Search

Phytophthora hibernalis, P. syringaeand P. cambivoraare devastating quarantine pathogens to many economically important plants worldwide. They often co-infect the same plant, causing rots and leading to great loss in yield and even to the death of the plant. Conventionally, the detection of the three pathogens has been based on time-consuming and laborious morphological methods and, although PCR-based detection is much better, it is still thermal cycler- and electrophoresis-dependent. A high throughput, rapid, convenient and cost-effective detection is required for quarantine detection at the port of entry. In this study, based on the novel LAMP technique, three sets of four LAMP primers, PH-F3/PH-B3/PH-FIP/PH-BIP, PS-F3/PS-B3/PS-FIP/PS-BIP and PC-F3/PC-B3/PC-FIP/PC-BIP, designed on the sequences of enolase (Enol), internal transcribed spacers (ITS) and ras-like protein Ypt1genes of P. hibernalis, P. syringaeand P. cambivora, were designed. Through the optimization of the reaction system and conditions, a highly specific and extremely sensitive LAMP detection system was developed for P. hibernalis, P. syringaeand P. cambivora. The LAMP products showed the typical DNA ladders upon agarose gel electrophoresis, the visual turbidity of the reaction solution and the white precipitate of byproduct magnesium pyrophosphate upon centrifugation were observed; the LAMP solution turned light green from orange upon the addition of SYBR Green I. No cross reactions with the other closely related Phytophthoraspecies were found, whereas specificity and sensitivity were very high, for the detection of 0.02 fg of genomic DNA was more than 105times higher than the corresponding conventional PCR. To the best of our knowledge, this represents the first detection of the three quarantine Phytophthorapathogens by LAMP, a technique with a great potential for the identification of other Phytophthorapathogens.

In order to develop a simultaneous and specific molecular detection of the three quarantine Phytophthorapathogens, Phytophthora hibernalis, Phytophthora cambivoraand Phytophthora syringaethat infect Malus Miller, three pairs of real-time PCR primers (PH-F/PH-R, PC-F/PC-R and PS-F/PS-R) and three probes (PH-Pr, PC-Pr and PS-Pr) labeled with HEX, FAM and ROX, respectively, were designed for P. hibernalis, P. cambivoraand P. syringaeby alignment analyses of enolase (Enol), ras-like protein Ypt1and HSP90gene sequences with other Phytophthoraspp. Black Hole Quencher 1 (BHQ1) was used for P. hibernalisand P. cambivoraand BHQ2 for P. syringae. Through the optimization of the reaction conditions, a triplex real-time PCR simultaneous detection for the three Phytophthoraspecies infecting Malus Millerwas developed. It could achieve simultaneous and specific detection with a sensitivity of 2 × 10−4, 2 × 10−4and 2 × 10−2ng/μL genomic DNA, respectively, for P. hibernalis, P. cambivoraand P. syringae.

Duchenne's muscular dystrophy (DMD) is a lethal muscle disease caused by a lack of dystrophin expression at the sarcolemma of muscle fibers. We investigated retroviral vector delivery of dystrophin in dystrophin-deficient DMDmdx (hereafter referred to as mdx) mice via an ex vivo approach using mdx muscle-derived stem cells (MDSCs). We generated a retrovirus carrying a functional human mini-dystrophin (RetroDys3999) and used it to stably transduce mdx MDSCs obtained by the preplate technique (MD3999). These MD3999 cells expressed dystrophin and continued to express stem cell markers, including CD34 and Sca-1. MD3999 cells injected into mdx mouse skeletal muscle were able to deliver dystrophin. Though a relatively low number of dystrophin-positive myofibers was generated within the gastrocnemius muscle, these fibers persisted for up to 24 weeks postinjection. The injection of cells from additional MDSC/Dys3999 clones into mdx skeletal muscle resulted in varying numbers of dystrophin-positive myofibers, suggesting a differential regenerating capacity among the clones. At 2 and 4 weeks postinjection, the infiltration of CD4- and CD8-positive lymphocytes and a variety of cytokines was detected within the injected site. These data suggest that the transplantation of retrovirally transduced mdx MDSCs can enable persistent dystrophin restoration in mdx skeletal muscle; however, the differential regenerating capacity observed among the MDSC/Dys3999 clones and the postinjection immune response are potential challenges facing this technology.

Efficient adenoviral gene delivery to mature skeletal muscle has been hindered by different factors. The low levels of adenoviral attachment receptor (CAR) that have been reported in this tissue may be a limiting factor. Therefore, adenoviral transduction of mature muscle may be improved by extending the tropism of the adenoviral vectors to attachment receptors that are highly expressed in mature myofibers. In this study, we have investigated whether an extended tropism adenoviral vector which additionally attaches to the broadly expressed heparan-containing receptors (AdPK) can bypass the maturation-dependent adenoviral transducibility of mouse skeletal muscle. The adenoviral vector AdPK carrying the LacZ gene was evaluated as a gene delivery vehicle in mouse skeletal muscle at different maturities in vitro and in vivo. The viral transduction efficiencies were determined by histochemical and ONPG analysis of the β-galactosidase activity level. Higher transduction efficiencies were detected in immature muscle from normal mice, and in mature muscle from merosin-deficient dy/dy mice (carrying myofibers with an impaired extracellular matrix) and dystrophin-deficient mdx mice (showing a high level of myoblast activity) when compared to mature muscle from normal mice. Despite the enhanced attachment characteristics, the extended tropism adenoviral vector is, similarly to the wild-type adenoviral vector in previous studies, still hindered by both a protective extracellular matrix and the diminished myoblast-mediation in mature muscle. Copyright © 1999 John Wiley & Sons, Ltd.