Rapid detection of goose megrivirus using TaqMan real-time PCR technology.

The aim of this study was to develop an efficient and accurate platform for the detection of the newly identified goose megrivirus (GoMV). To achieve this goal, we developed a TaqMan real-time PCR technology for the rapid detection and identification of GoMV. Our data showed that the established TaqMan real-time PCR assay had high sensitivity, with the lowest detection limit of 67.3 copies/µL. No positive signal can be observed from other goose origin viruses (including AIV, GPV, GoCV, GHPyV, and GoAstV), with strong specificity. The coefficients of variation of repeated intragroup and intergroup tests were all less than 1.5%, with excellent repeatability. Clinical sample investigation data from domestic Minbei White geese firstly provided evidence that GoMV can be transmitted both horizontally and vertically. In conclusion, since the TaqMan real-time PCR method has high sensitivity, specificity, and reproducibility, it can be a useful candidate tool for GoMV epidemiological investigation.

Simultaneous detection and differentiation of DuCV-1 and DuCV-2 by high-resolution melting analysis.

Birds infected with duck circovirus (DuCV) can potentially cause immunosuppression by damaging lymphoid tissues, causing great losses in the duck breeding industry. Duck circovirus can be divided into two genotypes (DuCV-1 and DuCV-2), but simultaneous detection and differentiation of DuCV-1 and DuCV-2 by high-resolution melting (HRM) analysis is still lacking. Here, we designed specific primers according to the sequence characteristics of the newly identified ORF3 gene and then established a PCR-HRM method for the simultaneous detection and differentiation of DuCV-1 and DuCV-2 via high-resolution melting analysis. Our data showed that the established PCR-HRM assay had the advantages of specificity, with the lowest detection limits of 61.9 copies/µL (for DuCV-1) and 60.6 copies/µL (for DuCV-2). The melting curve of the PCR-HRM results indicated that the amplification product was specific, with no cross-reaction with common waterfowl origin pathogens and a low coefficient of variation less than 1.50% in both intra-batch and inter-batch repetitions, indicating the advantages of repeatability. We found that the percentage of DuCV-2-positive ducks was higher than that of DuCV-1-positive ducks, with 8.62% rate of DuCV-1 and DuCV-2 coinfection. In addition, we found DuCV-2-positive in geese firstly. In conclusion, this study provides a candidate PCR-HRM assay for the detection and accurate differentiation of DuCV-1 and DuCV-2 infection, which will help us for further epidemiological surveillance of DuCVs.

Rapid detection of pigeon Megrivirus using TaqMan real-time PCR technology.

Megriviruses have been identified from fecal samples in wild pigeons in Hong Kong (China) and Hungary. In this study, the genomic sequences of pigeon Megriviruses (PiMeVs) were downloaded from GenBank and compared. Based on the genetic comparison results, a pair of primers and TaqMan probe were designed based on the conserved sequences of the 3C gene (located in the P3 gene coding region), and a TaqMan real-time PCR method (TaqMan-qPCR) was established. The standard curve of the TaqMan-qPCR had an axial intercept of 39.74 and a slope of -3.2475 with a linear correlation (R2) of 1.00 and an efficiency of 103.2%. No cross-amplification signal was found from other pigeon viruses (such as avian influenza virus, pigeon paramyxovirus type I, pigeon torque teno virus, pigeon adenovirus, and pigeon circovirus). The limit of detection concentration was 53.6 copies/µL. The intra- and interassay results were less than 1.0% based on the reproducibility test. Furthermore, field samples investigation by the established TaqMan-qPCR method showed that positive signals can be found from racing pigeon fecal samples and embryos. Thus, our data suggested that this visible TaqMan-qPCR method is sensitive, specific, and reproducible. Moreover, we first confirmed the presence of pigeon Megrivirus infection in racing pigeon embryos, indicating that the virus may be vertically transmitted. This study provides a reference basis for further understanding the epidemiology of PiMeVs.

"Don't eat me/eat me"-combined apoptotic body analogues for efficient targeted therapy of triple-negative breast cancer.

For the purpose of efficient targeted therapies, suppressing phagocytosis by a mononuclear phagocyte system (MPS), enhancing the "active" targeted delivery, and meeting clinical production criteria are extremely critical for engineering strategies of novel drug delivery systems. Herein, we used a chemically-induced membrane blebbing and extrusion combined method to induce triple-negative breast cancer (TNBC) cell apoptosis to secrete apoptotic body analogue (ABA) vesicles on a large scale for therapeutic drug delivery. After optimization, the ABAs have a desirable size, good biocompatibility, and long-term colloidal stability. Furthermore, ABAs present anti-phagocytosis ("don't eat me") and specific homologous targeting ("eat me") capacities because of their inheritance of membrane proteins such as CD47 and cellular adhesion molecules from parent cells. After loading with toxic protein saporin and anti-twist siRNA, ABAs can significantly inhibit the growth and lung metastasis of TNBC in an orthotopic metastasis model due to their reduced clearance of immune organs, long circulation time, and enhanced targeted accumulation at the tumor sites. These results suggest the great potential of ABAs for targeted drug delivery therapy, in particular efficient TNBC treatment.