Rapid and Visual Detection of the Emerging Novel Duck Reovirus by Using a Specific and Sensitive Reverse Transcription Recombinase Polymerase Amplification Method

Background Duck spleen necrosis disease (DSND) caused by Novel Duck Reovirus (NDRV), is an emerging infectious disease that causes severely threaten to duck industry. Currently, the popular conventional PCR technique for detecting NDRV is time consuming. So, it is essential to develop a rapid and accurate molecular diagnosis techniques of viral pathogens for the purpose to prevent further disease transmission or outbreaks. Recombinase polymerase Amplification (RPA) is a new generation of simple, rapid and cost-effective molecular diagnosis technology, which has been applied to the molecular detection of various pathogens. Methods In our study, a simple, rapid and reliable detection method was developed target NDRV by an isothermal reverse transcription-recombinase polymerase amplification (RT-RPA). The RT-RPA primers were designed based on the S3 gene of NDRV, and a series of other waterfowl-origin pathogens were detected by RT-RPA. A total of 20 field and experimental infected samples were tested by RT-RPA and compared with the results of conventional RT-PCR and quantitative RT-PCR simultaneously. Results The RT-RPA method proved to be repeatable and could detect as little as 3.48 × 10− 6 ng/µl of the standard plasmid DNA inserted with the viral S3 gene. This was a 10 × higher sensitivity rate than that of conventional RT-PCR. The major advantage of this RT-RPA method is that it could be performed as an isothermal reaction at 37 ℃ and completed within 20 min. In addition, no cross-reactivity was detected with other waterfowl-origin viruses. Also, the amplified products could be visualized faster, without the gel electrophoresis, by adding the SYBR Green I and observing them under an ultraviolet light. Conclusions This newly developed RT-RPA method offers a simple, rapid and accurate for rapid detection of NDRV, which especially useful in on-site facilities and resource-limited areas.

Expression of the PLS3 Gene in Circulating Cells in Patients with Colorectal Cancer

Circulating tumor cells (CTC) are cells in circulating blood that have the antigen and gene features of tumor cells of a specific type. Since they can be potentially used in diagnostics and monitoring of treatment of many tumors, they have been attracting attention of researchers worldwide. Plastin-3 (PL S3) is one of such markers of CTC.The aim of the study was to assess expression of PL S3 in CTC in patients with colorectal cancer, to conduct a statistical analysis and to demonstrate a link between expression of PL S3 and progress of the disease, level of CEA and Ca19-9 markers, gender and age of the patients.Material and methods. A group of 85 patients of the Department of General and Colorectal Surgery of the Medical University in Łódź were enrolled in this study. Circulating tumor cells were isolated from whole blood of patients with colorectal cancer and an analysis of PL S3 gene expression in CTC was conducted. The next step was to conduct a statistical analysis and to demonstrate a link between expression of PL S3 in patients’ CTC and progress of the disease, level of CEA and Ca19-9 markers, gender and age of the patients.Results. PL S3 is a marker which can be potentially used in prediction and monitoring of colorectal cancer. A link between expression of PL S3 in CTC of patients with colorectal cancer and metastasis to lymph nodes has been demonstrated. It may be of key importance how PL S3 could impact the qualification to supplementary cancer treatment in patients with stage II colorectal cancer. A link between expression of PL S3 gene in CTC and gender requires further in-depth studies. It is beyond doubt that PL S3 must be further investigated to determine its role in diagnostics, prediction, treatment and monitoring of treatment of colorectal cancer