A novel method for high-sensitivity detection of SARS-CoV-2 using dual double-quenched fluorescence probes.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected many people around the world; fast and accurate detection of the virus can help control the spread of the virus. Reverse transcription-polymerase chain reaction (RT-PCR) is the gold standard method for SARS-CoV-2 detection. In this study, we improved the RT-PCR by proposing a novel method using dual double-quenched fluorescence probes. We used the improved probes to detect the plasmid DNA and RNA reference materials of SARS-CoV-2, respectively. The results show that, the background fluorescence intensity reduced by 50%, the fluorescence increment increased to 2.8 folds, and the Ct value significantly reduced by 3 or more, indicating that the detection sensitivity increased at least 8 times. In addition, we demonstrated that the improved probes have well performance in detecting SARS-CoV-2, with the minimum concentration of 6.2 copies/µL. This study will help biological companies develop better products for SARS-CoV-2 and other clinical pathogen infection.

Synthesis of uniform Pickering microspheres doped with quantum dot by microfluidic technology and its application in tumor marker.

Tumor markers play a significant role in early cancer diagnosis, evaluation of the extent of the disease, and monitoring of therapy response. In this study, we described the Pickering emulsion polymerization method to synthesize uniform magnetic/fluorescent microspheres. A Pickering-structure composed of a lot silica nanoparticle closely covered onto the quantum dot-encoded magnetic microbeads is designed and synthesized. The uniform magnetic/fluorescent microspheres were prepared using a microfluidic device and the performance of the microspheres synthesized by the instruments was evaluated by flow cytometry. To avoid fluorescence quenching and intrinsic toxicity, CdSe/ZnS core-shell quantum dot and Fe3O4 nanoparticle were successfully encapsulated into MFM microspheres using the microfluidic technology. Using this structure enables the facile realization of a theoretical 4 × 4 barcoding matrix combining two colors and four fluorescence intensity levels. Then, different optical codes were prepared by simple changing the emission wavelength and the intensity of the quantum dots. The resulting microsphere are combined with flow cytometer using two lasers for decoding of multiplex tumor markers. Moreover, the stability testing of microspheres demonstrated good performance for further application in detection of tumor markers as well. When applied for the high-throughput ultrasensitive detection of three tumor markers (CEA, CA125 and CA199) in a single sample, the detection limits of 0.027 ng/mL for CEA, 1.48 KU/L for CA125 and 1.09 KU/L for CA199 are achieved, which exhibit superior detection performance. Thus, Pickering-structure magnetic/fluorescent microspheres are promising for application in tumor markers.

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity.

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by Sertoli cell junctions at the blood-testis barrier (BTB), which is the tightest tissue barrier in the mammalian body and segregates the seminiferous epithelium into two compartments, a basal and an adluminal. The BTB creates a unique microenvironment for germ cells in meiosis I/II and for the development of postmeiotic spermatids into spermatozoa via spermiogenesis. Here, we describe a reliable assay to monitor BTB integrity of mouse testis in vivo. An intact BTB blocks the diffusion of FITC-conjugated inulin from the basal to the apical compartment of the seminiferous tubules. This technique is suitable for studying gene candidates, viruses, or environmental toxicants that may affect BTB function or integrity, with an easy procedure and a minimal requirement of surgical skills compared to alternative methods.

A germline-specific role for the mTORC2 component Rictor in maintaining spermatogonial differentiation and intercellular adhesion in mouse testis.

STUDY QUESTION: What is the physiological role of Rictor in spermatogenic cells? SUMMARY ANSWER: Germline expression of Rictor regulates spermatogonial differentiation and has an essential role in coordinating germ cells and Sertoli cells in maintaining intact cell-cell adhesion dynamics and cytoskeleton-based architecture in the seminiferous epithelium. WHAT IS KNOWN ALREADY: The mechanistic target of rapamycin (mTOR) resides in its functions as the catalytic subunits of the structurally and functionally distinct mTORC1 and mTORC2 complexes. In the mammalian testis, mTORC1 regulates spermatogonial stem cell self-renewal and differentiation, whereas mTORC2 is required for Sertoli cell function. In contrast to mTORC1, mTORC2 has been much less well studied. Rictor is a distinct component of the mTORC2 complex. STUDY DESIGN, SIZE, DURATION: We investigated the effects of germ cell-specific ablation of Rictor on testicular development by using a mouse model of germline-specific ablation of Rictor. PARTICIPANTS/MATERIALS, SETTING, METHODS: We analyzed the in-vivo functions of Rictor through different methods including histology, immunofluorescent staining, chromosome spreads, blood-testis barrier (BTB) integrity assays and RNA sequencing. MAIN RESULTS AND THE ROLE OF CHANCE: Mutant mice did not show a defect in meiotic synapsis or recombination, but exhibited compromised spermatogonial differentiation potential, disorganized cell-cell junctions, impaired BTB dynamics and defective spermiogenesis. Concomitantly, RNA-seq profiling revealed that many genes involved in adhesion and migration were expressed inappropriately. LARGE SCALE DATA: RNA-seq data are published in the SRA database (PRJNA419273). LIMITATIONS REASONS FOR CAUTION: A detailed analysis of the mechanisms underlying the phenotype needs further investigations. WIDER IMPLICATIONS OF THE FINDINGS: Our work provides previously unidentified in-vivo evidence that germline expression of Rictor plays a role in maintaining spermatogonial differentiation and cell-cell adhesion. These findings are important for understanding the regulation of spermatogenesis and have clinical implications for the effect of mTOR inhibitors on human fertility. STUDY FUNDING AND COMPETING INTEREST(S): This study was supported by National Key R&D Program of China (2016YFA0500902), National Natural Science Foundation of China (31471228 and 31771653), Jiangsu Science Foundation for Distinguished Young Scholars (BK20150047), and Natural Science Foundation of Jiangsu Province (BK20140897, 14KJA180005 and 14KJB310004) to K.Z. The authors declare no competing or financial interests.