In order to realize simultaneous quantitative detection of various enteroviruses from water samples, a real-time reverse transcriptionpolymerase chain reaction （real-time RT-PCR） method was developed with universal ...In order to realize simultaneous quantitative detection of various enteroviruses from water samples, a real-time reverse transcriptionpolymerase chain reaction （real-time RT-PCR） method was developed with universal primer pairs designed based on the highly conserved non-coding region sequences of genome targeting poliovirus, coxsackievirus and enterovirus 71. The recombinant plasmid was constructed as enterovirus DNA standard by cloning poliovirus cDNA into a pMD18-T vector. The real-time RT-PCR method utilizing SYBR Green I was optimized. As a result of a series of examinations, the detection limit of the method was found to be 2.31 genome equivalent copy （GEC）/μL, the intraand inter-assay variations were lower than 2% and 5%, respectively, and enteroviruses were well distinguished from other microorganisms. There was a good linear relationship （r 2 = 0.997） between the logarithm of viral density and cycle threshold in a wide range of 2.31 × 10 0 to 2.31 × 10 9 GEC/μL. The validity of the method was further proved by its application for the detection of enteroviruses from various practical water samples.展开更多
Influenza viruses are major respiratory pathogens known to infect human and a variety of animals and are widely prevalent worldwide.Genome structure of influenza D virus(IDV)is identical to that of influenza C virus(I...Influenza viruses are major respiratory pathogens known to infect human and a variety of animals and are widely prevalent worldwide.Genome structure of influenza D virus(IDV)is identical to that of influenza C virus(ICV),and phylogenetic analyses suggest that IDV and ICV share a common ancestry and high homology.To date,the prevalence of ICV and IDV in China is unclear,but these viruses represent a potential threat to public health due to cross-species transmission and zoonotic potential.To efficiently monitor ICV and IDV,it is necessary to establish a dual detection method to understand their prevalence and conduct in-depth research.A duplex real-time PCR method for the simultaneous detection of ICV and IDV was developed.TaqMan fluorescent probes and specific primers targeting NP gene of ICV and PB1 gene of IDV were designed.This method exhibited good specificity and sensitivity,and the detection limit reached 1 × 10^(1) copies/pL of plasmid standards of each pathogen.Thirty-one clinical swine samples and 10 clinical cattle samples were analyzed using this method.One positive sample of IDV was detected,and the accuracy of clinical test results was verified by conventional PCR and DNA sequencing.The duplex real-time PCR detection method represents a sensitive and specific tool to detect IG/and IDV,It provides technical support for virus research and clinical diagnosis of ICV and IDV.This information will benefit animal and human health.展开更多
Mycoplasma hyopneumoniae is an important pathogen causing Mycoplasmal pneumonia of swine, which generally causes secondary infections and mixed infections, thus seriously threats the development of swine industry and ...Mycoplasma hyopneumoniae is an important pathogen causing Mycoplasmal pneumonia of swine, which generally causes secondary infections and mixed infections, thus seriously threats the development of swine industry and resulting in huge economic losses. Using PCR technology has very important significance to the correct diagnosis of Mycoplasmal pneumonia at the early stage. In this paper, specific target genes of Mycoplasma hyopneumoniae, methods for clinical sample collection, key technical factors of DNA sample processing method, and the research progress, main advantages and disadvantages, and application of general PCR technology, multiple PCR technology, nested-PCR technology, real-time fluorescence quantitative PCR technology, gene chip detection technology and loop-mediated isothermal amplification in detection of Mycoplasma hyopneumoniae were summarized, which provided convenience for the effective diagnosis and prevention of Mycoplasmal pneumonia of swine.展开更多
We developed one species-specific PCR assays for rapid and accurate detection of the pathogenic fungi Verticillium albo-atrum in diseased plant tissues and soil. Based on differences in internal transcribed spacer （...We developed one species-specific PCR assays for rapid and accurate detection of the pathogenic fungi Verticillium albo-atrum in diseased plant tissues and soil. Based on differences in internal transcribed spacer （ITS） sequences of Verticillium spp., a pair of species-specific primers, Vaal/Vaa2, was synthesized. After screening 17 isolates of V. alboatrum, 121 isolates from the Ascomycota, B asidiomycota, Deuteromycota, and Oomycota, the Vaal/Vaa2 primers amplified only a single PCR band of approximately 330 bp from V. albo-atrum. The detection sensitivity with primers Vaal/Vaa2 was 10 fg of genomic DNA. Using ITS1/ITS4 as the first-round primers, combined with Vaa1/Vaa2, the nested PCR procedures were developed, and the detection sensitivity increased 1 000-fold to 10 ag. The detection sensitivity for the soil pathogens was 100-conidiag^-1 soil. The PCR-based methods developed here could simplify both plant disease diagnosis and pathogen monitoring as well as guide plant disease management.展开更多
In recent years,advancements in autonomous vehicle technology have accelerated,promising safer and more efficient transportation systems.However,achieving fully autonomous driving in challenging weather conditions,par...In recent years,advancements in autonomous vehicle technology have accelerated,promising safer and more efficient transportation systems.However,achieving fully autonomous driving in challenging weather conditions,particularly in snowy environments,remains a challenge.Snow-covered roads introduce unpredictable surface conditions,occlusions,and reduced visibility,that require robust and adaptive path detection algorithms.This paper presents an enhanced road detection framework for snowy environments,leveraging Simple Framework forContrastive Learning of Visual Representations(SimCLR)for Self-Supervised pretraining,hyperparameter optimization,and uncertainty-aware object detection to improve the performance of YouOnly Look Once version 8(YOLOv8).Themodel is trained and evaluated on a custom-built dataset collected from snowy roads in Tromsø,Norway,which covers a range of snow textures,illumination conditions,and road geometries.The proposed framework achieves scores in terms of mAP@50 equal to 99%and mAP@50–95 equal to 97%,demonstrating the effectiveness of YOLOv8 for real-time road detection in extreme winter conditions.The findings contribute to the safe and reliable deployment of autonomous vehicles in Arctic environments,enabling robust decision-making in hazardous weather conditions.This research lays the groundwork for more resilient perceptionmodels in self-driving systems,paving the way for the future development of intelligent and adaptive transportation networks.展开更多
Objective Shellfish are recognized as important vehicles of norovirus-associated gastroenteritis. The present study aimed to monitor norovirus contamination in oysters along the farm-to-fork continuum in Guangxi, a ma...Objective Shellfish are recognized as important vehicles of norovirus-associated gastroenteritis. The present study aimed to monitor norovirus contamination in oysters along the farm-to-fork continuum in Guangxi, a major oyster production area in Southwestern China. Methods Oyster samples were collected monthly from farms, markets, and restaurants, from January to December 2016. Norovirus was detected and quantified by one-step reverse transcription-droplet digital polymerase chain reaction（RT-ddPCR）. Results A total of 480 oyster samples were collected and tested for norovirus genogroups I and II. Norovirus was detected in 20.7% of samples, with genogroup II predominating. No significant difference was observed in norovirus prevalence among different sampling sites. The norovirus levels varied widely, with a geometric mean of 19,300 copies/g in digestive glands. Both norovirus prevalence and viral loads showed obvious seasonality, with a strong winter bias. Conclusion This study provides a systematic analysis of norovirus contamination ‘from the farm to the fork＇ in Guangxi. RT-ddPCR can be a useful tool for detection and quantification of low amounts of norovirus in the presence of inhibitors found particularly in foodstuffs. This approach will contribute to the development of strategies for controlling and reducing the risk of human illness resulting from shellfish consumption.展开更多
Detailed analysis of Land Use/Land Cover （LULC） using remote sensing data in complex irrigated basins provides complete profile for better water resource management and planning. Using remote sensing data, this stud...Detailed analysis of Land Use/Land Cover （LULC） using remote sensing data in complex irrigated basins provides complete profile for better water resource management and planning. Using remote sensing data, this study provides detailed land use maps of the Lower Chenab Canal irrigated region of Pakistan from 2005 to 2012 for LULC change detection. Major crop types are demarcated by identifying temporal profiles of NDVI using MODIS 250 m × 250 m spatial resolution data. Wheat and rice are found to be major crops in rabi and kharif seasons, respectively. Accuracy assessment of prepared maps is performed using three dif- ferent techniques： error matrix approach, comparison with ancillary data and with previous study. Producer and user accuracies for each class are calculated along with kappa coeffi- cients （K）. The average overall accuracies for rabi and kharif are 82.83% and 78.21%, re- spectively. Producer and user accuracies for individual class range respectively between 72.5% to 77% and 70.1% to 84.3% for rabi and 76.6% to 90.2% and 72% to 84.7% for kharif. The K values range between 0.66 to 0.77 for rabi with average of 0.73, and from 0.69 to 0.74 with average of 0.71 for kharif. LULC change detection indicates that wheat and rice have less volatility of change in comparison with both rabi and kharif fodders. Transformation be- tween cotton and rice is less common due to their completely different cropping conditions. Results of spatial and temporal LULC distributions and their seasonal variations provide useful insights for establishing realistic LULC scenarios for hydrological studies.展开更多
During the past decade, great efforts have been made to boost the land use trans- formation in the Loess Plateau, especially for reducing soil erosion by vegetation restoration measures. The Grain-for-Green project （...During the past decade, great efforts have been made to boost the land use trans- formation in the Loess Plateau, especially for reducing soil erosion by vegetation restoration measures. The Grain-for-Green project （GFG） is the largest ecological rehabilitation program in China, which has a positive impact on the vegetation restoration and sustainable devel- opment for the ecologically fragile region of west China. Based on the Landsat TM/ETM im- ages for three time periods （2000, 2005 and 2010）, this study applied the GIS technology and a hill-slope analytical model to reveal the spatio-temporal evolutional patterns of returning slope farmland to grassland or woodland in Baota District, Yan＇an city of Shaanxi province. Results showed that： （1） from 2000 to 2010, the area of farmland decreased by approximately 35,030 ha, which is the greatest decrease among all the land-use types, whereas grassland, woodland and construction land increased, of which grassland expanded rapidly by 26,380 ha （2） The annual variation rate of land-use dynamics was 1.98% during the period 2000-2010, of which the rate was 1.05% for the 2000-2005 period and 2.92% for the 2005-2010 period, respectively. Over the past decade, returning farmland to woodland or pastures was the main source of increased grassland and woodland, and the reduction of farmland contributed to the increase in grassland and woodland by 97.39% and 85.28%, respectively. （3） As the terrain slope increases, farmland decreased and woodland and grassland increased significantly. Areas with a slope ranging from 15° to 25° and less than 15° were the focus of the GFG project, accounting for 85% of the total area of farmland reduction. Meanwhile, the reduction in farmland was significant and spatially correlated with the increase in woodland and grass- land. （4） Between 2000 and 2010, the area of destruction of grass and trees in grasslands and woodlands for the reclamation of farmland was approximately 4596 ha. The area subject to the GFG policy was 4456 ha with a slope greater than 25° over the decade, but the area of farmland was still 10,357 ha in 2010. Our results indicate that there has still a great potential for returning the steep-slope farmlands to woodlands or grasslands in the Loess Plateau.展开更多
Bacterial fruit blotch caused by Acidovorax citrulli is a serious threat to cucurbit industry worldwide.The pathogen is seedtransmitted,so seed detection to prevent distribution of contaminated seed is crucial in dise...Bacterial fruit blotch caused by Acidovorax citrulli is a serious threat to cucurbit industry worldwide.The pathogen is seedtransmitted,so seed detection to prevent distribution of contaminated seed is crucial in disease management.In this study,we adapted a quantitative real-time PCR(qPCR)assay to droplet digital PCR(ddPCR)format for A.citrulli detection by optimizing reaction conditions.The performance of ddPCR in detecting A.citrulli pure culture,DNA,infested watermelon/melon seed and commercial seed samples were compared with multiplex PCR,qPCR,and dilution plating method.The lowest concentrations detected(LCD)by ddPCR reached up to 2 fg DNA,and 102 CFU mL–1 bacterial cells,which were ten times more sensitive than those of the qPCR.When testing artificially infested watermelon and melon seed,0.1%infestation level was detectable using ddPCR and dilution plating method.The 26 positive samples were identified in 201 commercial seed samples through ddPCR,which was the highest positive number among all the methods.High detection sensitivity achieved by ddPCR demonstrated a promising technique for improving seed-transmitted pathogen detection threshold in the future.展开更多
Objective:To study virulence and regulatory genes(hlyA,ctxB,tcpI) in clinical strains of Vibrio ckolerae(V.cholerae),simultaneously.Methods:Three important genes,tepI,hlyA and ctxB were used for detection of toxigenic...Objective:To study virulence and regulatory genes(hlyA,ctxB,tcpI) in clinical strains of Vibrio ckolerae(V.cholerae),simultaneously.Methods:Three important genes,tepI,hlyA and ctxB were used for detection of toxigenic and pathogenic V.cholera by chain reaction assay method. Results:According to the results of the PCR,the incidence of hlyA,tcpI,and ctxB genes in clinical isolates was obtained as 94.7%(72 sample),90.8%(69 sample),and 92.1%(70 sample), respectively.Five strains possessed all genes except ctxB,six strains possessed all genes except tcpI,four strains possessed all genes except hlyA,one strain possessed only hlyA and 60 strains contained a combination of three genes.Including hlyA,ctxB and tcpI,Conclusions:Result show that this method could be reliable to detect toxigenic-pathogenic strains of V.cholerae in Iran.展开更多
Objective This study is aimed to develop a two-tube melting curve-based multiplex real time PCR assay （MCMRT-PCR） for the simultaneous detection of six common foodborne pathogenic bacteria （diarrhoeagenic Escherich...Objective This study is aimed to develop a two-tube melting curve-based multiplex real time PCR assay （MCMRT-PCR） for the simultaneous detection of six common foodborne pathogenic bacteria （diarrhoeagenic Escherichia coli, Salmonella, and 5higella in tube 1, Staphylococcus aureus, Vibrio parahaemolyticus, and Listeria monocytogenes in tube 2）. Methods A two-tube MCMRT-PCR assay was performed on 7900HT Fast Real-Time PCR System {Applied Biosystems, USA）. Amplification by PCR was optimized to obtain high efficiency. The sensitivity and specificity of assays were investigated. Results The detection limit of optimized MCMRT-PCR assay was 3.9x102 CFU/mLfor S. aureus, 4.4x102 CFU/mL for L. monocytogenes, 3.0x102 CFU/mL for Salmonella, 2.5x102 CFU/mL for Shigella, 2.1x102 CFU/mL for V. parahaemolyticus, and 1.2x102 CFU/mL for E. coll. The feasibility of MCMRT-PCR was further evaluated using artificially contaminated milk, the sensitivity was at the level of 10s CFU/mL. Conclusion A two-tube MCMRT-PCR assay using six primer sets was developed for detection of multiple pathogens. Our findings demonstrates that the proposed two-tube assay is reliable, useful and rapid for simultaneous detection of six foodborne pathogenic bacteria with an intended application in provincial Centers for Diseases Control and Prevention （CDC）.展开更多
Edwardsiella tarda is one of the most important emerging pathogens in tile global aquaculture industries. As such, an accurate diagnosis and quantitative analytical methods are urgently needed for this bacterium. In t...Edwardsiella tarda is one of the most important emerging pathogens in tile global aquaculture industries. As such, an accurate diagnosis and quantitative analytical methods are urgently needed for this bacterium. In this study, primers and a TaqMan probe specific to the conservative sequences of the 16S rRNA gene of E. tarda were designed. The concentration of primers and TaqMan probe were optimized to 200 nmol/L and 120 nmol/L, respectively. The detection sensitivity of the FQ- PCR assay was determined to be as low as five copies of the target sequence per reaction using the pGEM-16S rDNA recombinant plasmid as a template, which was 100 times more sensitive than conventional PCR. A standard curve by plotting the threshold cycle values （y） against the common logarithmic copies （logl0n~ as x; n~ is copy number） of pGEM-16S rDNA was generated. The results of intra- and inter-assay variability tests demonstrate that the established FQ-PCR method was highly reproducible. The assay was specific for E. tarda as it showed that there was no cross-reactivity to eight additional bacterial pathogen strains in aquaculture. Thus, the FQ-PCR assay has the potential for diagnostic purposes and for other applications, especially for the rapid detection and quantification of low-grade E. tarda infections.展开更多
Objective To develop an in situ PCR in combination with flow cytometry （ISPCR-FCM） for monitoring cholera toxin positive Vibrio cholerae. Methods In running this method, 4% paraformaldehyde was used to fix the Vibri...Objective To develop an in situ PCR in combination with flow cytometry （ISPCR-FCM） for monitoring cholera toxin positive Vibrio cholerae. Methods In running this method, 4% paraformaldehyde was used to fix the Vibrio cholerae cells and 1 mg/mL lysozyme for 20 min to permeabilize the cells. Before the PCR thermal cycling, 2.5% glycerol was added into the PCR reaction mixture in order to protect the integrality of the cells. Results A length of 1037bp DNA sequence was amplified, which is specific for the cholera toxin gene （ctxAB gene）. Cells subjected to ISPCR showed the presences of ctxAB gene both in epifluorescence microscopy and in flow cytometric analysis. The specificity and sensitivity of the method were investigated. The sensitivity was relatively low （10^5 cells/mL）, while the specificity was high. Conclusion We have successfully developed a new technique for detection of toxigenic Vibrio cholerae strains. Further study is needed to enhance its sensitivities. ISPCR-FCM shows a great promise in monitoring specific bacteria and their physiological states in environmental samples.展开更多
To develop a nested PCR assay for the detection of cattle-derived Cyclospora sp., two pairs of primers were designed on the basis of the cattle-derived Cyclospora sp. sequences. These primers selectively amplified a 1...To develop a nested PCR assay for the detection of cattle-derived Cyclospora sp., two pairs of primers were designed on the basis of the cattle-derived Cyclospora sp. sequences. These primers selectively amplified a 168-bp DNA fragment of the 18S rRNA gene of cattle-derived Cyclospora sp. With these primers, a nested PCR assay for the detection of cattlederived Cyclospora sp. was developed. The nested PCR assay was specific and there is no cross-reaction with other parasites, such as Eimeria spp., Cryptosporidium spp., Giardia sp., Toxoplasma sp., Trichuris sp. and cattle ciliate. The assay was able to detect as low as 2.85 × 10^-2 fg of the control positive DNA. The results of the detection of clinical samples indicated that the assay coincided with microscopic examination. The results show that the nested PCR assay will be an effective tool for the detection of Cyclospora sp. in cattle feces.展开更多
Due to inappropriate planning and management, accelerated urban growth and tremendous loss in land, especially cropland, have become a great challenge for sustainable urban development in China, especially in develope...Due to inappropriate planning and management, accelerated urban growth and tremendous loss in land, especially cropland, have become a great challenge for sustainable urban development in China, especially in developed urban area in the coastal regions; therefore, there is an urgent need to effectively detect and monitor the land use changes and provide accurate and timely information for planning and management. In this study a method combining principal component analysis (PCA) of multisensor satellite images from SPOT (systeme pour l'observation de la terre or earth observation satellite)-5 multispectral (XS) and Landsat-7 enhanced thematic mapper (ETM) panchromatic (PAN) data, and supervised classification was used to detect and analyze the dynamics of land use changes in the city proper of Hangzhou. The overall accuracy of the land use change detection was 90.67% and Kappa index was 0.89. The results indicated that there was a considerable land use change (10.03% of the total area) in the study area from 2001 to 2003, with three major types of land use conversions: from cropland into built-up land, construction site, and water area (fish pond). Changes from orchard land into built-up land were also detected. The method described in this study is feasible and useful for detecting rapid land use change in the urban area.展开更多
A real-time RT-PCR （RT-qPCR） assay for the detection of Tahyna virus was developed to monitor Tahyna virus infection in field-collected vector mosquito samples. The targets selected for the assay were S segment sequ...A real-time RT-PCR （RT-qPCR） assay for the detection of Tahyna virus was developed to monitor Tahyna virus infection in field-collected vector mosquito samples. The targets selected for the assay were S segment sequences encoding the nucleocapsid protein from the Tahyna virus. Primers and probes were selected in conserved regions by aligning genetic sequences from various Tahyna virus strains available from GenBank. The sensitivity of the RT-qPCR approach was compared to that of a standard plaque assay in BHK cells. RT-qPCR assay can detect 4.8 PFU of titrated Tahyna virus. Assay specificities were determined by testing a battery of arboviruses, including representative strains of Tahyna virus and other arthropod-borne viruses from China. Seven strains of Tahyna virus were confirmed as positive; the other seven species of arboviruses could not be detected by RT-qPCR. Additionally, the assay was used to detect Tahyna viral RNA in pooled mosquito samples. The RT-qPCR assay detected Tahyna virus in a sensitive, specific, and rapid manner; these findings support the use of the assay in viral surveillance.展开更多
Objective:To analyse molecular detection of coliforms and shorten the time of PCR.Methods:Rapid detection of coliforms by amplification of lacZ and uidA genes in a multiplex PCR reaction was designed and performed in ...Objective:To analyse molecular detection of coliforms and shorten the time of PCR.Methods:Rapid detection of coliforms by amplification of lacZ and uidA genes in a multiplex PCR reaction was designed and performed in comparison with most probably number(MPN)method for 16 artificial and 101 field samples.The molecular method was also conducted on isolated coliforms from positive MPN samples;standard sample for verification of microbial method certificated reference material;isolated strains from certificated reference material and standard bacteria.The PCR and electrophoresis parameters were changed for reducing the operation time.Results:Results of PCR for lacZ and uidA genes were similar in all of standard,operational and artificial samples and showed the 876 bp and 147 bp bands of lacZ and uidA genes by multiplex PCR.PCR results were confirmed by MPN culture method by sensitivity 86%(95%CI:0.71-0.93).Also the total execution time,with a successful change of factors,was reduced to less than two and a half hour.Conclusions:Multiplex PCR method with shortened operation time was used for the simultaneous detection of total coliforms and Escherichia coli in distribution system of Arak city.It's recommended to be used at least as an initial screening test,and then the positive samples could be randomly tested by MPN.展开更多
Pseudoalteromonas marina is one of the potential pathogens that cause green spot disease(GSD)in Pyropia yezoensis.To prevent GSD from development and spread,an effective method to detect the pathogen at early GSD infe...Pseudoalteromonas marina is one of the potential pathogens that cause green spot disease(GSD)in Pyropia yezoensis.To prevent GSD from development and spread,an effective method to detect the pathogen at early GSD infection stages need to be established.In this research,PCR methods were established targeting the dnaA gene(encoding chromosome replication initiator protein)and the dnaN gene(encodingβsliding clamp of DNA polymeraseⅢprotein)to detect P.marina with three primer pairs pws-dnaA2(Forward,5'-ACCGCATTAACGAACTACTCGTG-3';Reverse,5'-TGCCATTACCTACAGCATGG-3'),pcs-dnaN2(Forward,5'-CTTACAACGTTATCAGCGGC-3';Reverse,5'-GTTGAGTATTAAGTGATTGAGTAAGC-3')or pws-dnaN3(Forward,5'-ACTTACAA-CGTTATCAGCGGC-3';Reverse,5'-ACTGCTGTTTGAGTCTGCTAAC-3').Three PCR methods corresponding to the three primer pairs sufficiently distinguished P.marina from 22 bacterial species,thus resulting in detection limits of 4 to 4×10^2 CFU cells or 2.37×10^1 to 2.37×10^3 fg of P.marina DNA per PCR reaction.In an artificial infection experiment ofP.yezoensis infected with P.marina,all established PCRs successfully detected P.marina at early GSD infection stages.The results show that the established PCRs are specific and sensitive,and are potential for applications in early diagnosis of GSD in Pyropia.展开更多
Edwardsiella tarda has become one of the most important emerging pathogens in aquaculture industry. Therefore, a rapid, reproducible, and sensitive method for detection and quantification of this pathogen is needed ur...Edwardsiella tarda has become one of the most important emerging pathogens in aquaculture industry. Therefore, a rapid, reproducible, and sensitive method for detection and quantification of this pathogen is needed urgently. To achieve this purpose, we developed a TaqMan-based real-time PCR assay for detection and quantification orE. tarda. The assay targets the hemolysin activator HlyB domain protein of E. tarda. Our optimized TaqMan assay is capable of detecting as little as 40 fg of genomic DNA per reaction. A standard curve was generated from the threshold cycle values （y） against log10 （E. tarda genomic DNA concentration） as x. The intra- and inter-assay coefficient of variation （CV） values were less than 2.06% and 1.05% respectively, indicating that the assay had good reproducibility. This method is highly specific to E. tarda strains, as it shows no cross-reactivity to Edwardsiella ictaluri, a member of the same genus, or to nine other fish-pathogenic bacteria species belonging to three other genera. This sensitive and specific real-time PCR assay provides a valuable tool for diagnostic quantitation of E. tarda in clinical samples.展开更多
AIM: To compare the ligase detection reaction (LDR) and real-time PCR for detection of low abundant YMDD mutants in patients with chronic hepatitis B infection.METHODS: Mixtures of plasmids and serum samples from 52 c...AIM: To compare the ligase detection reaction (LDR) and real-time PCR for detection of low abundant YMDD mutants in patients with chronic hepatitis B infection.METHODS: Mixtures of plasmids and serum samples from 52 chronic hepatitis B patients with low abundant lamivudine-resistant mutations were tested with LDR and real-time PCR. Time required and reagent cost for both assays were evaluated.RESULTS: Real-time PCR detected 100, 50, 10, 1 and 0.1% of YIDD plasmid, whereas LDR detected 100, 50, 10, 1, 0.1, and 0.01% of YIDD plasmid, in mixtures with YMDD plasmid of 106 copies/mL. Among the 52 clinical serum samples, completely concordant results were obtained for all samples by both assays, and 39 YIDD, 9 YVDD, and 4 YIDD/YVDD were detected. Cost and time required for LDR and real-time PCR are 60/80 CNY (8/10.7 US dollars) and 4.5/2.5 h, respectively.CONCLUSION: LDR and real-time PCR are both sensitive and inexpensive methods for monitoring low abundant YMDD mutants during lamivudine therapy in patients with chronic hepatitis B. LDR is more sensitive and less expensive, while real-time PCR is more rapid.展开更多
基金supported by the National Natural Sci-ence Foundation of China (No. 50908185)the National Program of Water Pollution Control (No. 2008ZX07317-004)+2 种基金the Basic Research Foundation of Xi’an University of Architecture and Technology (No. JC0910)the Research Foundation for Talented Scholars of Xi’an University of Architecture and Technology (No. RC0824)the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT0853)
文摘In order to realize simultaneous quantitative detection of various enteroviruses from water samples, a real-time reverse transcriptionpolymerase chain reaction （real-time RT-PCR） method was developed with universal primer pairs designed based on the highly conserved non-coding region sequences of genome targeting poliovirus, coxsackievirus and enterovirus 71. The recombinant plasmid was constructed as enterovirus DNA standard by cloning poliovirus cDNA into a pMD18-T vector. The real-time RT-PCR method utilizing SYBR Green I was optimized. As a result of a series of examinations, the detection limit of the method was found to be 2.31 genome equivalent copy （GEC）/μL, the intraand inter-assay variations were lower than 2% and 5%, respectively, and enteroviruses were well distinguished from other microorganisms. There was a good linear relationship （r 2 = 0.997） between the logarithm of viral density and cycle threshold in a wide range of 2.31 × 10 0 to 2.31 × 10 9 GEC/μL. The validity of the method was further proved by its application for the detection of enteroviruses from various practical water samples.
基金This work was financially supported by the National Key Research and Development Program of China(2017YFD0500101)the Fundamental Research Funds for the Central Universities(Y0201900459).
文摘Influenza viruses are major respiratory pathogens known to infect human and a variety of animals and are widely prevalent worldwide.Genome structure of influenza D virus(IDV)is identical to that of influenza C virus(ICV),and phylogenetic analyses suggest that IDV and ICV share a common ancestry and high homology.To date,the prevalence of ICV and IDV in China is unclear,but these viruses represent a potential threat to public health due to cross-species transmission and zoonotic potential.To efficiently monitor ICV and IDV,it is necessary to establish a dual detection method to understand their prevalence and conduct in-depth research.A duplex real-time PCR method for the simultaneous detection of ICV and IDV was developed.TaqMan fluorescent probes and specific primers targeting NP gene of ICV and PB1 gene of IDV were designed.This method exhibited good specificity and sensitivity,and the detection limit reached 1 × 10^(1) copies/pL of plasmid standards of each pathogen.Thirty-one clinical swine samples and 10 clinical cattle samples were analyzed using this method.One positive sample of IDV was detected,and the accuracy of clinical test results was verified by conventional PCR and DNA sequencing.The duplex real-time PCR detection method represents a sensitive and specific tool to detect IG/and IDV,It provides technical support for virus research and clinical diagnosis of ICV and IDV.This information will benefit animal and human health.
基金Supported by National Natural Science Foundation of China(31100135)Agricultural Independent Innovation Fund of Jiangsu Province[CX(11)4038]~~
文摘Mycoplasma hyopneumoniae is an important pathogen causing Mycoplasmal pneumonia of swine, which generally causes secondary infections and mixed infections, thus seriously threats the development of swine industry and resulting in huge economic losses. Using PCR technology has very important significance to the correct diagnosis of Mycoplasmal pneumonia at the early stage. In this paper, specific target genes of Mycoplasma hyopneumoniae, methods for clinical sample collection, key technical factors of DNA sample processing method, and the research progress, main advantages and disadvantages, and application of general PCR technology, multiple PCR technology, nested-PCR technology, real-time fluorescence quantitative PCR technology, gene chip detection technology and loop-mediated isothermal amplification in detection of Mycoplasma hyopneumoniae were summarized, which provided convenience for the effective diagnosis and prevention of Mycoplasmal pneumonia of swine.
基金We thank Dr Chen Qinghe,Dr Ko Wenhong,Dr Ho Hanhin,Dr Hu Baishi,Dr Peng Jinghuo and China General Microbiological Culture Collection Center(CGMCC)for providing some isolates.This work was supported by the China National 863 Program(2003AA249020).
文摘We developed one species-specific PCR assays for rapid and accurate detection of the pathogenic fungi Verticillium albo-atrum in diseased plant tissues and soil. Based on differences in internal transcribed spacer （ITS） sequences of Verticillium spp., a pair of species-specific primers, Vaal/Vaa2, was synthesized. After screening 17 isolates of V. alboatrum, 121 isolates from the Ascomycota, B asidiomycota, Deuteromycota, and Oomycota, the Vaal/Vaa2 primers amplified only a single PCR band of approximately 330 bp from V. albo-atrum. The detection sensitivity with primers Vaal/Vaa2 was 10 fg of genomic DNA. Using ITS1/ITS4 as the first-round primers, combined with Vaa1/Vaa2, the nested PCR procedures were developed, and the detection sensitivity increased 1 000-fold to 10 ag. The detection sensitivity for the soil pathogens was 100-conidiag^-1 soil. The PCR-based methods developed here could simplify both plant disease diagnosis and pathogen monitoring as well as guide plant disease management.
文摘In recent years,advancements in autonomous vehicle technology have accelerated,promising safer and more efficient transportation systems.However,achieving fully autonomous driving in challenging weather conditions,particularly in snowy environments,remains a challenge.Snow-covered roads introduce unpredictable surface conditions,occlusions,and reduced visibility,that require robust and adaptive path detection algorithms.This paper presents an enhanced road detection framework for snowy environments,leveraging Simple Framework forContrastive Learning of Visual Representations(SimCLR)for Self-Supervised pretraining,hyperparameter optimization,and uncertainty-aware object detection to improve the performance of YouOnly Look Once version 8(YOLOv8).Themodel is trained and evaluated on a custom-built dataset collected from snowy roads in Tromsø,Norway,which covers a range of snow textures,illumination conditions,and road geometries.The proposed framework achieves scores in terms of mAP@50 equal to 99%and mAP@50–95 equal to 97%,demonstrating the effectiveness of YOLOv8 for real-time road detection in extreme winter conditions.The findings contribute to the safe and reliable deployment of autonomous vehicles in Arctic environments,enabling robust decision-making in hazardous weather conditions.This research lays the groundwork for more resilient perceptionmodels in self-driving systems,paving the way for the future development of intelligent and adaptive transportation networks.
文摘Objective Shellfish are recognized as important vehicles of norovirus-associated gastroenteritis. The present study aimed to monitor norovirus contamination in oysters along the farm-to-fork continuum in Guangxi, a major oyster production area in Southwestern China. Methods Oyster samples were collected monthly from farms, markets, and restaurants, from January to December 2016. Norovirus was detected and quantified by one-step reverse transcription-droplet digital polymerase chain reaction（RT-ddPCR）. Results A total of 480 oyster samples were collected and tested for norovirus genogroups I and II. Norovirus was detected in 20.7% of samples, with genogroup II predominating. No significant difference was observed in norovirus prevalence among different sampling sites. The norovirus levels varied widely, with a geometric mean of 19,300 copies/g in digestive glands. Both norovirus prevalence and viral loads showed obvious seasonality, with a strong winter bias. Conclusion This study provides a systematic analysis of norovirus contamination ‘from the farm to the fork＇ in Guangxi. RT-ddPCR can be a useful tool for detection and quantification of low amounts of norovirus in the presence of inhibitors found particularly in foodstuffs. This approach will contribute to the development of strategies for controlling and reducing the risk of human illness resulting from shellfish consumption.
文摘Detailed analysis of Land Use/Land Cover （LULC） using remote sensing data in complex irrigated basins provides complete profile for better water resource management and planning. Using remote sensing data, this study provides detailed land use maps of the Lower Chenab Canal irrigated region of Pakistan from 2005 to 2012 for LULC change detection. Major crop types are demarcated by identifying temporal profiles of NDVI using MODIS 250 m × 250 m spatial resolution data. Wheat and rice are found to be major crops in rabi and kharif seasons, respectively. Accuracy assessment of prepared maps is performed using three dif- ferent techniques： error matrix approach, comparison with ancillary data and with previous study. Producer and user accuracies for each class are calculated along with kappa coeffi- cients （K）. The average overall accuracies for rabi and kharif are 82.83% and 78.21%, re- spectively. Producer and user accuracies for individual class range respectively between 72.5% to 77% and 70.1% to 84.3% for rabi and 76.6% to 90.2% and 72% to 84.7% for kharif. The K values range between 0.66 to 0.77 for rabi with average of 0.73, and from 0.69 to 0.74 with average of 0.71 for kharif. LULC change detection indicates that wheat and rice have less volatility of change in comparison with both rabi and kharif fodders. Transformation be- tween cotton and rice is less common due to their completely different cropping conditions. Results of spatial and temporal LULC distributions and their seasonal variations provide useful insights for establishing realistic LULC scenarios for hydrological studies.
基金Foundation： National Natural Science Foundation of China, No.41130748
文摘During the past decade, great efforts have been made to boost the land use trans- formation in the Loess Plateau, especially for reducing soil erosion by vegetation restoration measures. The Grain-for-Green project （GFG） is the largest ecological rehabilitation program in China, which has a positive impact on the vegetation restoration and sustainable devel- opment for the ecologically fragile region of west China. Based on the Landsat TM/ETM im- ages for three time periods （2000, 2005 and 2010）, this study applied the GIS technology and a hill-slope analytical model to reveal the spatio-temporal evolutional patterns of returning slope farmland to grassland or woodland in Baota District, Yan＇an city of Shaanxi province. Results showed that： （1） from 2000 to 2010, the area of farmland decreased by approximately 35,030 ha, which is the greatest decrease among all the land-use types, whereas grassland, woodland and construction land increased, of which grassland expanded rapidly by 26,380 ha （2） The annual variation rate of land-use dynamics was 1.98% during the period 2000-2010, of which the rate was 1.05% for the 2000-2005 period and 2.92% for the 2005-2010 period, respectively. Over the past decade, returning farmland to woodland or pastures was the main source of increased grassland and woodland, and the reduction of farmland contributed to the increase in grassland and woodland by 97.39% and 85.28%, respectively. （3） As the terrain slope increases, farmland decreased and woodland and grassland increased significantly. Areas with a slope ranging from 15° to 25° and less than 15° were the focus of the GFG project, accounting for 85% of the total area of farmland reduction. Meanwhile, the reduction in farmland was significant and spatially correlated with the increase in woodland and grass- land. （4） Between 2000 and 2010, the area of destruction of grass and trees in grasslands and woodlands for the reclamation of farmland was approximately 4596 ha. The area subject to the GFG policy was 4456 ha with a slope greater than 25° over the decade, but the area of farmland was still 10,357 ha in 2010. Our results indicate that there has still a great potential for returning the steep-slope farmlands to woodlands or grasslands in the Loess Plateau.
基金supported by the the National Key Research and Development Program of China (2017YFD0201602)the National Natural Science Foundation of China (31401704)the Beijing Academy of Agriculture and Forestry Foundation, China (KJCX20180203)
文摘Bacterial fruit blotch caused by Acidovorax citrulli is a serious threat to cucurbit industry worldwide.The pathogen is seedtransmitted,so seed detection to prevent distribution of contaminated seed is crucial in disease management.In this study,we adapted a quantitative real-time PCR(qPCR)assay to droplet digital PCR(ddPCR)format for A.citrulli detection by optimizing reaction conditions.The performance of ddPCR in detecting A.citrulli pure culture,DNA,infested watermelon/melon seed and commercial seed samples were compared with multiplex PCR,qPCR,and dilution plating method.The lowest concentrations detected(LCD)by ddPCR reached up to 2 fg DNA,and 102 CFU mL–1 bacterial cells,which were ten times more sensitive than those of the qPCR.When testing artificially infested watermelon and melon seed,0.1%infestation level was detectable using ddPCR and dilution plating method.The 26 positive samples were identified in 201 commercial seed samples through ddPCR,which was the highest positive number among all the methods.High detection sensitivity achieved by ddPCR demonstrated a promising technique for improving seed-transmitted pathogen detection threshold in the future.
文摘Objective:To study virulence and regulatory genes(hlyA,ctxB,tcpI) in clinical strains of Vibrio ckolerae(V.cholerae),simultaneously.Methods:Three important genes,tepI,hlyA and ctxB were used for detection of toxigenic and pathogenic V.cholera by chain reaction assay method. Results:According to the results of the PCR,the incidence of hlyA,tcpI,and ctxB genes in clinical isolates was obtained as 94.7%(72 sample),90.8%(69 sample),and 92.1%(70 sample), respectively.Five strains possessed all genes except ctxB,six strains possessed all genes except tcpI,four strains possessed all genes except hlyA,one strain possessed only hlyA and 60 strains contained a combination of three genes.Including hlyA,ctxB and tcpI,Conclusions:Result show that this method could be reliable to detect toxigenic-pathogenic strains of V.cholerae in Iran.
基金supported by the China Mega Project for Infectious Disease(2013ZX10004-001,2012ZX10004-215,2013ZX10004-202,and 2013ZX10004804-007)
文摘Objective This study is aimed to develop a two-tube melting curve-based multiplex real time PCR assay （MCMRT-PCR） for the simultaneous detection of six common foodborne pathogenic bacteria （diarrhoeagenic Escherichia coli, Salmonella, and 5higella in tube 1, Staphylococcus aureus, Vibrio parahaemolyticus, and Listeria monocytogenes in tube 2）. Methods A two-tube MCMRT-PCR assay was performed on 7900HT Fast Real-Time PCR System {Applied Biosystems, USA）. Amplification by PCR was optimized to obtain high efficiency. The sensitivity and specificity of assays were investigated. Results The detection limit of optimized MCMRT-PCR assay was 3.9x102 CFU/mLfor S. aureus, 4.4x102 CFU/mL for L. monocytogenes, 3.0x102 CFU/mL for Salmonella, 2.5x102 CFU/mL for Shigella, 2.1x102 CFU/mL for V. parahaemolyticus, and 1.2x102 CFU/mL for E. coll. The feasibility of MCMRT-PCR was further evaluated using artificially contaminated milk, the sensitivity was at the level of 10s CFU/mL. Conclusion A two-tube MCMRT-PCR assay using six primer sets was developed for detection of multiple pathogens. Our findings demonstrates that the proposed two-tube assay is reliable, useful and rapid for simultaneous detection of six foodborne pathogenic bacteria with an intended application in provincial Centers for Diseases Control and Prevention （CDC）.
基金The Special Fund for Agro-scientific Research in the Public Interest under contract No.201103034Construction Special Fund of Modern Agriculture and Industrial Technology Research System under contract No.CARS-47
文摘Edwardsiella tarda is one of the most important emerging pathogens in tile global aquaculture industries. As such, an accurate diagnosis and quantitative analytical methods are urgently needed for this bacterium. In this study, primers and a TaqMan probe specific to the conservative sequences of the 16S rRNA gene of E. tarda were designed. The concentration of primers and TaqMan probe were optimized to 200 nmol/L and 120 nmol/L, respectively. The detection sensitivity of the FQ- PCR assay was determined to be as low as five copies of the target sequence per reaction using the pGEM-16S rDNA recombinant plasmid as a template, which was 100 times more sensitive than conventional PCR. A standard curve by plotting the threshold cycle values （y） against the common logarithmic copies （logl0n~ as x; n~ is copy number） of pGEM-16S rDNA was generated. The results of intra- and inter-assay variability tests demonstrate that the established FQ-PCR method was highly reproducible. The assay was specific for E. tarda as it showed that there was no cross-reactivity to eight additional bacterial pathogen strains in aquaculture. Thus, the FQ-PCR assay has the potential for diagnostic purposes and for other applications, especially for the rapid detection and quantification of low-grade E. tarda infections.
基金This work was supported by the Natural Sciences Foundation of China (Grant No. NSFC. 40176036).
文摘Objective To develop an in situ PCR in combination with flow cytometry （ISPCR-FCM） for monitoring cholera toxin positive Vibrio cholerae. Methods In running this method, 4% paraformaldehyde was used to fix the Vibrio cholerae cells and 1 mg/mL lysozyme for 20 min to permeabilize the cells. Before the PCR thermal cycling, 2.5% glycerol was added into the PCR reaction mixture in order to protect the integrality of the cells. Results A length of 1037bp DNA sequence was amplified, which is specific for the cholera toxin gene （ctxAB gene）. Cells subjected to ISPCR showed the presences of ctxAB gene both in epifluorescence microscopy and in flow cytometric analysis. The specificity and sensitivity of the method were investigated. The sensitivity was relatively low （10^5 cells/mL）, while the specificity was high. Conclusion We have successfully developed a new technique for detection of toxigenic Vibrio cholerae strains. Further study is needed to enhance its sensitivities. ISPCR-FCM shows a great promise in monitoring specific bacteria and their physiological states in environmental samples.
基金the National Natural Science Foundation of China （30371082, 30671577） Natural Science Foundation of Guangdong Province, China （32286）.
文摘To develop a nested PCR assay for the detection of cattle-derived Cyclospora sp., two pairs of primers were designed on the basis of the cattle-derived Cyclospora sp. sequences. These primers selectively amplified a 168-bp DNA fragment of the 18S rRNA gene of cattle-derived Cyclospora sp. With these primers, a nested PCR assay for the detection of cattlederived Cyclospora sp. was developed. The nested PCR assay was specific and there is no cross-reaction with other parasites, such as Eimeria spp., Cryptosporidium spp., Giardia sp., Toxoplasma sp., Trichuris sp. and cattle ciliate. The assay was able to detect as low as 2.85 × 10^-2 fg of the control positive DNA. The results of the detection of clinical samples indicated that the assay coincided with microscopic examination. The results show that the nested PCR assay will be an effective tool for the detection of Cyclospora sp. in cattle feces.
基金supported by the National Natural Science Foundation of China (NSFC) (No.30571112).
文摘Due to inappropriate planning and management, accelerated urban growth and tremendous loss in land, especially cropland, have become a great challenge for sustainable urban development in China, especially in developed urban area in the coastal regions; therefore, there is an urgent need to effectively detect and monitor the land use changes and provide accurate and timely information for planning and management. In this study a method combining principal component analysis (PCA) of multisensor satellite images from SPOT (systeme pour l'observation de la terre or earth observation satellite)-5 multispectral (XS) and Landsat-7 enhanced thematic mapper (ETM) panchromatic (PAN) data, and supervised classification was used to detect and analyze the dynamics of land use changes in the city proper of Hangzhou. The overall accuracy of the land use change detection was 90.67% and Kappa index was 0.89. The results indicated that there was a considerable land use change (10.03% of the total area) in the study area from 2001 to 2003, with three major types of land use conversions: from cropland into built-up land, construction site, and water area (fish pond). Changes from orchard land into built-up land were also detected. The method described in this study is feasible and useful for detecting rapid land use change in the urban area.
基金supported by the National Science and Technology Major Project of the Ministry of Science and Technology of China(No.2013ZX10004-101)
文摘A real-time RT-PCR （RT-qPCR） assay for the detection of Tahyna virus was developed to monitor Tahyna virus infection in field-collected vector mosquito samples. The targets selected for the assay were S segment sequences encoding the nucleocapsid protein from the Tahyna virus. Primers and probes were selected in conserved regions by aligning genetic sequences from various Tahyna virus strains available from GenBank. The sensitivity of the RT-qPCR approach was compared to that of a standard plaque assay in BHK cells. RT-qPCR assay can detect 4.8 PFU of titrated Tahyna virus. Assay specificities were determined by testing a battery of arboviruses, including representative strains of Tahyna virus and other arthropod-borne viruses from China. Seven strains of Tahyna virus were confirmed as positive; the other seven species of arboviruses could not be detected by RT-qPCR. Additionally, the assay was used to detect Tahyna viral RNA in pooled mosquito samples. The RT-qPCR assay detected Tahyna virus in a sensitive, specific, and rapid manner; these findings support the use of the assay in viral surveillance.
基金Supported by the Ministry of Power of I.R.Iran(Grant No.201)
文摘Objective:To analyse molecular detection of coliforms and shorten the time of PCR.Methods:Rapid detection of coliforms by amplification of lacZ and uidA genes in a multiplex PCR reaction was designed and performed in comparison with most probably number(MPN)method for 16 artificial and 101 field samples.The molecular method was also conducted on isolated coliforms from positive MPN samples;standard sample for verification of microbial method certificated reference material;isolated strains from certificated reference material and standard bacteria.The PCR and electrophoresis parameters were changed for reducing the operation time.Results:Results of PCR for lacZ and uidA genes were similar in all of standard,operational and artificial samples and showed the 876 bp and 147 bp bands of lacZ and uidA genes by multiplex PCR.PCR results were confirmed by MPN culture method by sensitivity 86%(95%CI:0.71-0.93).Also the total execution time,with a successful change of factors,was reduced to less than two and a half hour.Conclusions:Multiplex PCR method with shortened operation time was used for the simultaneous detection of total coliforms and Escherichia coli in distribution system of Arak city.It's recommended to be used at least as an initial screening test,and then the positive samples could be randomly tested by MPN.
基金Supported by the China Agriculture Research System(No.CARS-50)the National High-Tech R&D Program of China(No.2012AA10A406)+1 种基金the National Science and Technology Infrastructure Platform Construction(No.2018DKA30470)the Aoshan Technology Innovation Program of Qingdao National Laboratory for Marine Science and Technology(No.2015ASKJ02)
文摘Pseudoalteromonas marina is one of the potential pathogens that cause green spot disease(GSD)in Pyropia yezoensis.To prevent GSD from development and spread,an effective method to detect the pathogen at early GSD infection stages need to be established.In this research,PCR methods were established targeting the dnaA gene(encoding chromosome replication initiator protein)and the dnaN gene(encodingβsliding clamp of DNA polymeraseⅢprotein)to detect P.marina with three primer pairs pws-dnaA2(Forward,5'-ACCGCATTAACGAACTACTCGTG-3';Reverse,5'-TGCCATTACCTACAGCATGG-3'),pcs-dnaN2(Forward,5'-CTTACAACGTTATCAGCGGC-3';Reverse,5'-GTTGAGTATTAAGTGATTGAGTAAGC-3')or pws-dnaN3(Forward,5'-ACTTACAA-CGTTATCAGCGGC-3';Reverse,5'-ACTGCTGTTTGAGTCTGCTAAC-3').Three PCR methods corresponding to the three primer pairs sufficiently distinguished P.marina from 22 bacterial species,thus resulting in detection limits of 4 to 4×10^2 CFU cells or 2.37×10^1 to 2.37×10^3 fg of P.marina DNA per PCR reaction.In an artificial infection experiment ofP.yezoensis infected with P.marina,all established PCRs successfully detected P.marina at early GSD infection stages.The results show that the established PCRs are specific and sensitive,and are potential for applications in early diagnosis of GSD in Pyropia.
基金Supported by the Special Fund for Agro-scientific Research in the Public Interest(No.201103034)the Construction Special Fund of Modern Agriculture and Industrial Technology Research System(No.CARS-47)
文摘Edwardsiella tarda has become one of the most important emerging pathogens in aquaculture industry. Therefore, a rapid, reproducible, and sensitive method for detection and quantification of this pathogen is needed urgently. To achieve this purpose, we developed a TaqMan-based real-time PCR assay for detection and quantification orE. tarda. The assay targets the hemolysin activator HlyB domain protein of E. tarda. Our optimized TaqMan assay is capable of detecting as little as 40 fg of genomic DNA per reaction. A standard curve was generated from the threshold cycle values （y） against log10 （E. tarda genomic DNA concentration） as x. The intra- and inter-assay coefficient of variation （CV） values were less than 2.06% and 1.05% respectively, indicating that the assay had good reproducibility. This method is highly specific to E. tarda strains, as it shows no cross-reactivity to Edwardsiella ictaluri, a member of the same genus, or to nine other fish-pathogenic bacteria species belonging to three other genera. This sensitive and specific real-time PCR assay provides a valuable tool for diagnostic quantitation of E. tarda in clinical samples.
文摘AIM: To compare the ligase detection reaction (LDR) and real-time PCR for detection of low abundant YMDD mutants in patients with chronic hepatitis B infection.METHODS: Mixtures of plasmids and serum samples from 52 chronic hepatitis B patients with low abundant lamivudine-resistant mutations were tested with LDR and real-time PCR. Time required and reagent cost for both assays were evaluated.RESULTS: Real-time PCR detected 100, 50, 10, 1 and 0.1% of YIDD plasmid, whereas LDR detected 100, 50, 10, 1, 0.1, and 0.01% of YIDD plasmid, in mixtures with YMDD plasmid of 106 copies/mL. Among the 52 clinical serum samples, completely concordant results were obtained for all samples by both assays, and 39 YIDD, 9 YVDD, and 4 YIDD/YVDD were detected. Cost and time required for LDR and real-time PCR are 60/80 CNY (8/10.7 US dollars) and 4.5/2.5 h, respectively.CONCLUSION: LDR and real-time PCR are both sensitive and inexpensive methods for monitoring low abundant YMDD mutants during lamivudine therapy in patients with chronic hepatitis B. LDR is more sensitive and less expensive, while real-time PCR is more rapid.
