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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.
RESUMO
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.
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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.
