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
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.
