a “sterile” method) of obtaining T₂ transgenic lines has not evolved along with improvements in transformation technology as it continues to be laborious and time-consuming. In this study, we report on the development of an improved protocol (a “nonsterile” method) for selecting and growing A. thaliana transformants (T₂ transgenic lines) resistant to kanamycin under nonsterile conditions. This protocol involves the use of a simple yet specialized device that will aid in solium selection of T₂ transgenic lines that can be rapidly grown in a hydroponic system. The “nonsterile” method reduces labor and time involved as compared to the “sterile” method; moreover, it is easy to set up and maintain. This method may also be applicable to other selecting agents, and perhaps to other plants.     

Evaluation of antiserum raised againstPestalotiopsis theœ for the detection of grey blight of tea by ELISA

Polyclonal antiserum was raised against the mycelial extract ofPestalotiopsis theœ and immunoglobulin fractions were purified by ammonium sulfate fractionation and chromatography on DEAE-Sephadex. In enzyme-linked immunosorbent assay, antiserum dilution up to 1∶16000 detected homologous antigen at a 5 mg/L concentration, and at 1∶125 antiserum dilution fungal antigens could be detected at a concentration as low as 25 μg/L. In fifteen varieties of tea tested, originating from Darjeeling, UPASI and Tocklai breeding stations, absorbance values of infected leaf extracts were significantly higher than those of healthy extracts at a concentration of 40 mg/L in indirect ELISA. ELISA-positive material was detected in tea leaves as early as 12 h after inoculation withP. theœ. At antiserum dilutions up to 1∶125, the pathogen could be detected in inoculated leaf extracts up to antigen concentration of 2 mg/L. The antiserum reacted with two other isolates ofP. theœ tested but not with the antigens from mycelial extracts ofGlomerella cingulata andCorticium invisum or with extracts of tea leaves inoculated with these pathogens. The results demonstrate that ELISA can be used for early detection ofP. theœ in leaf tissues even at a very low level of infection.     

In vitro cytocidal effect of novel lytic peptides on Plasmodium falciparum and Trypanosoma cruzi

Plasmodium falciparum and Trypanosoma cruzi were killed by two novel lytic peptides (SB-37 and Shiva-1) in vitro. Human erythrocytes infected with P. falciparum, and Vero cells infected with T. cruzi, were exposed to these peptides. The result, in both cases, was a significant decrease in the level of parasite infection. Furthermore, the peptides had a marked cytocidal effect on trypomastigote stages of T. cruzi in media, whereas host eukaryotic cells were unaffected by the treatments. In view of the worldwide prevalence of these protozoan diseases and the lack of completely suitable treatments, lytic peptides may provide new and unique chemotherapeutic agents for the treatment of these infections.     

A Rapid and Simple PCR-based Method for Analysis of Transgenic Fish using a Restricted Amount of Fin Tissue

The protocol described in this paper offers a simple and rapid method for PCR analysis of transgenes using a restricted amount of fin tissue from small-sized transgenic fish. A simple preparation of fin lysate using a buffer containing a low concentration of an ionic detergent, SDS (0.01%), followed by neutralization with a second buffer containing higher concentrations of non-ionic detergents NP40 (2%) and Tween 20 (2%) consistently provides a reliable quantity of high-quality DNA template for PCR amplification of transgenes. Based on this protocol, transgenic fish can be clearly distinguished from non-transgenic fish using PCR in a rapid and reproducible manner. Tedious DNA purifications are avoided while fidelity of amplification and efficient identification of transgenic fish are maintained.     

Klebsiella pneumoniae haemolysin adsorption to red blood cells

It was necessary to incubate the Klebsiella pneumoniae haemolysin with erythrocytes at 37°C to produce the whole lytic action. The amount of attached klebolysin at 4°C increased as its concentration in the medium increased, until the erythrocyte surface was saturated. Treatment with 2-mercaptoethanol was necessary to permit the adsorption; it was inhibited by low concentrations of cholesterol. Klebsolysin was immunogenic and its antiserum neutralized its own haemolytic effect and streptolysin O. Anti-streptolysin serum also neutralized klebsolysin. Streptolysin O attachment to erythrocytes impeded the posterior klebolysin adsorption in the same way that klebsolysin adsorption interfered with streptolysin O attachment.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of pineapple var. Queen using a novel encapsulation-based antibiotic selection technique

A protocol for Agrobacterium-mediated transformation of a local ‘elite’ Indian variety (Queen) of pineapple [Ananus comosus (L.) Merr, family Bromeliaceae] has been established using a standard transformation vector (pCAMBIA 1304). High transformation efficiency, expressed as the mean percentage of transgenic micro-shoots regenerated from initial callus explants (20.6%) was achieved using a novel encapsulation-based, antibiotic selection procedure. The Agrobacterium-infected micro-shoots derived from callus explants survived selection in high concentration of hygromycin (60 mg l⁻¹ and beyond) in encapsulated alginate beads. The integration of transgene in hygromycin-resistant shoots and plants was confirmed by histochemical GUS assay, PCR amplification and Southern hybridization. It is possible to eliminate false antibiotic positives in pineapple transformation program to a large extent following this procedure.     

Klebsiella pneumoniae haemolysin adsorption to red blood cells

It was necessary to incubate the Klebsiella pneumoniae haemolysin with erythrocytes at 37 degrees C to produce the whole lytic action. The amount of attached klebolysin at 4 degrees C increased as its concentration in the medium increased, until the erythrocyte surface was saturated. Treatment with 2-mercaptoethanol was necessary to permit the adsorption; it was inhibited by low concentrations of cholesterol. Klebsolysin was immunogenic and its antiserum neutralized its own haemolytic effect and streptolysin O. Anti-streptolysin serum also neutralized klebsolysin. Streptolysin O attachment to erythrocytes impeded the posterior klebolysin adsorption in the same way that klebsolysin adsorption interfered with streptolysin O attachment.     

In vitro pollen functionality of attacin-transgenic “Royal Gala” apple plants and apples transformed with 1-aminocyclopropane-1-carboxylic acid synthase (ACS)-antisense vector

Abstract

To assess pollen functionality of transgenic apple trees, in vitro pollen germination and tube growth were evaluated. Flowers of transgenic “Royal Gala” apple lines containing attacin E gene to confer resistance to fire blight (Erwinia amylovora), or antisense 1-aminocyclopropane-1-carboxylic acid synthase (ACS) construct to improve fruit storage life, were collected, and pollen was harvested. Amongst the 19 transgenic lines, pollen from three lines transformed with an ACS-antisense vector consistently had significantly lower germination rate compared to “Royal Gala”; however, no correlation between ACS level in fruit and pollen germination rate was observed. Western blots showed that the amounts of the lytic protein, attacin, varied in pollen of the four attacin-transgenic lines sampled. There was no significant correlation between attacin level in the pollen and pollen germination rate or pollen tube growth. The addition of boric acid to the germination buffer enhanced germination in attacin-transgenic lines, as well as in lines down-regulated in ethylene synthesis and control “Royal Gala”. This initial study suggests that the majority of transgenic lines tested do not differ from the control “Royal Gala” in pollen germination, and that attacin or down-regulation of ethylene does not influence in vitro pollen functionality.     

Purification of recombinant antigen BmSXP used in panLF rapid kit for lymphatic filariasis

A rapid antibody detection test is very useful for the detection of lymphatic filariasis, especially for certification and surveillance of post-mass drug administration. panLF Rapid kit is suitable for this purpose since it can detect all species of lymphatic filaria. It is based on the detection of anti-filarial IgG4 antibodies that react with recombinant B. malayi antigens, BmR1 and BmSXP. There is an increase demand for the test due to its attributes of being rapid, sensitive and specific results, as well as its field-applicability. The main aim of this paper is to obtain high recovery and purity of recombinant antigen BmSXP via a modified method of immobilized metal affinity chromatography (IMAC). The highest product yield of 11.82 mg/g dry cell weight (DCW) was obtained when IMAC was performed using the optimized protocol of 10 mM imidazole concentration in lysis buffer, 30 mM imidazole concentration in wash buffer, and 10 column volume wash buffer containing 300 mM salt concentration. This gave a 54% protein recovery improvement over the manufacturer's protocol which recorded a product yield of only 7.68 mg/g DCW. The recovered BmSXP recombinant antigen showed good western blot reactivity, high sensitivity (31/32, 97%) and specificity (32/32, 100%) in ELISA, thus attesting to its good purity and quality.     

An Improved Antiserum for Sensitive Serologic Detection of Chickpea chlorotic dwarf virus

A Syrian chickpea isolate of Chickpea chlorotic dwarf virus (CpCDV; genus Mastrevirus, family Geminiviridae) was purified and yielded 0.6–0.8 mg of purified virus per kg of infected chickpea tissue. The purified preparations were injected into a rabbit and an antiserum of good quality was obtained and used to evaluate different serological tests for the detection of CpCDV in infected chickpea leaf tissue and extracts. CpCDV was detected in sap dilutions of 1/640 by double‐antibody sandwich enzyme‐linked immunosorbent assay (DAS‐ELISA) and dot‐blot ELISA, and in sap dilutions of 1/1280 by direct antigen‐coating (DAC)‐ELISA using CpCDV immunoglobulin G (IgG) at 0.5 μg/ml. The antiserum was also able to detect the capsid protein of CpCDV by Western blot using raw antiserum at a dilution of 1/2000. The CpCDV raw antiserum (third bleeding) produced had a titre of 1/320 000 when determined by tissue‐blot immunoassay (TBIA); whereas, coating ELISA plates with CpCDV IgG at a concentration of 0.004 μg/ml was enough to detect the virus by DAS‐ELISA in a sap dilution of 1/20 using an enzyme conjugate at a dilution of 1/2000.     

Generation of trangenic Xenopus laevis using the Sleeping Beauty transposon system

Using the Sleeping Beauty (SB) transposon system, we have developed a simple method for the generation of Xenopus laevis transgenic lines. The transgenesis protocol is based on the co-injection of the SB transposase mRNA and a GFP-reporter transposon into one-cell stage embryos. Transposase-dependent reporter gene expression was observed in cell clones and in hemi-transgenic animals. We determined an optimal ratio of transposase mRNA versus transposon-carrying plasmid DNA that enhanced the proportion of hemi-transgenic tadpoles. The transgene is integrated into the genome and may be transmitted to the F1 offspring depending on the germline mosaicism. Although the transposase is necessary for efficient generation of transgenic Xenopus, the integration of the transgene occurred by an non-canonical transposition process. This was observed for two transgenic lines analysed. The transposon-based technique leads to a high transgenesis rate and is simple to handle. For these reasons, it could present an attractive alternative to the classical Restriction Enzyme Mediated Integration (REMI) procedure.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Transgene Detection by Digital Droplet PCR

Somatic gene therapy is a promising tool for the treatment of severe diseases. Because of its abuse potential for performance enhancement in sports, the World Anti-Doping Agency (WADA) included the term ‘gene doping’ in the official list of banned substances and methods in 2004. Several nested PCR or qPCR-based strategies have been proposed that aim at detecting long-term presence of transgene in blood, but these strategies are hampered by technical limitations. We developed a digital droplet PCR (ddPCR) protocol for Insulin-Like Growth Factor 1 (IGF1) detection and demonstrated its applicability monitoring 6 mice injected into skeletal muscle with AAV9-IGF1 elements and 2 controls over a 33-day period. A duplex ddPCR protocol for simultaneous detection of Insulin-Like Growth Factor 1 (IGF1) and Erythropoietin (EPO) transgenic elements was created. A new DNA extraction procedure with target-orientated usage of restriction enzymes including on-column DNA-digestion was established. In vivo data revealed that IGF1 transgenic elements could be reliably detected for a 33-day period in DNA extracted from whole blood. In vitro data indicated feasibility of IGF1 and EPO detection by duplex ddPCR with high reliability and sensitivity. On-column DNA-digestion allowed for significantly improved target detection in downstream PCR-based approaches. As ddPCR provides absolute quantification, it ensures excellent day-to-day reproducibility. Therefore, we expect this technique to be used in diagnosing and monitoring of viral and bacterial infection, in detecting mutated DNA sequences as well as profiling for the presence of foreign genetic material in elite athletes in the future.     

Self-fertile transgenic wheat plants regenerated from isolated scutellar tissues following microprojectile bombardment with two distinct gene constructs†

A system for enhanced induction of somatic embryo-genesis and regeneration of plants from isolated scutellar tissue of wheat has been developed. This system has been successfully used in the development of a simple and reproducible protocol for the production of self-fertile transgenic wheat plants. The procedure is rapid resulting in the production of transgenic plantlets within 12 weeks from initiation of cultures and it avoids the need for establishing long-term callus, cell suspension or protoplast cultures. Somatic embryos regenerated from scutella bombarded with plasmid pBARGUS were selected on L-phosphinothricin (L-PPT) to obtain herbicide-resistant self-fertile transgenic plants. Phosphinothricin acetyltransferase (PAT) activity was observed at varying levels in 50% of the plants selected on L-PPT whereas none of the plants showed β-glucuronidase (GUS) activity. Molecular analysis of PAT-positive plants confirmed stable integration of both bar and gus genes in R₀ and R₁ progeny plants. Segregation of the PAT activity and herbicide resistance in R₁ progeny plants confirmed the Mendelian inheritance of the bar gene. Additionally, isolated scutella bombarded with plasmid DNA containing a gus::nptII fusion gene driven by a rice actin promoter and its first intron were selected in the presence of geneticin to obtain fully fertile transgenic plants. Functional expression of the fusion gene was demonstrated in transgenic plants by GUS and neomycin phospho-transferase (NPTII) enzyme assays. Southern blot analysis confirmed the integration of transgenes into the wheat genome. Histochemical GUS staining showed transmission of the fusion gene to floral organs of primary transformants and confirmed Mendelian segregation of the transgene in R₁ progeny.     

Cherry fruit development: The use of a microassay for studying indoleacetic acid oxidase

An improved procedure is described for extraction and assay of indoleacetic acid oxidase from seeds of sour cherry (Prunus cerasus L.). The extraction procedure was optimized for pH, buffer, polyvinylpolypyrrolidone (PVP) and tissue: buffer ratio. Greatest extraction efficiency was obtained at pH 4.0, 0.2 M acetate buffer, tissue: PVP ratio of 1:2.5 and tissue: buffer ratio of 50 ml per g of seed. The enzyme was assayed at 30°C using indoleacetic acid-1-¹⁴C as substrate and radioassaying the ¹⁴CO₂ evolved. Mn²⁺ and 2,4-dichlorophenol enhanced enzyme activity but were not obligatory. A minimum substrate concentration of 60 M was needed for quantitative evaluation. This assay was sensitive and reproducible, enzyme activity being demonstrated in as little as 0.8 mg of seed tissue with a coefficient of variation of 1 to 9%.     

Development of an immunofluorescence technique for detecting <Emphasis Type="Italic">Prorocentrum donghaiense</Emphasis> Lu

Prorocentrum donghaiense Lu is a key harmful algal bloom (HAB) species which is widespread along the China Sea coast. In this study, P. donghaiense-specific antiserum was developed, and the detection method, based on immunofluorescence (IF), was optimized. The antiserum was raised using 0.5% paraformaldehyde-fixed whole cells (WC) as antigen. The titer and the specificity were examined using whole-cell IF. Results showed that ethanol was an effective decolorization reagent, and 80% ethanol was able to minimize autofluorescence of cells. Samples preserved by freezing at −20°C or −80°C remained above 85% detection efficiency after 1-month storage. The antiserum against WC had a high titer (1:10000), and exhibited high specificity at species level. The antiserum showed a weak cross reaction with the closely related species P. dentatum HK, P. dentatum CCMP1517 and P. minimum HK only at very low dilution (1:5). However, it did not cross-react with the species from the same genus or other phytoplankton species when the dilution reached or exceeded 1:100. Results from different P. donghaiense samples collected at different growth phases or grown under different nutrient conditions showed no significant difference in IF intensity. In addition, the antiserum exhibited high specific binding to P. donghaiense in both mixed phytoplankton samples and field samples. The results indicate that the technique is a potentially useful tool for the rapid identification of P. donghaiense and can facilitate the analysis of various natural samples.