Background Real-time reverse transcription-PCR (rRT-PCR) has been the most effective and widely implemented diagnostic technology since the beginning of the COVID-19 pandemic. However, fuzzy rRT-PCR readouts with high Ct values are frequently encountered, resulting in uncertainty in diagnosis. Methods A Specific Enhancer for PCR-amplified Nucleic Acid (SENA) was developed based on the Cas12a trans -cleavage activity, which is specifically triggered by the rRT-PCR amplicons of the SARS-CoV-2 Orf1ab ( O ) and N fragments. SENA was first characterized to determine its sensitivity and specificity, using a systematic titration experiment with pure SARS-CoV-2 RNA standards, and was then verified in several hospitals, employing a couple of commercial rRT-PCR kits and testing various clinical specimens under different scenarios. Findings The ratio (10 min/5 min) of fluorescence change (FC) with mixed SENA reaction (mix- FCratio ) was defined for quantitative analysis of target O and N genes, and the Limit of Detection (LoD) of mix- FCratio with 95% confidence interval was 1.2≤1.6≤2.1. Totally, 295 clinical specimens were analyzed, among which 21 uncertain rRT-PCR cases as well as 4 false negative and 2 false positive samples were characterized by SENA and further verified by next-generation sequencing (NGS). The cut-off values for mix- FCratio were determined as 1.145 for positive and 1.068 for negative. Interpretation SENA increases both the sensitivity and the specificity of rRT-PCR, solving the uncertainty problem in COVID-19 diagnosis and thus providing a simple and low-cost companion diagnosis for combating the pandemic. Funding Detailed funding information is available at the end of the manuscript.

Background: Congenital ectopia lentis (EL) refers to the congenital dysplasia or weakness of the lens suspensory ligament, resulting in an abnormal position of the crystalline lens, which can appear as isolated EL or as an ocular manifestation of a syndrome, such as the Marfan syndrome. The fibrillin-1 protein encoded by the FBN1 gene is an essential component of the lens zonules. Mutations in FBN1 are the leading causes of congenital EL and Marfan syndrome. Owing to the complexity and individual heterogeneity of FBN1 gene mutations, the correlation between FBN1 mutation characteristics and various clinical phenotypes remains unclear.Methods: This study describes the clinical characteristics and identifies possible causative genes in eight families with Marfan syndrome or isolated EL using Sanger and whole-exome sequencing.Results: Eight FBN1 mutations were identified in these families, of which three (c.5065G > C, c.1600 T > A, and c.2210G > C) are reported for the first time. Based on in silico analyses, we hypothesized that these mutations may be pathogenic by affecting the fibrillin-1 protein structure and function. Conclusion:These findings expand the number of known mutations involved in EL and provide a reference for the research on their genotype and phenotype associations.

High Ct-values falling in the grey zone are frequently encountered in SARS-CoV-2 detection by real-time reverse transcription PCR (rRT-PCR) and have brought urgent challenges in diagnosis of samples with low viral load. Based on the single-stranded DNA reporter trans-cleavage activity by Cas12a upon target DNA recognition, we create a Specific Enhancer for detection of PCR-amplified Nucleic Acids (SENA) to confirm SARS-CoV-2 detection through specifically targeting its rRT-PCR amplicons. SENA is highly sensitive, with its limit of detection being at least 2 copies/reaction lower than that of the corresponding rRT-PCR, and highly specific, which identifies both false-negative and false-positive cases in clinic applications. SENA provides effective confirmation for nucleic acid amplification-based molecular diagnosis, and may immediately eliminate the uncertainty problems of rRT-PCR in SARS-CoV-2 clinic detection.

<p>Top 100 genes that have similar expression patterns with TIMP3 in ccRCC by GEPIA2</p>

<p>Supplementary Figure 2 shows the efficiency of endothelial cell depletion in immunomagnetic cell separation, and TIMP3-overexpression in HUVECs suppressed the growth of 786O cancer cells implanted in the subcutaneous tissue of nude mice.</p>