Development and Evaluation of Droplet Digital PCR Assay for the Detection of Reticuloendotheliosis Virus in Attenuated Vaccines

Background: The use of Reticuloendotheliosis virus (REV) from contaminated live virus vaccine is suspected to be one of the most important causes of massive outbreaks of Reticuloendotheliosis in China. Methods: In this study, we established a droplet digital PCR (ddPCR) detection method for REV and compared its sensitivity to different methods to detect REV contamination in a vaccine. Results: The results indicated that both quantitative PCR and dot-blot methods could detect REV contamination at a dose of 1 TCID50/1,000 feathers, whereas ddPCR could detect REV contamination at a dose of 0.1 TCID50/1,000 feathers, which is 1,000-fold more sensitive than conventional polymerase chain reaction detection (102 TCID50/1000 feathers). ddPCR not only exhibited the highest sensitivity but also proved extremely intuitive, especially to detect REV contamination in vaccines.Conclusions: The ddPCR method established in this study to detect REV contamination in vaccines can effectively detect and quantify low-dose REV contamination. This provides a new method for the rapid detection of REV contamination in various samples, especially vaccines.

Broad but restricted detection of malacosporeans in a Neotropical cradle of diversification

This study undertook the first investigation of malacosporean infections in Neotropical fish. We used polymerase chain reaction detection with a primer set generally targeting known malacosporeans to assay for infection in the kidney of 146 fish in 21 species belonging to 12 families collected from two areas in the Amazon Basin. Infections were found in 13 fish variously belonging to seven species in six families and included the first identification of a malacosporean infection in cartilaginous fish (a freshwater stingray). Based on ssrDNA, all infections represented a single Buddenbrockia species (Buddenbrockia sp. E) that demonstrates an exceptionally broad range of fish species infected, and countered our expectations of high Neotropical malacosporean diversity. Infections were characterized at varying and often high prevalences in fish species but sample sizes were small. Ascertaining whether highly divergent malacosporeans have not been detected by current primers, and more comprehensive sampling may reveal whether malacosporeans are truly as species poor in the Amazon Basin as present data suggest. Our results prompt speculations about evolutionary scenarios including introduction via marine incursions and patterns of host use over time.

Quantitative Polymerase Chain Reaction Detection of Microchimerism in Female Transplant Renal Recipients

<b><i>Introduction:</i></b> Microchimerism (MC) is the presence of a small amount of foreign cells or DNA within a person’s circulation or tissues. It has been identified also in recipients of solid organ transplants where it seems to be critical for the development and maintenance of immunological tolerance. Nevertheless, natural and/or iatrogenic MC can be acquired prior to transplantation, through pregnancy and/or blood transfusion. <b><i>Objective:</i></b> The aim of this study was to detect the presence of MC in women after renal transplantation from male cadaveric donors and its relationship with graft outcomes. <b><i>Methods:</i></b> We studied by qPCR the presence of the DYS14 gene sequence of the Y chromosome in 12 females who received a kidney graft from a male donor before transplantation (T0), after 15 days (T1) and 1 year of transplantation (T2). We found the sequence in all recipients after renal transplantation. <b><i>Results:</i></b> All the women were negative for this sequence prior to transplantation (T0). Mean (SD) Y-related DNA quantity was 0.80 (0.69) ng/mL plasma and 0.15 (0.26) ng/mL plasma at T1 and T2, respectively. No acute rejection was observed, and mean (SD) estimated Cr clearance was 68.8 (16.9) mL/min within 1 year from transplantation. <b><i>Conclusions:</i></b> Presence of MC was associated with good kidney graft outcomes after 1 year of transplantation, but further studies will be needed to investigate the relationship between clinical outcomes and the development of MC in renal transplant recipient.