Canine parvovirus type 2(CPV-2) infection is the most lethal disease of dogs with higher mortality in puppies worldwide.In today’s world,dogs are an integral part of our communities as well as dogs breeding and reari...Canine parvovirus type 2(CPV-2) infection is the most lethal disease of dogs with higher mortality in puppies worldwide.In today’s world,dogs are an integral part of our communities as well as dogs breeding and rearing has become a lucrative business.Therefore,a fast,accurate,portable,and costeffective CPV-2 detection method with the ability for on-site detection is highly desired.In this study,we for the first time proposed a nanosystem for CPV-2 DNA detection with RNA-guided RNA endonuclease Cas13 a,which upon activation results in collateral RNA degradation.We expressed LwCasl3 a in prokaryotic expression system and purified it through nickel column.Activity of Cas13 a was verified by RNA-bound fluorescent group while using a quenched fluorescent probe as signals.Further Cas13 a was combined with Recombinase polymerase amplification(RPA) and T7 transcription to establish molecular detection system termed specific high-sensitivity enzymatic reporter un-locking(SHERLOCK) for sensitive detection of CPV-2 DNA.This nanosystem can detect 100 amol/L CPV-2 DNA within 30 min.The proposed nanosystem exhibited high specificity when tested for CPV-2 and other dog viruses.This CRISPR-Cas13 a mediated sensitive detection approach can be of formidable advantage during CPV-2 outbreaks because it is time-efficient,less laborious and does not involve the use of sophisticated instruments.展开更多
【目的】建立一种基于成簇的规律间隔短回文重复序列及其相关蛋白13a(clustered regularly interspaced short palindromic repeats/associated protein 13a,CRISPR/Cas13a),针对马尔堡病毒核酸的快速检测方法。【方法】根据马尔堡病毒...【目的】建立一种基于成簇的规律间隔短回文重复序列及其相关蛋白13a(clustered regularly interspaced short palindromic repeats/associated protein 13a,CRISPR/Cas13a),针对马尔堡病毒核酸的快速检测方法。【方法】根据马尔堡病毒核蛋白(nucleoprotein,NP)基因保守区序列设计合成特异性的逆转录酶重组酶介导链替换核酸扩增(reverse transcription recombinase aided amplification,RT-RAA)引物及CRISPR RNA(crRNA),利用RT-RAA等温扩增技术对靶序列进行扩增,通过CRISPR/Cas13a系统检测扩增产物,并结合消线法(easy-readout and sensitive enhanced,ERASE)侧流层析试纸进行结果判读。最后利用国家标准品对建立的新方法的灵敏度和特异性进行评估。【结果】筛选出一组针对马尔堡病毒NP基因的高效扩增引物和crRNA,建立了用于马尔堡病毒检测的CRISPR-ERASE方法,可在1 h内检测出浓度为1 copy/μL目标核酸,并与其他多种病原体无交叉反应。【结论】本研究基于CRISPR/Cas13a建立了一种快速、简便、高灵敏度和高特异性的马尔堡病毒核酸检测方法。展开更多
基金supported by the National Key Research and Development Program of China (No.2017YFA0205301)National Natural Science Foundation of China (Nos.81902153,61527806 and 81430055)+2 种基金Key Research and Development Project of Jiangsu Province (No.BE2019761)Programs for Changjiang Scholars and Innovative Research Team in University (No.IRT_15R13)open Funding of State Key Laboratory of Oral Diseases (No.SKLOD20190F03)
文摘Canine parvovirus type 2(CPV-2) infection is the most lethal disease of dogs with higher mortality in puppies worldwide.In today’s world,dogs are an integral part of our communities as well as dogs breeding and rearing has become a lucrative business.Therefore,a fast,accurate,portable,and costeffective CPV-2 detection method with the ability for on-site detection is highly desired.In this study,we for the first time proposed a nanosystem for CPV-2 DNA detection with RNA-guided RNA endonuclease Cas13 a,which upon activation results in collateral RNA degradation.We expressed LwCasl3 a in prokaryotic expression system and purified it through nickel column.Activity of Cas13 a was verified by RNA-bound fluorescent group while using a quenched fluorescent probe as signals.Further Cas13 a was combined with Recombinase polymerase amplification(RPA) and T7 transcription to establish molecular detection system termed specific high-sensitivity enzymatic reporter un-locking(SHERLOCK) for sensitive detection of CPV-2 DNA.This nanosystem can detect 100 amol/L CPV-2 DNA within 30 min.The proposed nanosystem exhibited high specificity when tested for CPV-2 and other dog viruses.This CRISPR-Cas13 a mediated sensitive detection approach can be of formidable advantage during CPV-2 outbreaks because it is time-efficient,less laborious and does not involve the use of sophisticated instruments.
