[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
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
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
Classical swine fever virus （CSFV） and porcine reproductive and respiratory syndrome virus （PRRSV） have caused immense economic loss in the pig industry and are considered to be the two most important infectious d...Classical swine fever virus （CSFV） and porcine reproductive and respiratory syndrome virus （PRRSV） have caused immense economic loss in the pig industry and are considered to be the two most important infectious diseases of pigs in the world A multiplex reverse transcription polymerase chain reaction （multiplex RT-PCR） was developed for CSFV and PRRSV co-infections or infections, respectively. A set of two pairs of primer was designed based on the sequence of nonstructural protein NS54B of CSFV and ORF7 gene of PRRSV. The diagnostic accuracy of multiplex RT-PCR assay was evaluated by using 56 field clinical samples by multiplex RT-PCR, single RT-PCR and sequence analysis; and the specificity of multiplex PCR was verified by using constructed plasmids containing the specific viral target fragments of PRRSV and CSFV, respectively. The results indicated that this assay could reliably differentiate PRRSV and CSFV in co-infection samples. The multiplex RT-PCR developed in this study might provide a new avenue to the rapid the detection of CSFV and PRRSV in one reaction.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 identify waterborne enteric viruses in Korean surface water. Methods Integrated cell culture（ICC）multiplex reverse transcription-polymerase chain reaction （RT-PCR） was simultaneously designed to detec...Objective To identify waterborne enteric viruses in Korean surface water. Methods Integrated cell culture（ICC）multiplex reverse transcription-polymerase chain reaction （RT-PCR） was simultaneously designed to detect coxsackieviruses （CV）, polioviruses （PV）, and reoviruses （RV）. ICC-multiplex RT-PCR and phylogenetic analysis were conducted using 21 total culturable virus assay （TCVA）-positive sample-inoculated cell cultures. Results CV and RV were detected in 9 samples each, and 3 samples were positive for both CV and RV. PV was not detected in any sample. Molecular phylogenetic analysis of the VP1 gene sequences revealed that CV types B2 and B4 predominated in Korean surface water, and the nucleotide sequences of CV type B2 were clustered with those of CVs isolated from China and Japan. The results suggested that the evolution of these viruses occurred in a region-specific manner. Conclusion CV and RV are detectable in Korean surface water, with a predominance of CV type B2, and the evolution of CV type B2 occur in a region-specific manner.展开更多
AIM: To develop a multiplex reverse transcription polymerase chain reaction (RT-PCR) method detecting cir-culating tumor cells in the peripheral blood of colorectal cancer (CRC) patients. METHODS: Peripheral blood sam...AIM: To develop a multiplex reverse transcription polymerase chain reaction (RT-PCR) method detecting cir-culating tumor cells in the peripheral blood of colorectal cancer (CRC) patients. METHODS: Peripheral blood samples were collected from 88 CRC patients and 40 healthy individuals from the blood donors' clinic and subsequently analyzed by multiplex RT-RCR for the expression of carcinoembryonic antigen (CEA), cytokeratin 20 (CK20) and epidermal growth factor receptor (EGFR) mRNA. The analysis involved determining the detection rates of CEA, CK20 and EGFR transcripts vs disease stage and overall survival. Median follow-up period was 19 mo (range 8-28 mo). RESULTS: Rates of CEA, CK20 and EGFR detection in CRC patients were 95.5%, 78.4% and 19.3%, respectively. CEA transcripts were detected in 3 healthy volunteer samples (7.5%), whereas all control samples were tested negative for CK20 and EGFR transcripts. The increasing number of positive detections for CEA, CK20 and EGFR transcripts in each blood sample was positively correlated with Astler-Coller disease stage (P< 0.001) and preoperative serum levels of CEA (P=0.029) in CRC patients. Data analysis using Kaplan-Meier estimator documented signif icant differences in the overall survival of the different CRC patient groups as formed according to the increasing number of positivity for CEA, CK20 and EGFR transcripts. CONCLUSION: These data suggest that multiplex RTPCR assay can provide useful information concerning disease stage and overall survival of CRC patients.展开更多
The specific primers were designed according to conserved sequences of coat protein （CP） gene of Freesia mosaic virus （FreMV）, Cucumber mosaic vi- rus （CMV） and Bean yellow mosaic virus （BYMV） published in Gen...The specific primers were designed according to conserved sequences of coat protein （CP） gene of Freesia mosaic virus （FreMV）, Cucumber mosaic vi- rus （CMV） and Bean yellow mosaic virus （BYMV） published in GenBank, and a multiplex PCR protocol for simultaneous detection of these three viruses in freesia was developed. Three specific fragments were simultaneously amplified in a single PCR reaction. Their lengths were determined to be 340,628 and 212 bp, respec-tively. The sequence analysis indicated that three viruses shared at least 97% of homology with reference sequence. Sensitivity test showed that these three viruses could be detected out in the infected plant tissue greater than 10^-2 mg.展开更多
The aim of this study was to detect the expression of 4 clinically-important efflux pumps in the Resistance-Nodulation-Cell Division (RND) family including MexAB-OprM, MexXY, MexCD-OprJ and MexEF-OprN in Pseudomonas a...The aim of this study was to detect the expression of 4 clinically-important efflux pumps in the Resistance-Nodulation-Cell Division (RND) family including MexAB-OprM, MexXY, MexCD-OprJ and MexEF-OprN in Pseudomonas aeruginosa using a combination of resistance-phenotypic markers and multiplex RT-PCR (mRT-PCR). The antibiotic substrates specific for each Mex systems were used as phenotypic markers including carbenicillin, MexAB-OprM, erythromycin, MexCD-OprJ, norfloxacin and imipenem, MexEF-OprN and gentamicin, MexXY-OprM. The methods were validated with reference strains with known genotypes of the Mex systems and the potential applicability in clinical practice was tested with clinical isolates. The results for the reference strains support that the combination of resistance phenotype and mRT-PCR is a potential-attractive method for diagnosis of efflux-mediated resistance in P. aeruginosa. Further development to make it more practical for clinical use and study in a larger number of clinical isolates is required.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: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
Arboviral encephalitis is a group of animal and human illness that is mostly caused by several distinct families of viruses including orthobunya virus, phlebovirus, flaviviruses, and the alphaviruses. Although specifi...Arboviral encephalitis is a group of animal and human illness that is mostly caused by several distinct families of viruses including orthobunya virus, phlebovirus, flaviviruses, and the alphaviruses. Although specific signs and symptoms vary by the type of central nervous system (CNS), initial signs and symptoms are very similar. Therefore rapid immunologic and molecular tools for differential diagnosis of arboviral encephalitis viruses are important for effective case management and control of the spread of encephalitis. The qRT-PCR assay, especially multiplex PCR, has the potential to produce considerable savings in time and resources in the laboratory detection. Meanwhile, the use of IC can prevent false negatives effectively by monitoring the processes of nucleic acid extraction and amplification. This report describes the development of a panel of internally controlled multiplex one-step real-time RT-PCR assays in which two virus specific-probe sets were used in the same reaction for the detection of 15 species arboviral encephalitis viruses: the comparative sensitivity of multiplex one-step qRT-PCR assays to single plex one-step qRT-PCR assays as well as one-step RT-PCR assays for detection of each viral species. And total of 150 human serum samples were detected to evaluate the multiplex one-step qRT-PCR assays. These multiplex one-step real-time RT-PCR assays with IC were evaluated in terms of sensitivity, linearity, precision, specificity, and also field samples including serum and vector. These assays can detect and differentiate arboviral encephalitis viruses by high throughput, sensitive, and specific way. It is useful for clinical management and outbreak control of arboviral encephalitis viruses and vector surveillance.展开更多
Advancements in mode-division multiplexing(MDM)techniques,aimed at surpassing the Shannon limit and augmenting transmission capacity,have garnered significant attention in optical fiber communica-tion,propelling the d...Advancements in mode-division multiplexing(MDM)techniques,aimed at surpassing the Shannon limit and augmenting transmission capacity,have garnered significant attention in optical fiber communica-tion,propelling the demand for high-quality multiplexers and demultiplexers.However,the criteria for ideal-mode multiplexers/demultiplexers,such as performance,scalability,compatibility,and ultra-compactness,have only partially been achieved using conventional bulky devices(e.g.,waveguides,grat-ings,and free space optics)—an issue that will substantially restrict the application of MDM techniques.Here,we present a neuro-meta-router(NMR)optimized through deep learning that achieves spatial multi-mode division and supports multi-channel communication,potentially offering scalability,com-patibility,and ultra-compactness.An MDM communication system based on an NMR is theoretically designed and experimentally demonstrated to enable simultaneous and independent multi-dataset transmission,showcasing a capacity of up to 100 gigabits per second(Gbps)and a symbol error rate down to the order of 104,all achieved without any compensation technologies or correlation devices.Our work presents a paradigm that merges metasurfaces,fiber communications,and deep learning,with potential applications in intelligent metasurface-aided optical interconnection,as well as all-optical pat-tern recognition and classification.展开更多
Multiple quantum well(MQW) Ⅲ-nitride diodes that can simultaneously emit and detect light feature an overlapping region between their electroluminescence and responsivity spectra, which allows them to be simultaneous...Multiple quantum well(MQW) Ⅲ-nitride diodes that can simultaneously emit and detect light feature an overlapping region between their electroluminescence and responsivity spectra, which allows them to be simultaneously used as both a transmitter and a receiver in a wireless light communication system. Here, we demonstrate a mobile light communication system using a time-division multiplexing(TDM) scheme to achieve bidirectional data transmission via the same optical channel.Two identical blue MQW diodes are defined by software as a transmitter or a receiver. To address the light alignment issue, an image identification module integrated with a gimbal stabilizer is used to automatically detect the locations of moving targets;thus, underwater audio communication is realized via a mobile blue-light TDM communication mode. This approach not only uses a single link but also integrates mobile nodes in a practical network.展开更多
Introduction: Arbovirus diseases such as dengue and chikungunya threaten public health worldwide. Early and rapid diagnosis and surveillance of dengue virus (DENV) and chikungunya virus (CHIKV) infections are essentia...Introduction: Arbovirus diseases such as dengue and chikungunya threaten public health worldwide. Early and rapid diagnosis and surveillance of dengue virus (DENV) and chikungunya virus (CHIKV) infections are essential to the control of these diseases. In this study, we evaluate the diagnostic performance of our new in-house multiplex RT-qPCR method for detecting DENV serotypes and CHIKV in an external laboratory. Methodology: The evaluation study was conducted on 200 clinical samples of suspected patients for arbovirus disease infection, collected in Centre de Recherche Biomoléculaire Pietro Annigoni (CERBA), Ouagadougou, Burkina Faso. Our new multiplex RT-qPCR was compared to the commercial kit, the Zika, Dengue, and Chikungunya (ZDC) Real-Time PCR Assays kit (Bio-Rad, California, USA). Results and Conclusions: Among 200 samples, 21.5% (43/200) were DENV-positive by multiplex RT-qPCR, and 21.5% (43/200) were also DENV-positive by reference real-time RT-PCR. 157 (78.5%) samples tested negative for DENV by both tests (new mRT-qPCR and reference test). The sensitivity and specificity of mRT-qPCR were 100%. The DENV serotypes detected were DENV-1 60.5% (26/43) and DENV-3 39.5% (17/43). CHIKV was not detected in this study. Our new mRT-qPCR is sensitive, cost-effective, simple, and can be used in developing country laboratories.展开更多
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 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.
文摘Classical swine fever virus （CSFV） and porcine reproductive and respiratory syndrome virus （PRRSV） have caused immense economic loss in the pig industry and are considered to be the two most important infectious diseases of pigs in the world A multiplex reverse transcription polymerase chain reaction （multiplex RT-PCR） was developed for CSFV and PRRSV co-infections or infections, respectively. A set of two pairs of primer was designed based on the sequence of nonstructural protein NS54B of CSFV and ORF7 gene of PRRSV. The diagnostic accuracy of multiplex RT-PCR assay was evaluated by using 56 field clinical samples by multiplex RT-PCR, single RT-PCR and sequence analysis; and the specificity of multiplex PCR was verified by using constructed plasmids containing the specific viral target fragments of PRRSV and CSFV, respectively. The results indicated that this assay could reliably differentiate PRRSV and CSFV in co-infection samples. The multiplex RT-PCR developed in this study might provide a new avenue to the rapid the detection of CSFV and PRRSV in one reaction.
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.
文摘Objective To identify waterborne enteric viruses in Korean surface water. Methods Integrated cell culture（ICC）multiplex reverse transcription-polymerase chain reaction （RT-PCR） was simultaneously designed to detect coxsackieviruses （CV）, polioviruses （PV）, and reoviruses （RV）. ICC-multiplex RT-PCR and phylogenetic analysis were conducted using 21 total culturable virus assay （TCVA）-positive sample-inoculated cell cultures. Results CV and RV were detected in 9 samples each, and 3 samples were positive for both CV and RV. PV was not detected in any sample. Molecular phylogenetic analysis of the VP1 gene sequences revealed that CV types B2 and B4 predominated in Korean surface water, and the nucleotide sequences of CV type B2 were clustered with those of CVs isolated from China and Japan. The results suggested that the evolution of these viruses occurred in a region-specific manner. Conclusion CV and RV are detectable in Korean surface water, with a predominance of CV type B2, and the evolution of CV type B2 occur in a region-specific manner.
基金Supported by The Ministry of Development of the Greek Government (GGET-AKMON)
文摘AIM: To develop a multiplex reverse transcription polymerase chain reaction (RT-PCR) method detecting cir-culating tumor cells in the peripheral blood of colorectal cancer (CRC) patients. METHODS: Peripheral blood samples were collected from 88 CRC patients and 40 healthy individuals from the blood donors' clinic and subsequently analyzed by multiplex RT-RCR for the expression of carcinoembryonic antigen (CEA), cytokeratin 20 (CK20) and epidermal growth factor receptor (EGFR) mRNA. The analysis involved determining the detection rates of CEA, CK20 and EGFR transcripts vs disease stage and overall survival. Median follow-up period was 19 mo (range 8-28 mo). RESULTS: Rates of CEA, CK20 and EGFR detection in CRC patients were 95.5%, 78.4% and 19.3%, respectively. CEA transcripts were detected in 3 healthy volunteer samples (7.5%), whereas all control samples were tested negative for CK20 and EGFR transcripts. The increasing number of positive detections for CEA, CK20 and EGFR transcripts in each blood sample was positively correlated with Astler-Coller disease stage (P< 0.001) and preoperative serum levels of CEA (P=0.029) in CRC patients. Data analysis using Kaplan-Meier estimator documented signif icant differences in the overall survival of the different CRC patient groups as formed according to the increasing number of positivity for CEA, CK20 and EGFR transcripts. CONCLUSION: These data suggest that multiplex RTPCR assay can provide useful information concerning disease stage and overall survival of CRC patients.
基金Supported by Major Projects of Science and Technology Plan of Fujian Province (2010NZ0003)Finance Special Project of Fujian Province "Project of Science and Technology Innovation Team Building of Fujian Academy of Agriculture" (CXTD2011-20)
文摘The specific primers were designed according to conserved sequences of coat protein （CP） gene of Freesia mosaic virus （FreMV）, Cucumber mosaic vi- rus （CMV） and Bean yellow mosaic virus （BYMV） published in GenBank, and a multiplex PCR protocol for simultaneous detection of these three viruses in freesia was developed. Three specific fragments were simultaneously amplified in a single PCR reaction. Their lengths were determined to be 340,628 and 212 bp, respec-tively. The sequence analysis indicated that three viruses shared at least 97% of homology with reference sequence. Sensitivity test showed that these three viruses could be detected out in the infected plant tissue greater than 10^-2 mg.
文摘The aim of this study was to detect the expression of 4 clinically-important efflux pumps in the Resistance-Nodulation-Cell Division (RND) family including MexAB-OprM, MexXY, MexCD-OprJ and MexEF-OprN in Pseudomonas aeruginosa using a combination of resistance-phenotypic markers and multiplex RT-PCR (mRT-PCR). The antibiotic substrates specific for each Mex systems were used as phenotypic markers including carbenicillin, MexAB-OprM, erythromycin, MexCD-OprJ, norfloxacin and imipenem, MexEF-OprN and gentamicin, MexXY-OprM. The methods were validated with reference strains with known genotypes of the Mex systems and the potential applicability in clinical practice was tested with clinical isolates. The results for the reference strains support that the combination of resistance phenotype and mRT-PCR is a potential-attractive method for diagnosis of efflux-mediated resistance in P. aeruginosa. Further development to make it more practical for clinical use and study in a larger number of clinical isolates is required.
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 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.
文摘Arboviral encephalitis is a group of animal and human illness that is mostly caused by several distinct families of viruses including orthobunya virus, phlebovirus, flaviviruses, and the alphaviruses. Although specific signs and symptoms vary by the type of central nervous system (CNS), initial signs and symptoms are very similar. Therefore rapid immunologic and molecular tools for differential diagnosis of arboviral encephalitis viruses are important for effective case management and control of the spread of encephalitis. The qRT-PCR assay, especially multiplex PCR, has the potential to produce considerable savings in time and resources in the laboratory detection. Meanwhile, the use of IC can prevent false negatives effectively by monitoring the processes of nucleic acid extraction and amplification. This report describes the development of a panel of internally controlled multiplex one-step real-time RT-PCR assays in which two virus specific-probe sets were used in the same reaction for the detection of 15 species arboviral encephalitis viruses: the comparative sensitivity of multiplex one-step qRT-PCR assays to single plex one-step qRT-PCR assays as well as one-step RT-PCR assays for detection of each viral species. And total of 150 human serum samples were detected to evaluate the multiplex one-step qRT-PCR assays. These multiplex one-step real-time RT-PCR assays with IC were evaluated in terms of sensitivity, linearity, precision, specificity, and also field samples including serum and vector. These assays can detect and differentiate arboviral encephalitis viruses by high throughput, sensitive, and specific way. It is useful for clinical management and outbreak control of arboviral encephalitis viruses and vector surveillance.
基金supported by the National Key Research and Development Program of China(2023YFB2804704)the National Natural Science Foundation of China(12174292,12374278,and 62105250).
文摘Advancements in mode-division multiplexing(MDM)techniques,aimed at surpassing the Shannon limit and augmenting transmission capacity,have garnered significant attention in optical fiber communica-tion,propelling the demand for high-quality multiplexers and demultiplexers.However,the criteria for ideal-mode multiplexers/demultiplexers,such as performance,scalability,compatibility,and ultra-compactness,have only partially been achieved using conventional bulky devices(e.g.,waveguides,grat-ings,and free space optics)—an issue that will substantially restrict the application of MDM techniques.Here,we present a neuro-meta-router(NMR)optimized through deep learning that achieves spatial multi-mode division and supports multi-channel communication,potentially offering scalability,com-patibility,and ultra-compactness.An MDM communication system based on an NMR is theoretically designed and experimentally demonstrated to enable simultaneous and independent multi-dataset transmission,showcasing a capacity of up to 100 gigabits per second(Gbps)and a symbol error rate down to the order of 104,all achieved without any compensation technologies or correlation devices.Our work presents a paradigm that merges metasurfaces,fiber communications,and deep learning,with potential applications in intelligent metasurface-aided optical interconnection,as well as all-optical pat-tern recognition and classification.
基金jointly supported by the National Natural Science Foundation of China (U21A20495)Natural Science Foundation of Jiangsu Province (BG2024023)+1 种基金National Key Research and Development Program of China (2022YFE0112000)111 Project (D17018)。
文摘Multiple quantum well(MQW) Ⅲ-nitride diodes that can simultaneously emit and detect light feature an overlapping region between their electroluminescence and responsivity spectra, which allows them to be simultaneously used as both a transmitter and a receiver in a wireless light communication system. Here, we demonstrate a mobile light communication system using a time-division multiplexing(TDM) scheme to achieve bidirectional data transmission via the same optical channel.Two identical blue MQW diodes are defined by software as a transmitter or a receiver. To address the light alignment issue, an image identification module integrated with a gimbal stabilizer is used to automatically detect the locations of moving targets;thus, underwater audio communication is realized via a mobile blue-light TDM communication mode. This approach not only uses a single link but also integrates mobile nodes in a practical network.
文摘Introduction: Arbovirus diseases such as dengue and chikungunya threaten public health worldwide. Early and rapid diagnosis and surveillance of dengue virus (DENV) and chikungunya virus (CHIKV) infections are essential to the control of these diseases. In this study, we evaluate the diagnostic performance of our new in-house multiplex RT-qPCR method for detecting DENV serotypes and CHIKV in an external laboratory. Methodology: The evaluation study was conducted on 200 clinical samples of suspected patients for arbovirus disease infection, collected in Centre de Recherche Biomoléculaire Pietro Annigoni (CERBA), Ouagadougou, Burkina Faso. Our new multiplex RT-qPCR was compared to the commercial kit, the Zika, Dengue, and Chikungunya (ZDC) Real-Time PCR Assays kit (Bio-Rad, California, USA). Results and Conclusions: Among 200 samples, 21.5% (43/200) were DENV-positive by multiplex RT-qPCR, and 21.5% (43/200) were also DENV-positive by reference real-time RT-PCR. 157 (78.5%) samples tested negative for DENV by both tests (new mRT-qPCR and reference test). The sensitivity and specificity of mRT-qPCR were 100%. The DENV serotypes detected were DENV-1 60.5% (26/43) and DENV-3 39.5% (17/43). CHIKV was not detected in this study. Our new mRT-qPCR is sensitive, cost-effective, simple, and can be used in developing country laboratories.
