University of Maryland Biotechnology Institute

About: University of Maryland Biotechnology Institute is a based out in . It is known for research contribution in the topics: Gene & Population. The organization has 1565 authors who have published 2458 publications receiving 171434 citations. The organization is also known as: UMBI.

Species-specificity and sensitivity of a PCR-based assay for Perkinsus marinus in the eastern oyster, Crassostrea virginica: a comparison with the fluid thioglycollate assay.

Abstract: We examined the species-specificity and sensitivity of a polymerase chain reaction (PCR)-based assay for Perkinsus marinus and compared its overall performance with the fluid thioglycollate medium (FTM) assay on oyster (Crassostrea virginica) hemolymph, mantle, and rectum samples. Our results indicated that the PCR-based methodology is species-specific because Perkinsus olseni, Perkinsus atlanticus, and Perkinsus spp. DNAs were not amplified with the PCR primers developed for P. marinus diagnosis. The sensitivity of the PCR method, as assessed through spike/recovery experiments, was established by the detection of as few as 1 cell of P. marinus in 30 mg of oyster tissue. Tissue samples from naturally infected oysters analyzed both by the FTM and PCR assay suggested that the latter was more sensitive for the diagnosis of P. marinus. Positive results for P. marinus infection ranged from 70% to 83% by FTM and from 92% to 100% by PCR, depending on the tissue examined. Therefore, species-specificity and sensitivity of the NTS-based PCR assay validate its use as a tool for assessment of P. marinus in mollusks.

Expression of green fluorescent protein in insect larvae and its application for heterologous protein production.

Abstract: Many eukaryotic proteins have been successfully expressed in insect cells infected with a recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus (AcNPV). There are, however, disadvantages with this cell-based system when carried out in suspension cultures at high bioreactor volume (e.g., limited oxygen transfer, susceptibility to contamination, high cost). These problems can be avoided by using whole larvae as the "reactors." There are, however, other problems encountered with larvae, one being their inaccessibility for product sampling. To combat this problem, we have investigated the expression of green fluorescent protein (GFP) as a reporter molecule in Trichoplusia ni insect larvae. A high production level of GFPuv (1.58 mg per larva, 26% of total protein) was obtained, enabling the rapid and non-invasive monitoring of GFP. Bright green light was emitted directly from the large opaque carcasses ( approximately 30mm) after illumination with UV light. Based on the green light intensity and a correlation between intensity and GFP mass, we determined the optimal harvest time (c.a. approximately 3 days post-infection). In parallel experiments, we expressed human interleukin-2 (IL-2) from another recombinant baculovirus with an almost identical expression profile. Since both GFP and IL-2 were rapidly degraded by protease activity during the fourth day post-infection (another disadvantage with larvae), we found an accurate determination of harvest time was critical. Correspondingly, our results demonstrated that GFP was an effective on-line marker for expression of heterologous protein in insect larvae.