Objective:Based on the specific cleavage and non-specific "trans-cleavage" activities of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein(CRISPR/Cas13), we established a visually amplification-free rapid detection technique of 2019-nCoV nucleic acid. This technique is easily processed with a low detection limit and good specificity.Methods:According to the 2019-nCoV gene sequence, specific CRISPR RNAs were screened and designed by bioinformatics analysis, and then synthesized as universal signal-strained RNA transcription targets in vitro to establish and optimize the reaction system. Moreover, the 2019-nCoV pseudoviral nucleic acid was used as a standard substance to evaluate the detection limit. A total of 65 positive samples were collected from various 2019-nCoV variants, while 48 negative samples included other clinically common respiratory pathogens, such as influenza A virus, influenza B virus, human parainfluenza virus, Klebsiella pneumonia, etc. All samples were tested by quantitative PCR (qPCR), digital PCR, and the method established in this study. The sensitivity and specificity of the newly established method were analyzed and evaluated. Results:With the newly established technique, the detection time for 2019-nCoV nucleic acid could be minimized to 6 minutes. In addition, the detection limit was 14 copies/μl when assisted by the displaying instrument, whereas it increased to 28 copies/μl with the naked eye. This technique had a sensitivity and specificity of 98.5% (66/67) and 100% (46/46) respectively, showing no statistically significant difference compared to the gold standard qPCR( P=1). Conclusions:This study has successfully established a CRISPR/Cas13a-based visually rapid detection technique for 2019-nCoV nucleic acid. This technique offers the advantages of a simple process, convenient operation, low environmental operating requirements, a detection limit close to qPCR, and a strong potential for on-site testing applications.

Objective: To understand the epidemiologic features of the rabies in Xishuang banna prefecture of Yunnan province, China in 2008-2017 and the viral molecular-evolution characteristics. Methods: The data of rabies case questionnaire were collected. The brain tissue samples from mad dogs, suspicious sick dogs and human brain tissue, saliva and cerebrospinal fluid samples from rabies patients were collected in Xishuangbanna. Coding region of nucleoprotein and glycoprotein genes were amplified by RT-PCR and sequenced. Homology and phylogenetic analysis were performed using the relevant bioinformatics software. Results: A total of 62 cases of human rabies were occurred in 28 districts of the 3 counties, Xishuangbanna prefecture in 2008-2017. Of them, 37 cases in Jinghong county, 15 in Menghai county and 10 in Mengla county. In which 48 cases were bitten by domestic dogs (77.42%), 11 cases were bitten by wild dogs (17.74%). Rabies case was occurred every year in the past decade. The seasonal incidence was not obvious. The majority of patients were aged from 30 to 59 years-old, with the youngest 1 year-old and the eldest 91 year-old. The male to female ratio was 1.70∶1, most cases were farmers. The nucleotide sequences of nucleoprotein gene of 9 virus strains (7 from Jinghong, 1 from Menghai and 1 from Mengla) were obtained from the samples of dogs and patients. Homology and phylogenetic analyses indicated that the 5 strains belonged to clade China-Ⅰ, 3 clade China-Ⅱ and 1 clade China-Ⅵ. The nucleotide sequences of glycoprotein gene of 5 virus strains (3 from Jinghong, 1 from Menghai and 1 from Mengla) were obtained from these positive samples, and all were clade China-Ⅰ, it is same with nucleoprotein genes analysis result from these 5 virus strains. These China-Ⅰ and China-Ⅱ strains from Xishuangbanna have a closer genetic relationship with same clade strains isolated from Pu’er and other prefectures of Yunnan province as well as Sichuan, Guizhou and Guangxi. The China-Ⅵ strain from Xishuangbanna share high homology and genetic relationship with China-Ⅵ strains isolated from southwestern Yunnan and neighbouring countries such as Myanmar, Laos and Vietnam in recent years. Conclusions In Xishuangbanna, rabies mainly occurred in rural area and domestic dog was the main source of transmission. These RABV clades China-Ⅰ, China-Ⅱ and China-Ⅵ were found in this region and the China-Ⅰ was principal clade. The transmission source of China-Ⅰ and China-Ⅱ were from adjacent areas in the province and China-Ⅵ was from Myanmar and Laos.