A CRISPR-Cas12a-based specific enhancer for more sensitive detection of SARS-CoV-2 infection

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. One Sentence SummaryCRISPR-Cas12a-based COVID-19 diagnosis.

Sensitivity evaluation of 2019 novel coronavirus (SARS-CoV-2) RT-PCR detection kits and strategy to reduce false negative

An ongoing outbreak of pneumonia associated with SARS-CoV-2 has now been confirmed globally. In absence of effective vaccines, infection prevention and control through diagnostic testing and quarantine is critical. Early detection and differential diagnosis of respiratory infections increases the chances for successful control of COVID-19 disease. The nucleic acid RT-PCR test is regarded as the current standard for molecular diagnosis with high sensitivity. However, the highest specificity confirmation target ORF1ab gene is considered to be less sensitive than other targets in clinical application. In addition, a large amount of recent evidence indicates that the initial missed diagnosis of asymptomatic patients with SARS-CoV-2 and discharged patients with "re-examination positive" may be due to low viral load, and the ability of rapid mutation of coronavirus also increases the rate of false negative results. We aimed to evaluate the sensitivity of different nucleic acid detection kits so as to make recommendations for the selection of validation kit, and amplify the suspicious result to be reportable positive by means of simple continuous amplification, which is of great significance for the prevention and control of the current epidemic and the discharge criteria of low viral load patients.

Highly pathogenic coronavirus N protein aggravates lung injury by MASP-2-mediated complement over-activation

An excessive immune response contributes to SARS-CoV, MERS-CoV and SARS-CoV-2 pathogenesis and lethality, but the mechanism remains unclear. In this study, the N proteins of SARS-CoV, MERS-CoV and SARS-CoV-2 were found to bind to MASP-2, the key serine protease in the lectin pathway of complement activation, resulting in aberrant complement activation and aggravated inflammatory lung injury. Either blocking the N protein:MASP-2 interaction or suppressing complement activation can significantly alleviate N protein-induced complement hyper-activation and lung injury in vitro and in vivo. Complement hyper-activation was also observed in COVID-19 patients, and a promising suppressive effect was observed when the deteriorating patients were treated with anti-C5a monoclonal antibody. Complement suppression may represent a common therapeutic approach for pneumonia induced by these highly pathogenic coronaviruses. One Sentence SummaryThe lectin pathway of complement activation is a promising target for the treatment of highly pathogenic coronavirus induced pneumonia.

Rapid Detection of SARS-CoV-2 Using Reverse transcription RT-LAMP method

Corona Virus Disease 2019 (COVID-19) is a recently emerged life-threatening disease caused by SARS-CoV-2. Real-time fluorescent PCR (RT-PCR) is the clinical standard for SARS-CoV-2 nucleic acid detection. To detect SARS-CoV-2 early and control the disease spreading on time, a faster and more convenient method for SARS-CoV-2 nucleic acid detecting, RT-LAMP method (reverse transcription loop-mediated isothermal amplification) was developed. RNA reverse transcription and nucleic acid amplification were performed in one step at 63 {degrees}C isothermal conditions, and the results can be obtained within 30 minutes. ORF1ab gene, E gene and N gene were detected at the same time. ORF1ab gene was very specific and N gene was very sensitivity, so they can guarantee both sensitivity and specificity for SARS-CoV-2. The sensitivity of RT-LAMP assay is similar to RT-PCR, and specificity was 99% as detecting 208 clinical specimens. The RT-LAMP assay reported here has the advantages of rapid amplification, simple operation, and easy detection, which is useful for the rapid and reliable clinical diagnosis of SARS-CoV-2.

Genomic sequences analysis of human Norovirus GII.4 variant JN010 in Jinan / 中华实验和临床病毒学杂志

Objective@#To analyse the genomic characteristics of Novirus(NoV) GII.4 genotype JN010 strain isolated form Jinan in 2017.@*Methods@#Seven pairs of primers were designed and used to amplify the JN010 genome. Sequence analyses, alignment and phylogenetic trees of ORF1 and ORF2 genes were performed using the software Lasergene7.1 and MEGA5.2. At the same time the major protein VP1 amino acid mutations were analyzed.@*Results@#The 7 516 bp complete genome sequence of JN010 strain was obtained, the most mutation sites were located in P2 subdomain of VP1. Two substitutions I293N and H373N of VP1 were locate neighboring epitope A, and R297H mutation happened within epitope A and the site A that binding with histo-blood group antigens(HBGAs). The JN010 strain was GII.Pe/GII.4 genotype and genetically closest to the strains found in Osaka of Japan(GenBank accession number LC066046) and the strains in Zhongshan city of Guangdong (GenBank accession number KY407064) respectively according to ORF1 and ORF2 gene homologous and phylogenetic analysis.@*Conclusions@#The NoV GII.4 variant strain JN010 has occurred mutations in the key site of the epitope A and site A that bind with HBGAs, and maybe affect its antigenicity and interaction with HBGAs.

Characteristics of Coxsackievirus A16 VP1 gene in Jinan, 2017 / 中华实验和临床病毒学杂志

Objective@#To analyze the VP1 gene characteristics of Coxsackievirus A16 (CV-A16) isolated in(hand, foot and mouth disease, HFMD) patients of Jinan 2017.@*Methods@#The samples collected from patients with HFMD in Jinan city in 2017 were analyzed and some of the CV-A16 nucleic acid positive samples were choosen with simple random sampling method and pretreated to inoculate RD cells. The whole VP1 gene sequences of CV-A16 stains which had obvious cytopathic effect in RD cells were amplified and sequenced. MEGA5.2 software was used to constructed phylogenetic tree and Lasergene software was used to analysis the homology consistency of nucleotide and amino acid of VP1 gene.@*Results@#All the 10 CV-A16 strains isolated from Jinan were located 3 clusters belonged to B1b genotypes, with a nucleotide similarity of 94.9%~100% and deduced amino acid similarity of 99.3%~100%. The Jinan CV-A16 strains were nearest related with Shanghai strains KX871333(nucleotide similarity: 99.7%), Henan strains KM260134(99.0%), Jiangsu strains KP751580、KR138327(98.7%) and Guangdong strains MH004016(98.5%). Compared with some reference strains from our country, amino acid mutation were identified at positions 14, 59, and 145 in VP1 proteins of Jinan CV-A16 strains.@*Conclusions@#CV-A16 strains B1b genotypes were the strains prevailed in Jinan city, and 3 amino acid substitutions in VP1 proteins were happened in CV-A16 strains isolated from Jinan.