ABSTRACT
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ABSTRACT
Point of care (POC) diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are particularly significant for preventing transmission of coronavirus disease 2019 (COVID-19) by any user at any given time and place. CRISPR/Cas-assisted SARS-CoV-2 assays are viewed as supplemental to RT-PCR due to simple operation, convenient use and low cost. However, most current CRISPR molecular diagnostics based on fluorescence measurement increased the difficulty of POC test with need of the additional light sources. Some instrument-free visual detection with the naked eye has limitations in probe universality. Herein, we developed a universal, rapid, sensitive and specific SARS-CoV-2 POC test that combines the outstanding DNase activity of Cas12a with universal AuNPs strand-displacement probe. The oligo trigger, which is the switch the AuNPs of the strand-displacement probe, is declined as a result of Cas12a recognition and digestion. The amount of released AuNPs produced color change which can be visual with the naked eye and assessed by UV-Vis spectrometer for quantitative detection. Furthermore, a low-cost hand warmer is used as an incubator for the visual assay, enabling an instrument-free, visual SARS-CoV-2 detection within 20 min. A real coronavirus GX/P2V instead of SARS-CoV-2 were chosen for practical application validation. After rapid virus RNA extraction and RT-PCR amplification, a minimum of 2.7 × 102 copies/mL was obtained successfully. The modular design can be applied to many nucleic acid detection applications, such as viruses, bacteria, species, etc., by simply modifying the crRNA, showing great potential in POC diagnosis.
ABSTRACT
The CRISPR/Cas system is widely used for molecular diagnostics after the discovery of trans-cleavage activity, especially now with the COVID-19 outbreak. However, the majority of contemporary trans-cleavage activity-based CRISPR/Cas biosensors exploited standard single-strand DNA (ssDNA) reporters, which were based on the FRET principle from pioneering research. An in-depth comparison and understanding of various fluorescent readout types are essential to facilitate the outstanding analytical performance of CRISPR probes. We investigated various types of fluorescent reporters of Cas12a comprehensively. Results show that trans-cleavage of Cas12a is not limited to ssDNA and dsDNA reporters, but can be extended to molecular beacons (MB). And MB reporters can achieve superior analytical performance compared with ssDNA and ds DNA reporters at the same conditions. Accordingly, we developed a highly-sensitive SARS-CoV-2 detection with the sensitivity as low as 100 fM were successfully achieved without amplification strategy. The model target of ORF1a could robustly identify the current widespread emerging SARS-CoV-2 variants. A real coronavirus GX/P2V instead of SARS-CoV-2 were chosen for practical application validation. And a minimum of 27 copies/mL was achieved successfully. This inspiration can also be applied to other Cas proteins with trans-cleavage activity, which provides new perspectives for simple, highly-sensitive and universal molecular diagnosis in various applications.
ABSTRACT
The emergence of a new type of COVID-19 patients, who were retested positive after hospital discharge with long-term persistent SARS-CoV-2 infection but without COVID-19 clinical symptoms (hereinafter, LTPPs), poses novel challenges to COVID-19 treatment and prevention. Why was there such a contradictory phenomenon in LTPPs? To explore the mechanism underlying this phenomenon, we performed quantitative proteomic analyses using the sera of 12 LTPPs (Wuhan Pulmonary Hospital), with the longest carrying history of 132 days, and mainly focused on 7 LTPPs without hypertension (LTPPs-NH). The results showed differential serum protein profiles between LTPPs/LTPPs-NH and health controls. Further analysis identified 174 differentially-expressed-proteins (DEPs) for LTPPs, and 165 DEPs for LTPPs-NH, most of which were shared. GO and KEGG analyses for these DEPs revealed significant enrichment of "coagulation" and "immune response" in both LTPPs and LTPPs-NH. A unity of contradictory genotypes in the 2 aspects were then observed: some DEPs showed the same dysregulated expressed trend as that previously reported for patients in the acute phase of COVID-19, which might be caused by long-term stimulation of persistent SARS-CoV-2 infection in LTPPs, further preventing them from complete elimination; in contrast, some DEPs showed the opposite expression trend in expression, so as to retain control of COVID-19 clinical symptoms in LTPPs. Overall, the contrary effects of these DEPs worked together to maintain the balance of LTPPs, further endowing their contradictory steady-state with long-term persistent SARS-CoV-2 infection but without symptoms. Additionally, our study revealed some potential therapeutic targets of COVID-19. Further studies on these are warranted. IMPORTANCE This study reported a new type of COVID-19 patients and explored the underlying molecular mechanism by quantitative proteomic analyses. DEPs were significantly enriched in "coagulation" and "immune response". Importantly, we identified 7 "coagulation system"- and 9 "immune response"-related DEPs, the expression levels of which were consistent with those previously reported for patients in the acute phase of COVID-19, which appeared to play a role in avoiding the complete elimination of SARS-CoV-2 in LTPPs. On the contrary, 6 "coagulation system"- and 5 "immune response"-related DEPs showed the opposite trend in expression. The 11 inconsistent serum proteins seem to play a key role in the fight against long-term persistent SARS-CoV-2 infection, further retaining control of COVID-19 clinical symptom of LTPPs. The 26 proteins can serve as potential therapeutic targets and are thus valuable for the treatment of LTPPs; further studies on them are warranted.
ABSTRACT
Persistent infection and prolonged shedding of human bocavirus 1 (HBoV1) in children have been reported, and the role of HBoV1 as a sole causative pathogen in acute respiratory infection (ARI) is yet to be established. While the reported prevalence of HBoV infection varies due to different detection methods and sampling criteria, determining the viral and bacterial etiology of HBoV infection using multiplex real-time PCR is yet to be reported. Herein, we aimed to further explore the pathogenicity of HBoV in patients with ARI by screening the viral and bacterial infections in children with ARI in Qingdao and comparing the epidemiological, clinical characteristics, and etiological results. Human bocavirus was identified in 28.1% of the samples, and further sequencing analysis of the detected HBoV confirmed 96.4% as HBoV1. The rate of HBoV as a single viral infection was 75%, and the rate of coinfection with bacteria was 66.1%, suggesting the need for continued monitoring of HBoV in children with ARIs. Clinical characterization suggested that HBoV infection may affect the function of organs, such as the liver, kidney, and heart, and the blood acid-base balance. Additionally, it is essential to promote awareness about the importance of disinfection and sterilization of the hospital environment and standardizing operations. The interactions between HBoV and other pathogens remain to be investigated in further detail in the future.