为了实现对存在传入风险的南非2型(South African type 2, SAT2)口蹄疫病毒的早期发现、精准鉴定和有效预警,本研究基于SAT2型口蹄疫病毒毒株IRAN/2024和ETH/2022完整VP1基因序列,构建了pUC57-IRAN-VP1、pUC57-ETH-VP1质粒;参考SAT2型...为了实现对存在传入风险的南非2型(South African type 2, SAT2)口蹄疫病毒的早期发现、精准鉴定和有效预警,本研究基于SAT2型口蹄疫病毒毒株IRAN/2024和ETH/2022完整VP1基因序列,构建了pUC57-IRAN-VP1、pUC57-ETH-VP1质粒;参考SAT2型口蹄疫病毒VP1基因序列设计并筛选特异性引物,以所构建的质粒为模板,建立了SAT2型口蹄疫病毒特异性RT-PCR检测方法,并开展敏感性试验、特异性试验。敏感性试验结果显示,该方法可以检测质量浓度低至1 pg/mL的质粒DNA。特异性试验结果显示,该方法对伪狂犬病毒、猪繁殖与呼吸综合征病毒、乙型脑炎病毒、猪瘟病毒、1型蓝舌病病毒、牛病毒性腹泻病毒、山羊痘病毒、阿卡斑病毒、流行性出血热病毒等常见病毒的核酸,以及参试的O型和A型口蹄疫病毒(PanAsia、Cathay、Mya98、Ind2001-1、Ind2001-2、AKT-Ⅲ、Sea-97毒株)核酸均无交叉反应。应用该方法对2023年云南边境地区50份牛食道-咽部分泌物样品进行核酸检测,检测结果与RT-qPCR检测结果一致。本研究建立的SAT2型口蹄疫病毒特异性RT-PCR检测方法具有一定的实用性,为口蹄疫疫情防控提供技术支撑。展开更多
本研究以日本黄瓜绿斑驳花叶病毒(kyuri green mottle mosaic virus,KGMMV)为材料,设计1对常规RT-PCR检测引物KGCPN-F/KGCPN-R和1组引物/探针KGM-F/KGM-R/KGM-P,分析测试了2对已发表KGMMV常规RT-PCR检测引物KGCP-F/KGCP-R和KGMP-F/KGMP...本研究以日本黄瓜绿斑驳花叶病毒(kyuri green mottle mosaic virus,KGMMV)为材料,设计1对常规RT-PCR检测引物KGCPN-F/KGCPN-R和1组引物/探针KGM-F/KGM-R/KGM-P,分析测试了2对已发表KGMMV常规RT-PCR检测引物KGCP-F/KGCP-R和KGMP-F/KGMP-R,建立了KGMMV的常规和实时荧光RT-PCR检测方法。结果表明,引物KGCP-F/KGCP-R扩增KGMMV时出现预期大小的条带,扩增黄瓜绿斑驳花叶病毒(cucumber green mottle mosaic virus,CGMMV)时出现非常微弱的条带;引物KGCPN-F/KGCPN-R扩增KGMMV时出现预期大小的条带,扩增小西葫芦绿斑驳花叶病毒(zucchini green mottle mosaic virus,ZGMMV)时出现比预期稍大的条带,通过对PCR产物进行序列测定和分析比对可准确鉴定KGMMV。KGCP-F/KGCP-R和KGCPN-F/KGCPN-R的相对灵敏度分别为10^(-6)和10^(-5)稀释度,适用于KGMMV的常规RT-PCR检测。基于引物探针KGM-F/KGM-R/KGM-P建立的KGMMV实时荧光RT-PCR检测方法能特异性检出KGMMV,相对灵敏度达10^(-7)稀释度,分别比2对常规RT-PCR检测引物高10倍和100倍,适用于瓜类种子中KGMMV的快速检测。展开更多
The implementation of multiple pathogen testing is essential for a rapid response to future outbreaks and for reducing disease transmission.This study introduces a 96-channel microfluidic chip,fabricated through a mol...The implementation of multiple pathogen testing is essential for a rapid response to future outbreaks and for reducing disease transmission.This study introduces a 96-channel microfluidic chip,fabricated through a molding process,which enables the batch detection of pathogens.It explores the rapid lysis and elution processes of pathogens within the microfluidic chips to ensure that nucleic acid extraction,elution,and amplification are completed entirely within the chip.This chip can extract nucleic acids from samples in just 10 min,achieving an extraction efficiency comparable to that of traditional in-tube methods.An oil phase is pre-loaded into the chip to effectively prevent aerosol contamination.This approach allows for the simultaneous detection of 21 common respiratory pathogens,with a detection limit of 10 copies per reaction.Furthermore,applications involving clinical samples demonstrate significant practicality.Compared to many traditional in-tube pathogen detection methods and molecular biology technologies that utilize microfluidic chips,this detection chip not only enables simultaneous detection of multiple pathogens but also demonstrates high sensitivity.展开更多
Rice, a global staple food, is critical for food security. The cultivated Oryza sativa, domesticated from wild O. rufipogon, derives~80%of its 993 identified domestication-related genes from O. rufipogon and 20%from S...Rice, a global staple food, is critical for food security. The cultivated Oryza sativa, domesticated from wild O. rufipogon, derives~80%of its 993 identified domestication-related genes from O. rufipogon and 20%from South/Southeast Asian wild O. nivara(Jing et al., 2023). Genes like An-1, BH4, PROG1,SH4, Rc, Rd, and GS3—which regulate awn length, hull color,til er angle, seed shattering, pericarp color, seed length, and thousand-grain weight, respectively—were selected against during domestication to form modern O. sativa(Yu et al., 2021).However, domestication and yield-focused breeding eliminated wild rice's valuable genes(e.g., for disease resistance, stress tolerance, nutrition), narrowing genetic diversity and impeding efforts to meet growing societal demands.展开更多
[Objective] The research aimed to design primers that are suitable for detecting H5 and H7 subtypes of avian influenza virus （AIV） ; [Method] DNAStar was used to analyze the homology of the sequences of H5 and H7 su...[Objective] The research aimed to design primers that are suitable for detecting H5 and H7 subtypes of avian influenza virus （AIV） ; [Method] DNAStar was used to analyze the homology of the sequences of H5 and H7 subtypes of AIV accessed in GenBank, and design primers（ by Primer Premier 5.0） on high homologous region of these sequences, and then amplified by RT-PCR. [Result] The multiplex RT-PCR amplification, agarose gel electrophoresis and sequencing results showed that the self-designed primers are successful for detecting AIV. [Conclusion] It is feasible to rapidly diagnose AIV through this method.展开更多
Multiplex RT-PCR assays have been widely used tools for detection and differentiation of a panel of respiratory viral pathogens. In this study, we evaluated the Qiagen ResPlex lI V2.0 kit and explored factors influenc...Multiplex RT-PCR assays have been widely used tools for detection and differentiation of a panel of respiratory viral pathogens. In this study, we evaluated the Qiagen ResPlex lI V2.0 kit and explored factors influencing its sensitivity. Nasopharyngeal swab （NPS） specimens were prospectively collected from pediatric inpatients with lower respiratory tract infections at the time of admission in the Shenzhen Children＇s Hospital from May 2009 to April 2010. Total nucleic acids were extracted using the EZ1 system （Qiagen, Germany） and 17 respiratory viruses and genotypes including influenza A virus （FluA）, FluB, parainfluenza virus 1 （PIV1）, PIV2, PIV3, PIV4, respiratory syncytial virus （RSV）, human metapneumovirus （hMPV）, rhinoviruses （RhV）, enteroviruses （EnV）, human bocaviruses （hBoV）, adenoviruses （AdV）, four coronaviruses （229E, OC43, NL63 and HKU1）, and FluA 2009 pandemic H1NI（H1NI-p） were detected and identified by the ResPlex II kit. In parallel, 16 real-time TaqMan quantitative RT-PCR assays were used to quantitatively detect each virus except for RhV. Influenza and parainfluenza viral cultures were also performed. Among the total 438 NPS specimens collected during the study period, one or more viral pathogens were detected in 274 （62.6%） and 201（45.9%） specimens by monoplex TaqMan RT-PCR and multiplex ResPlex, respectively. When results from monoplex PCR or cell culture were used as the reference standard, the multiplex PCR possessed specificities of 92.9-100.0%. The sensitivity of multiplex PCR for PIV3, hMPV, PIV1 and BoV were 73.1%, 70%, 66.7% and 55.6%, respectively, while low sensitivities （11.1%-40.0%） were observed for FluA, EnV, OC43, RSV and H1N1. Among the seven viruses/genotypes detected with higher frequencies, multiplex PCR sensitivities were correlated significantly with viral loads determined by the TaqMan RT-PCR in FluA, H 1N 1-p and RSV （p=0.011-0.000） The Qiagen ResPlex II multiplex RT-PCR kit possesses excellent specificity for simultaneous detection of 17 viral pathogens in NPS specimens in pediatric inpatients at the time of admission. The sensitivity of multiplex RT-PCR was influenced by viral loads, specimen process methods, primer and probe design and amplification condition.展开更多
A multiplex reverse transcriptase-polymerase chain reaction(multiplex RT-PCR) assay was developed and subsequently evaluated for its efficacy in the detection of multiple viral infections simultaneously,in swine.Speci...A multiplex reverse transcriptase-polymerase chain reaction(multiplex RT-PCR) assay was developed and subsequently evaluated for its efficacy in the detection of multiple viral infections simultaneously,in swine.Specific primers for each of the 3 RNA viruses,North American genotype porcine reproductive and respiratory syndrome virus,Japanese encephalitis virus,and swine influenza virus,were used in the testing procedure.The assay was shown to be highly sensitive because it could detect as little as 10-5 ng of each of the respective amplicons in a single sample containing a composite of all 3 viruses.The assay was also effective in detecting one or more of the same viruses in various combinations in specimens,including lymph nodes,lungs,spleens,and tonsils,collected from clinically ill pigs and in spleen specimens collected from aborted pig fetuses.The results from the multiplex RT-PCR were confirmed by virus isolation.The relative efficiency(compared to the efficiency of separate assays for each virus) and apparent sensitivity of the multiplex RT-PCR method show that this method has potential for application in routine molecular diagnostic procedures.展开更多
This study developed a multiplex RT-PCR integrated with luminex technology to rapidly subtype simultaneously multiple influenza viruses. Primers and probes were designed to amplify NS and M genes of influenza A viruse...This study developed a multiplex RT-PCR integrated with luminex technology to rapidly subtype simultaneously multiple influenza viruses. Primers and probes were designed to amplify NS and M genes of influenza A viruses HA gene of ill, H3, H5, HT, H9 subtypes, and NA gene of the N1 and N2 subtypes. Universal super primers were introduced to establish a multiplex RT-PCR （GM RT-PCR）. It included three stages of RT-PCR amplification, and then the RT-PCR products were further tested by LiquiChip probe, combined to give an influenza virus （IV） rapid high throughput subtyping test, designated as GMPLex. The IV GMPLex rapid high throughput subtyping test presents the following features： high throughput, able to determine the subtypes of 9 target genes in H1, H3, H5, H7, H9, N1, and N2 subtypes of the influenza A virus at one time; rapid, completing the influenza subtyping within 6 hours; high specificity, ensured the specificity of the different subtypes by using two nested degenerate primers and one probe, no cross reaction occurring between the subtypes, no non-specific reactions with other pathogens and high sensitivity. When used separately to detect the product of single GM RT-PCR for single H5 or N1 gene, the GMPLex test showed a sensitivity of 10-5（= 280ELDs0） forboth tests and the Luminex qualitative ratio results were 3.08 and 3.12, respectively. When used to detect the product of GM RT-PCR for H5N1 strain at the same time, both showed a sensitivity of 10-4（=2800 ELD50）. The GMPLex rapid high throughput subtyping test can satisfy the needs of influenza rapid testing.展开更多
Objective:To develop diagnostic test for detection chikungunya virus(CHIKV and Dengue virus (DENV) infection.Methods:We have performed a rapid,accurate laboratory confirmative method to simultaneously detect,quantify ...Objective:To develop diagnostic test for detection chikungunya virus(CHIKV and Dengue virus (DENV) infection.Methods:We have performed a rapid,accurate laboratory confirmative method to simultaneously detect,quantify and differentiate CHIKV and DENV infection by single-step multiplex real-time RT-PCR.Results:The assay’s sensitivity was 97.65%,specificity was 92.59% and accuracy was 95.82%when compared to conventional RT-PCR.Additionally,there was no cross-reaction between CHIKV,DENV,Japanese encephalitis virus,hepatitis C,hepatitis A or hepatitis E virus.Conclusions:This rapid and reliable assay provides a means for simultaneous early diagnosis of CHIKV and DENV in a single-step reaction.展开更多
文摘本研究以日本黄瓜绿斑驳花叶病毒(kyuri green mottle mosaic virus,KGMMV)为材料,设计1对常规RT-PCR检测引物KGCPN-F/KGCPN-R和1组引物/探针KGM-F/KGM-R/KGM-P,分析测试了2对已发表KGMMV常规RT-PCR检测引物KGCP-F/KGCP-R和KGMP-F/KGMP-R,建立了KGMMV的常规和实时荧光RT-PCR检测方法。结果表明,引物KGCP-F/KGCP-R扩增KGMMV时出现预期大小的条带,扩增黄瓜绿斑驳花叶病毒(cucumber green mottle mosaic virus,CGMMV)时出现非常微弱的条带;引物KGCPN-F/KGCPN-R扩增KGMMV时出现预期大小的条带,扩增小西葫芦绿斑驳花叶病毒(zucchini green mottle mosaic virus,ZGMMV)时出现比预期稍大的条带,通过对PCR产物进行序列测定和分析比对可准确鉴定KGMMV。KGCP-F/KGCP-R和KGCPN-F/KGCPN-R的相对灵敏度分别为10^(-6)和10^(-5)稀释度,适用于KGMMV的常规RT-PCR检测。基于引物探针KGM-F/KGM-R/KGM-P建立的KGMMV实时荧光RT-PCR检测方法能特异性检出KGMMV,相对灵敏度达10^(-7)稀释度,分别比2对常规RT-PCR检测引物高10倍和100倍,适用于瓜类种子中KGMMV的快速检测。
基金supported by grants from the National Key Research and Development Program of China(Nos.2023YFA0915200,2023YFA0915204)the Equipment Research and Development Projects of the Chinese Academy of Sciences(No.PTYQ2024YZ0010)+3 种基金the Science and Technology Commission of Shanghai Municipality Project(No.XTCX-KJ-2024-038)the Natural Science Foundation of Hebei Province of China(No.H2024206249)the Postdoctoral Fellowship Program of CPSF(No.GZC20232838)Science and Technology Commission of Shanghai Municipality(No.22S31901700).
文摘The implementation of multiple pathogen testing is essential for a rapid response to future outbreaks and for reducing disease transmission.This study introduces a 96-channel microfluidic chip,fabricated through a molding process,which enables the batch detection of pathogens.It explores the rapid lysis and elution processes of pathogens within the microfluidic chips to ensure that nucleic acid extraction,elution,and amplification are completed entirely within the chip.This chip can extract nucleic acids from samples in just 10 min,achieving an extraction efficiency comparable to that of traditional in-tube methods.An oil phase is pre-loaded into the chip to effectively prevent aerosol contamination.This approach allows for the simultaneous detection of 21 common respiratory pathogens,with a detection limit of 10 copies per reaction.Furthermore,applications involving clinical samples demonstrate significant practicality.Compared to many traditional in-tube pathogen detection methods and molecular biology technologies that utilize microfluidic chips,this detection chip not only enables simultaneous detection of multiple pathogens but also demonstrates high sensitivity.
基金supported by the Biological BreedingMajor Projects(2023ZD04076)the National Natural Science Foundation of China(32300312)+2 种基金the Innovation Program of Chinses Academy of Agricultural Sciences(CAAS-CSIAF-202303)the Guangdong Basic and Applied Basic Research Foundation(2020B1515120086)the KeyArea Research and Development Program of Guangdong Province(2021B0707010006)。
文摘Rice, a global staple food, is critical for food security. The cultivated Oryza sativa, domesticated from wild O. rufipogon, derives~80%of its 993 identified domestication-related genes from O. rufipogon and 20%from South/Southeast Asian wild O. nivara(Jing et al., 2023). Genes like An-1, BH4, PROG1,SH4, Rc, Rd, and GS3—which regulate awn length, hull color,til er angle, seed shattering, pericarp color, seed length, and thousand-grain weight, respectively—were selected against during domestication to form modern O. sativa(Yu et al., 2021).However, domestication and yield-focused breeding eliminated wild rice's valuable genes(e.g., for disease resistance, stress tolerance, nutrition), narrowing genetic diversity and impeding efforts to meet growing societal demands.
基金Supported by Important Project of Jinlin Provincial Science and Technology Department(20065020)~~
文摘[Objective] The research aimed to design primers that are suitable for detecting H5 and H7 subtypes of avian influenza virus （AIV） ; [Method] DNAStar was used to analyze the homology of the sequences of H5 and H7 subtypes of AIV accessed in GenBank, and design primers（ by Primer Premier 5.0） on high homologous region of these sequences, and then amplified by RT-PCR. [Result] The multiplex RT-PCR amplification, agarose gel electrophoresis and sequencing results showed that the self-designed primers are successful for detecting AIV. [Conclusion] It is feasible to rapidly diagnose AIV through this method.
基金supported in part by Grant Name awarded to the State Key Lab of Respiratory Diseases,Guangzhou Medical College (2007DA780154F0910)
文摘Multiplex RT-PCR assays have been widely used tools for detection and differentiation of a panel of respiratory viral pathogens. In this study, we evaluated the Qiagen ResPlex lI V2.0 kit and explored factors influencing its sensitivity. Nasopharyngeal swab （NPS） specimens were prospectively collected from pediatric inpatients with lower respiratory tract infections at the time of admission in the Shenzhen Children＇s Hospital from May 2009 to April 2010. Total nucleic acids were extracted using the EZ1 system （Qiagen, Germany） and 17 respiratory viruses and genotypes including influenza A virus （FluA）, FluB, parainfluenza virus 1 （PIV1）, PIV2, PIV3, PIV4, respiratory syncytial virus （RSV）, human metapneumovirus （hMPV）, rhinoviruses （RhV）, enteroviruses （EnV）, human bocaviruses （hBoV）, adenoviruses （AdV）, four coronaviruses （229E, OC43, NL63 and HKU1）, and FluA 2009 pandemic H1NI（H1NI-p） were detected and identified by the ResPlex II kit. In parallel, 16 real-time TaqMan quantitative RT-PCR assays were used to quantitatively detect each virus except for RhV. Influenza and parainfluenza viral cultures were also performed. Among the total 438 NPS specimens collected during the study period, one or more viral pathogens were detected in 274 （62.6%） and 201（45.9%） specimens by monoplex TaqMan RT-PCR and multiplex ResPlex, respectively. When results from monoplex PCR or cell culture were used as the reference standard, the multiplex PCR possessed specificities of 92.9-100.0%. The sensitivity of multiplex PCR for PIV3, hMPV, PIV1 and BoV were 73.1%, 70%, 66.7% and 55.6%, respectively, while low sensitivities （11.1%-40.0%） were observed for FluA, EnV, OC43, RSV and H1N1. Among the seven viruses/genotypes detected with higher frequencies, multiplex PCR sensitivities were correlated significantly with viral loads determined by the TaqMan RT-PCR in FluA, H 1N 1-p and RSV （p=0.011-0.000） The Qiagen ResPlex II multiplex RT-PCR kit possesses excellent specificity for simultaneous detection of 17 viral pathogens in NPS specimens in pediatric inpatients at the time of admission. The sensitivity of multiplex RT-PCR was influenced by viral loads, specimen process methods, primer and probe design and amplification condition.
基金supported by a grant from the Out-standing Person Innovation Foundation of Henan,China(0621002100)
文摘A multiplex reverse transcriptase-polymerase chain reaction(multiplex RT-PCR) assay was developed and subsequently evaluated for its efficacy in the detection of multiple viral infections simultaneously,in swine.Specific primers for each of the 3 RNA viruses,North American genotype porcine reproductive and respiratory syndrome virus,Japanese encephalitis virus,and swine influenza virus,were used in the testing procedure.The assay was shown to be highly sensitive because it could detect as little as 10-5 ng of each of the respective amplicons in a single sample containing a composite of all 3 viruses.The assay was also effective in detecting one or more of the same viruses in various combinations in specimens,including lymph nodes,lungs,spleens,and tonsils,collected from clinically ill pigs and in spleen specimens collected from aborted pig fetuses.The results from the multiplex RT-PCR were confirmed by virus isolation.The relative efficiency(compared to the efficiency of separate assays for each virus) and apparent sensitivity of the multiplex RT-PCR method show that this method has potential for application in routine molecular diagnostic procedures.
基金The Basic Rasearch Project of Shenzhen(JC200903190778A)
文摘This study developed a multiplex RT-PCR integrated with luminex technology to rapidly subtype simultaneously multiple influenza viruses. Primers and probes were designed to amplify NS and M genes of influenza A viruses HA gene of ill, H3, H5, HT, H9 subtypes, and NA gene of the N1 and N2 subtypes. Universal super primers were introduced to establish a multiplex RT-PCR （GM RT-PCR）. It included three stages of RT-PCR amplification, and then the RT-PCR products were further tested by LiquiChip probe, combined to give an influenza virus （IV） rapid high throughput subtyping test, designated as GMPLex. The IV GMPLex rapid high throughput subtyping test presents the following features： high throughput, able to determine the subtypes of 9 target genes in H1, H3, H5, H7, H9, N1, and N2 subtypes of the influenza A virus at one time; rapid, completing the influenza subtyping within 6 hours; high specificity, ensured the specificity of the different subtypes by using two nested degenerate primers and one probe, no cross reaction occurring between the subtypes, no non-specific reactions with other pathogens and high sensitivity. When used separately to detect the product of single GM RT-PCR for single H5 or N1 gene, the GMPLex test showed a sensitivity of 10-5（= 280ELDs0） forboth tests and the Luminex qualitative ratio results were 3.08 and 3.12, respectively. When used to detect the product of GM RT-PCR for H5N1 strain at the same time, both showed a sensitivity of 10-4（=2800 ELD50）. The GMPLex rapid high throughput subtyping test can satisfy the needs of influenza rapid testing.
基金supported by the Center of Excellence in Clinical Virology.Chulalongkorn University,CU Centenary Academic Development ProjectKing Chulalongkorn Memorial Hospital,the National Research University Project of CHEthe Ratchadaphiseksonphot Endowment Fund(HR1155A)
文摘Objective:To develop diagnostic test for detection chikungunya virus(CHIKV and Dengue virus (DENV) infection.Methods:We have performed a rapid,accurate laboratory confirmative method to simultaneously detect,quantify and differentiate CHIKV and DENV infection by single-step multiplex real-time RT-PCR.Results:The assay’s sensitivity was 97.65%,specificity was 92.59% and accuracy was 95.82%when compared to conventional RT-PCR.Additionally,there was no cross-reaction between CHIKV,DENV,Japanese encephalitis virus,hepatitis C,hepatitis A or hepatitis E virus.Conclusions:This rapid and reliable assay provides a means for simultaneous early diagnosis of CHIKV and DENV in a single-step reaction.
