Droplet magnetic-controlled microfluidic chip integrated nucleic acid extraction and amplification for the detection of pathogens and tumor mutation sites.

Traditional nucleic acid extraction and detection is based on open operation, which may cause cross-contamination and aerosol formation. This study developed a droplet magnetic-controlled microfluidic chip integrated nucleic acid extraction, purification and amplification. The reagent is sealed in oil to form a droplet, and the nucleic acid is extracted and purified by controlling the movement of the magnetic beads (MBs) through a permanent magnet, ensuring a closed environment. This chip can automatically extract nucleic acid from multiple samples within 20 min, and can be directly placed in the in situ amplification instrument for amplification without further transfer of nucleic acid, characterized by simple, fast, time-saving and labor-saving. The results showed that the chip was able to detect <10 copies/test SARS-CoV-2 RNA, and EGFR exon 21 L858R mutations were detected in H1975 cells as low as 4 cells. In addition, on the basis of the droplet magnetic-controlled microfluidic chip, we further developed a multi-target detection chip, which used MBs to divide the nucleic acid of the sample into three parts. And the macrolides resistance mutations A2063G and A2064G, and the P1 gene of mycoplasma pneumoniae (MP) were successfully detected in clinical samples by the multi-target detection chip, providing the possibility for future application in the detection of multiple pathogens.

Effect of Disulfide Bridge on the Binding of SARS-CoV-2 Fusion Peptide to Cell Membrane: A Coarse-Grained Study.

In this paper, we present the parameterization of the CAVS coarse-grained (CG) force field for 20 amino acids, and our CG simulations show that the CAVS force field could accurately predict the amino acid tendency of the secondary structure. Then, we used the CAVS force field to investigate the binding of a severe acute respiratory syndrome-associated coronavirus fusion peptide (SARS-CoV-2 FP) to a phospholipid bilayer: a long FP (FP-L) containing 40 amino acids and a short FP (FP-S) containing 26 amino acids. Our CAVS CG simulations displayed that the binding affinity of the FP-L to the bilayer is higher than that of the FP-S. We found that the FP-L interacted more strongly with membrane cholesterol than the FP-S, which should be attributed to the stable helical structure of the FP-L at the C-terminus. In addition, we discovered that the FP-S had one major and two minor membrane-bound states, in agreement with previous all-atom molecular dynamics (MD) studies. However, we found that both the C-terminal and N-terminal amino acid residues of the FP-L can strongly interact with the bilayer membrane. Furthermore, we found that the disulfide bond formed between Cys840 and Cys851 stabilized the helices of the FP-L at the C-terminus, enhancing the interaction between the FP-L and the bilayer membrane. Our work indicates that the stable helical structure is crucial for binding the SARS-CoV-2 FP to cell membranes. In particular, the helical stability of FP should have a significant influence on the FP-membrane binding.

Different Binding Modes of SARS-CoV-1 and SARS-CoV-2 Fusion Peptides to Cell Membranes: The Influence of Peptide Helix Length.

Although the amino acid sequences of SARS-CoV-1 and SARS-CoV-2 fusion peptides (FPs) are highly conserved, the cryo-electron microscopy structures of the SARS-CoV-1 and SARS-CoV-2 spike proteins show that the helix length of SARS-CoV-1 FP is longer than that of SARS-CoV-2 FP. In this work, we simulated the membrane-binding models of SARS-CoV-1 and SARS-CoV-2 FPs and compared the binding modes of the FPs with the POPC/POPE/cholesterol bilayer membrane. Our simulation results show that the SARS-CoV-2 FP binds to the bilayer membrane more effectively than the SARS-CoV-1 FP. It is seen that the short N-terminal helix of SARS-CoV-2 FP is more favorable to insert into the target membrane than the long N-terminal helix of SARS-CoV-1 FP. Meanwhile, the potential of mean force calculations showed that the SARS-CoV-2 FP would prefer only one binding mode (N-terminal binding), whereas the SARS-CoV-1 FP has two favorable membrane-binding modes (C-terminal and N-terminal binding modes). Moreover, in the case of SARS-CoV-1 FP binding to the target membrane, the transition between the two binding modes is relatively fast. Finally, we discovered that the membrane-binding mode would influence the helix length of SARS-CoV-1 FP, while the helix length of SARS-CoV-2 FP could be stably maintained in the membrane-bound configurations. This work suggests that the short helix might endow the FP with high membrane-anchoring strength. In particular, the membrane-penetrating residues (Phe, Ile, and Leu) of short α-helix interact with the cell membrane more strongly than those of long α-helix.

Micro-PCR chip-based multifunctional ultrafast SARS-CoV-2 detection platform.

When dealing with infectious pathogens, the point-of-care screening and diagnosis strategy should be low-cost, simple, rapid and accurate. Here, we report a multifunctional rapid PCR platform allowing both simultaneous screening of suspected cases and accurate identification and quantification of the virus. Based on the platform, samples suspected of being infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are screened first, after which subsequent precise quantification of the virus (SARS-CoV-2) can be performed if necessary. This fast screening technique offers a detection limit of 10 nucleic acid copies per test during the entire running time of 15 minutes, with a throughput of 9 samples at a time. Besides, depending on a droplet microfluidic chip, this platform could also provide assays of nucleic acids across four orders of magnitude of concentration within less than 15 minutes. Additionally, we successfully use the platform to quickly distinguish between positive and negative cases in clinical samples and rapidly quantify the viral load in each sample, which is consistent with standard RT-qPCR tests. As such, we demonstrate a promising and versatile rapid PCR platform for point-of-care diagnosis of infectious diseases.

Ultrafast multiplexed detection of SARS-CoV-2 RNA using a rapid droplet digital PCR system.

We report the first combination of droplet digital and rapid PCR techniques for efficient, accurate, and quantitative detection of SARS-CoV-2 RNA. The presented rapid digital PCR system simultaneously detects two specific targets (ORF1ab and N genes) and one reference gene (RNase P) with a single PCR thermal cycling period around 7 s and the total running time less than 5 min. A clear positive signal could be identified within 115 s via the rapid digital RT-PCR, suggesting its efficiency for the end-point detection. In addition, benchmark tests with serial diluted reference samples of SARS-CoV-2 RNA reveal the excellent accuracy of our system (R2>0.99). More importantly, the rapid digital PCR system gives consistent and accurate detection of low-concentration reference samples, whereas qPCR yields Ct values with significant variations that could lead to false-negative results. Finally, we apply the rapid digital PCR system to analyze clinical samples with both positive and control cases, where results are consistent with qPCR test outcomes. By providing similar accuracy with qPCR while minimizing the detection time-consuming and the false-negative tendency, the presented rapid digital PCR system represents a promising improvement on the rapid diagnosis of COVID-19.