Dot-blot hybridization was applied in this paper to detect five viruses infecting cucurbitaceous crops, Zuccini yellow mosaic virus (ZYMV), Watermelon mosaic virus (WMV), Cucumber mosaic virus (CMV), Papaya ring...Dot-blot hybridization was applied in this paper to detect five viruses infecting cucurbitaceous crops, Zuccini yellow mosaic virus (ZYMV), Watermelon mosaic virus (WMV), Cucumber mosaic virus (CMV), Papaya ringspot viruswatermelon strain (PRSV-W) and Squash mosaic virus (SqMV), as a good alternative assay in seed health test and epidemiological and transgenic research. Digoxigenin-labelled cDNA probes of the five viruses were synthesized by PCR with the specific primers and applied in dot-blot hybridization to detect five viruses in crude extraction of the infected leaves. And three SqMV probes of different lengths (0.55, 1.6, and 2.7 kb, respectively) were designed to investigate the effect of hybridization. The results showed that the sensitivity for detecting the crude extraction of infected leaves by ZYMV, WMV, CMV, PRSV-W, and SqMV was down to 1:160, 1:160, 1:320, 1:160, and 1:320, respectively. Three SqMV probes of different length showed no differences on the sensitivity and specificity. The digoxigenin-labelled probes prepared by PCR could be used for accurate and rapid identification of 5 viruses infecting cucurbitaceous crops with good stabilities, sensitivities, specificity, and reproducibilifies.展开更多
Harmful algal blooms recently have been under the spotlight throughout the world, because of their nega- tive impact on the marine environment, aquaculture, fisheries as well as public health. The development of metho...Harmful algal blooms recently have been under the spotlight throughout the world, because of their nega- tive impact on the marine environment, aquaculture, fisheries as well as public health. The development of methods for rapid and precise identification and quantification of causative species is essential for the warning and monitoring of blooms, among which the techniques based on taxonomic probes are the most favored. In this study, two harmful algae, i.e., Prorocentrum minimum and Karenia mikimotoi were tak- en into consideration. The partial large subunit rDNA (D1-D2) of both species were firstly PCR-amplified, cloned and sequenced. The obtained sequences were then introduced to carry out alignment analysis for gene specific regions. Three respective candidate probes for each species were designed and used to screen the optimal probe by performing fluorescence in situ hybridization (FISH) tests. The results showed that the probes Pmin0443 and Kmik0602 displayed the best hybridization for P. minimum and K.. mikimotoi, respectively. Both the specific (taxonomic) (Pmin0443 and Kmik0602) and the control probes (UniC0512 and UniR0499) were used for cross-reactivity tests with other microalgae in our laboratory. The probes Pmin0443 and Kmik0602 are specific and could be served as taxonomic probes introduced into the tech- niques targeting rRNA, such as FISH, sandwich hybridization, and DNA-microarray assay of P minimum and K. mikimotoi in the future. Finally, FISH analyses with both probes were performed on the simulated field samples. The probes could hybridize exclusively with the target cells well, and no significant differ- ence (p 〉0.05) was observed in the cell densities of the samples determined by FISH and light microscopy (LM). All suggest that the probes are specific and could be introduced into FISH for the monitoring of both harmful algae.展开更多
The dinoflagellate Alexandrium minutum is often associated with harmful algal blooms (HABs). This species consists of many strains that differ in their ability to produce toxins but have similar morphology, making ide...The dinoflagellate Alexandrium minutum is often associated with harmful algal blooms (HABs). This species consists of many strains that differ in their ability to produce toxins but have similar morphology, making identification difficult. In this study, species-specific rRNA probes were designed for whole-cell fluorescence in situ hybridization (FISH) to distinguish A. minutum from two phylogenetic clades. We acquired the complete SSU to LSU rDNA sequences (GenBank accession numbers JF906989-JF906999) of 11 Alexandrium strains and used these to design rRNA targeted oligonucleotide probes. Three ribotype-specific probes, M-GC-1, M-PC-2, and M-PC-3, were designed. The former is specific for the GC clade ("Global clade") of A. minutum, the majority of which have been found non-toxic, and the latter two are specific for the PSP (paralytic shellfish poisoning)-producing PC clade ("Pacific clade"). The specificity of these three probes was confirmed by FISH. All cells in observed fields of view were fluorescently labeled when probes and target species were incubated under optimized FISH conditions. However, the accessibility of rRNA molecules in ribosomes varied among the probe binding positions. Thus, there was variation in the distribution of positive signals in labeled cells within nucleolus and cytosol (M-GC-1, M-PC-3), or just nucleolus (M-PC-2). Our results provide a methodological basis for studying the biogeography and population dynamics of A. minutum, and providing an early warning of toxic HABs.展开更多
HUMAN herpes simplex virus esophagitis (HSVE) was first reported in 1940 by Johnson. ^1HSVE usually occurs in immunocompromised patients,such as those with acquired immunodeficiency syndrome (AIDS), 2-4 malignanc...HUMAN herpes simplex virus esophagitis (HSVE) was first reported in 1940 by Johnson. ^1HSVE usually occurs in immunocompromised patients,such as those with acquired immunodeficiency syndrome (AIDS), 2-4 malignancies, cutaneous burns, connective tissue diseases, inflammatory bowel disease, those taking immuno-suppressive therapy, and those undergoing organ transplantation,5 etc. In the immunocompetent individuals, HSVE is rare, having been reported in 39 cases and mainly affecting young males^6,7 The aim of this study was to delineate the clinical experience in the diagnosis of HSVE using rapid in situ hybridization and assess the various detection methods.展开更多
Partial rDNA sequences of Prorocentrum minimum and Takayama pulchella were amplified, cloned and sequenced, and these sequence data were deposited in the GenBank. Eight oligonucleotide probes (DNA probes) were desig...Partial rDNA sequences of Prorocentrum minimum and Takayama pulchella were amplified, cloned and sequenced, and these sequence data were deposited in the GenBank. Eight oligonucleotide probes (DNA probes) were designed based on the sequence analysis. The probes were employed to detect and identify P. minimum and T. pulchella in unialgal and mixed algal samples with a fluorescence in situ hybridization method using flow cytometry. Epifluorescence micrographs showed that these specific probes labeled with fluorescein isothiocyanate entered the algal cells and bound to target sequences, and the fluorescence signal resulting from whole-cell hybridization varied from probe to probe. These DNA probes and the hybridization protocol we developed were specific and effective for P. minimum and T. pulchella, without any specific binding to other algal species. The hybridization efficiency of different probes specific to P. minimum was in the order: PM18S02 PM28S02 〉 PM28S01 〉PM18S01, and that of the probes specific to T. pulcheUa was TP18S02 TP28S01 〉 TP28S02 〉TP18S01. The different hybridization efficiency of the DNA probes could also be shown in the fluorescent signals between the labeled and unlabeled cells demonstrated using flow cytometry. The DNA probes PM18S02, PM28S02, TP18S02 and TP28S01, and the protocol, were also useful for the detection of Mgae in natural samples.展开更多
The receptor for autocrine motility factor (AMFR) is known to be involved in the process of AMF-mediated cell migration and metastasis. This paper describes the procedures of non-radioactive in situ hybridization (ISH...The receptor for autocrine motility factor (AMFR) is known to be involved in the process of AMF-mediated cell migration and metastasis. This paper describes the procedures of non-radioactive in situ hybridization (ISH) detection of AMFR mRNA in both paraffin-embedded surgical sections and cultured cells using either biotinylated oligonucleotide probes or digoxigenin-labeled RNA probes. The results showed that the AMFR mRNA was expressed at an enhanced level in hyperplaJstic and malignant tissues of breast and prostate cancer patient surgical specimens, indicating that the elevated AMFR expression was associated with the tissue malignancy Moreover, AMFR mRNA was detected in both normal and earcinoma cells when cultured at a subconfluent density. However, AMFR expression was inhibited in confluent normal (3T3-A31 murine fibroblast and FHs738BL human bladder) cells while it continued to express in carcinoma (J82 human bladder)and metastatic (3T3-M murine fibroblast) cells irrespective of cell density This suggested a cell-cell contact downregulation of AMFR mRNA expression in normal but not in cancer cells. The ISH data obtained in this study are closely consistent with the AMFR protein expression pattern previously reported, implying that the differential expression of AMFR gene may be regulated and controlled at the transcriptional level.展开更多
In this study, in situ hybridization (ISH) was developed to detect avian influenza'virus (AIV) in Madin-Darby canine kidney (MDCK) cells and formalin-fixed, paraffin-embedded chicken tissues. A cDNA probe corre...In this study, in situ hybridization (ISH) was developed to detect avian influenza'virus (AIV) in Madin-Darby canine kidney (MDCK) cells and formalin-fixed, paraffin-embedded chicken tissues. A cDNA probe corresponding to a region of AIV nucleoprotein (NP) gene was synthesized and labeled with digoxigenin. Probe specificity was determined by AIV infected MDCK cells in vitro and the results showed that strong cytoplasmic staining was only detected in AIV-infected cells. Various tissues were collected from 12 h to 35 days post-infection (PI) following inoculation with the H9N2 subtype A1V. AIV was localized in the epithelial cells of the duodenum and cartilage of the throat and trachea at 12 h PI. Tissues from uninfected chickens were negative. The finding of this study indicated ISH was a sensitive and specific technique to detect and localize AIV as well as to study AIV pathogenesis.展开更多
We have develoPed a simPle and economical method f0r Chlamydia trachomatisdetecting, called microtiter plate hybridization (PCR-MPH), which may replace stan-dard PCR. This method is similar to that of an ELISA. Brithe...We have develoPed a simPle and economical method f0r Chlamydia trachomatisdetecting, called microtiter plate hybridization (PCR-MPH), which may replace stan-dard PCR. This method is similar to that of an ELISA. Brithe, the PCR productslabeled at the 5'termini with biotin were hybridized with probes immobilized on a mi-crotiter well, and the bound PCR products were detected by streptavidin-c0njugatedenzymes followed by color development. Two inprovements have been made in immobi-lizing the probe to the microtiter wells, in terms of increasing both immobility and hy-bridization deciency. One is that singleustranded (ss )DNA, without the complemen-tary strand, is used. The other is that instead of a single copy, a tandem array of theprobe is used for immobilization and hybridization. Using of ssDNA containing abouta 5O-rePeat array of a relevant sequence as an immobilized probe, the sensitivity in-creased 1O-fold over that of a single oligonucleotide unit. We also found that the hy-brldizatlon condltions such as time, temPerature, and solution composition could be simplthed. The advantages of this microtiter plate-hybridization method for routinepathogens detecting are a short time assay, easy processing of large numbers of sansples, and the potential for automation.展开更多
To screen species-specific DNA probes for identification of Fallopia muhiflora, the genomic DNA (gDNA) suppression subtraction hybridization (SSH) between F. muhiflora and F. muhiflora var. ciliinervis was firstly...To screen species-specific DNA probes for identification of Fallopia muhiflora, the genomic DNA (gDNA) suppression subtraction hybridization (SSH) between F. muhiflora and F. muhiflora var. ciliinervis was firstly performed. The obtained differential gDNA fragments by SSH were then hybridized with gDNA ar- rays consisting of multiple whole genomes of several species (adulterants and/or closely related species of F. muhiflora) and four differential fragments were screened uniquely representing F. muhiflora, which could be used as F. muhiflora species-specific probes. The screened DNA probes were tested by reverse dot blot hybridization and the results demonstrated that these probes could be used reliably to identify F, muhiflora. The species-specific DNA probes obtained in this study exhibited broad application prospects in the preparation of gene chips for identifying Chinese traditional medicines and the authentication of germplasm re- sources and crude drugs of F. muhiflora.展开更多
Objective To explore if strand breaks of DNA in human early chorionic villus cells in uterus were induced by diagnostic ultrasound and to evaluate the method used for detection of single-stranded breaks and double-str...Objective To explore if strand breaks of DNA in human early chorionic villus cells in uterus were induced by diagnostic ultrasound and to evaluate the method used for detection of single-stranded breaks and double-stranded breaks in human DNA. Methods 60 normal pregnant women aged 20-30, who underwent artificial abortion during 6-8 weeks of gestation, were randomly divided into 2 experimental groups: All 30 cases were exposed to diagnostic ultrasound in uterus for 10 minutes, and 24 hours later chorionic villi were extracted; the other 30 cases were taken as the control group. Single-stranded DNA and double-stranded DNA in villus cells in all cases were isolated by the alkaline unwinding combined with hydroxylapatite chromatography, and were quantitatively detected using 32 P-labeled Alu probe for dot-blotting hybridization. Results There was no significant difference in quantity and percentage in single-stranded DNA and double-stranded DNA between 2 groups (P>0.05). 32 P-Alu probe could only hybridize with human DNA, and could detect DNA isolated from as few as 2.5×10 3 chorionic villus cells and 0.45ng DNA in human leukocytes. Conclusion The results suggested that there were no DNA strand damages in human chorionic villus cells when the uterus was exposed to diagnostic ultrasound for 10 minutes. The method,^(32)P-Alu probe for dot-blotting hybridization, was even more specific, sensitive and accurate than conventional approaches.展开更多
Systematic analysis of factors determining efficiency in discrimination of a point substitution (SNP) within specific DNA sequences was carried out in the context of hybridization approach. There are two types of sele...Systematic analysis of factors determining efficiency in discrimination of a point substitution (SNP) within specific DNA sequences was carried out in the context of hybridization approach. There are two types of selectivity that are critical for the rational design of highly specific oligonucleotides probes. The first type is the real selectivity of hybridization (fa) that is the ratio of association degrees of targets with an oligonucleotide probe upon the perfect and imperfect complex formation. This type of selectivity reflects the level of discrimination between matched and mismatched signals, which is determined both by experimental conditions and the thermodynamics of oligonucleotide hybridization. The second parameter characterizing the efficiency of SNP discrimination is the limit selectivity of hybridization, which determines the utmost value of fa at a given temperature. This value can be calculated as the ratio of corresponding equilibrium association constants of perfect and imperfect complex formation determined purely by thermodynamics. We have shown that the fa function is the most reliable characteristic describing the hybridization selectivity. For the analytical system designed to reveal any type of perturbation in DNA (e.g. SNP or modification), there is usually a temperature at which fa has its maximum value. The dependency of the fa maximum on different experimental parameters as well as the structural characteristics of a probe are described in details. The results allowed us to postulate points of principle to rationally design the most selective probes on the basis of oli- gonucleotides or their derivatives.展开更多
单分子荧光原位杂交(single-molecule fluorescence in situ hybridization,smFISH)技术是一种通过用偶联荧光基团的寡核苷酸探针,对固定细胞或组织中单个mRNA分子进行成像的方法。smFISH可对RNA进行定位、定量,以此对目标转录本进行实...单分子荧光原位杂交(single-molecule fluorescence in situ hybridization,smFISH)技术是一种通过用偶联荧光基团的寡核苷酸探针,对固定细胞或组织中单个mRNA分子进行成像的方法。smFISH可对RNA进行定位、定量,以此对目标转录本进行实时研究。sm FISH适用于细胞、组织切片等多种类型生物样本。近年来,多种基于基础smFISH的改进技术被发明,进一步促进了该技术的实际应用。smFISH良好的RNA单分子可视化能力,使得其在发育生物学、神经生物学及肿瘤生物学等基础生物学科中得到了广泛的应用。本文综述了smFISH技术基本原理、smFISH技术的局限性、smFISH衍生技术方法、smFISH在不同生物学科中的应用进展,并对smFISH技术的发展前景做出展望。展开更多
We propose a scheme that can generate tunable double optomechanically induced transparency in a hybrid optomechanical cavity system.In this system, the mechanical resonator of the optomechanical cavity is coupled with...We propose a scheme that can generate tunable double optomechanically induced transparency in a hybrid optomechanical cavity system.In this system, the mechanical resonator of the optomechanical cavity is coupled with an additional mechanical resonator and the additional mechanical resonator can be driven by a weak external coherently mechanical driving field.We show that both the intensity and the phase of the external mechanical driving field can control the propagation of the probe field, including changing the transmission spectrum from double windows to a single-window.Our study also provides an effective way to generate intensity-controllable, narrow-bandwidth transmission spectra, with the probe field modulated from excessive opacity to remarkable amplification.展开更多
A two-dimensional hybrid simulation scheme is proposed to study the characteristics of dual-frequency (DF) capacitively coupled plasma (CCP) discharge based on the geometry of real device. Given the experimental p...A two-dimensional hybrid simulation scheme is proposed to study the characteristics of dual-frequency (DF) capacitively coupled plasma (CCP) discharge based on the geometry of real device. Given the experimental parameters for argon plasma, the output from the fluid module such as ion density, number flux, electron temperature and the Monte-Carlo collision (MCC) results of ion energy distribution function (IEDF) as well as electron energy distribution function (EEDF) are obtained and discussed in detail. A novel complete floating double probe is designed to measure both density and temperature of electron and a quadrupole mass spectrometer is also equipped for IEDF investigations. The measurements on the density of bulk plasma, electron temperature and IEDF agree well, qualitatively, with the simulated results. A comparison with experimental results indicates that, since the structure of real device is taken into account, this model is capable of describing the global dynamic characteristics occurred in DF-CCP and presenting more reliable results than the model with an ideal chamber structure.展开更多
To study the technique of D-FISH and its application in the diagnosis of a 49, XXXXY chromosomal abnormality. Methods: Biotin-labeled alpha satellite X chromosome DNA (pBamX7) probe and digoxi-genin-labeled Y chromoso...To study the technique of D-FISH and its application in the diagnosis of a 49, XXXXY chromosomal abnormality. Methods: Biotin-labeled alpha satellite X chromosome DNA (pBamX7) probe and digoxi-genin-labeled Y chromosome long arm terminal repetitive sequence (pY3.4) probe in situ hybridized with pre-treated slides of peripheral blood chromosome and interphase nucleus. After washing, the slides were treated with avidin-FITC, rhodamine-FITC and anti-avidin, amplified with an additional layer and counter-stained with DAPI in an antifade solution. The hy bridization signals and chromosomal or interphase nucleus settings were observed respectively with WIB, WIG and WU filters under fluorescent microscope (Olympus AX-70) and the number of metaphase chromosome and interphase nucleus in the peripheral blood was counted. Results: The biotin-labeled pBamX7 probe showed 4 green hybridization signal and the digoxigenin-labeled pY3.4 probe showed 1 red hybridization signal. The chromosome or cytoplasm counter-stained with DAPI showed blue. The positive rate of X chromosome hybridization signal for the 350 metaphase chromosomes and interphase nucleus was 91.43 % and 92. 57 %, respectively, while that of the Y chromosome hybridization signal was 99.5 % and 99.8 %, respectively. Conclusion: D-FISH is a valuable technique in diagnosing 49, XXXXY chromosomal abnormality and other sex chromosomal abnormalities. [Reprod Contracep (in Chinese) 2002; 22: 287]展开更多
The Plasmodtum falctparum DNA fragment was isolated from a cloned recombinant plasmid pPF14. labelled with Digoxigenin (Dig) and used as a probe (pPF14-F-Dig)to detect 274 blood samples from the eqdemic area populatio...The Plasmodtum falctparum DNA fragment was isolated from a cloned recombinant plasmid pPF14. labelled with Digoxigenin (Dig) and used as a probe (pPF14-F-Dig)to detect 274 blood samples from the eqdemic area population in Hainan Province by dot hybridization.The results showed that out of 274 blood specimens one was positive when using microscopic examination and the positivity rate was 0.36 percent. Fifteen samples showed to be positive (including one positive specimen examined by microscopy) when this probe was applied to detect the 274 samples and its positivity rate was 5.47 percent.The positive coincidence rate between pPF14-F-Dig and microscopic examination was 1/1 and the negative coincidence rate was 94.87 percent. Since a piece of nitrocellulose membrane with the size of 9 by 12 square centimetres can accommodate blots of 96 samples,this probe is fit for large-scale epidemiological surveys.展开更多
文摘Dot-blot hybridization was applied in this paper to detect five viruses infecting cucurbitaceous crops, Zuccini yellow mosaic virus (ZYMV), Watermelon mosaic virus (WMV), Cucumber mosaic virus (CMV), Papaya ringspot viruswatermelon strain (PRSV-W) and Squash mosaic virus (SqMV), as a good alternative assay in seed health test and epidemiological and transgenic research. Digoxigenin-labelled cDNA probes of the five viruses were synthesized by PCR with the specific primers and applied in dot-blot hybridization to detect five viruses in crude extraction of the infected leaves. And three SqMV probes of different lengths (0.55, 1.6, and 2.7 kb, respectively) were designed to investigate the effect of hybridization. The results showed that the sensitivity for detecting the crude extraction of infected leaves by ZYMV, WMV, CMV, PRSV-W, and SqMV was down to 1:160, 1:160, 1:320, 1:160, and 1:320, respectively. Three SqMV probes of different length showed no differences on the sensitivity and specificity. The digoxigenin-labelled probes prepared by PCR could be used for accurate and rapid identification of 5 viruses infecting cucurbitaceous crops with good stabilities, sensitivities, specificity, and reproducibilifies.
基金Shandong Province Young and Middle-Aged Scientists Research Awards Fund under contract No. BS2010HZ002the National Natural Science Foundation of China under contract Nos 41106082 and 41176141+1 种基金Key Laboratory of Marine Ecology and Environmental Science and Engineering, SOA under contract No. MESE-2011-06the Basic Research of Harbin Institute of Technology Outstanding Talents Cultivation Plan of Class Ⅲ
文摘Harmful algal blooms recently have been under the spotlight throughout the world, because of their nega- tive impact on the marine environment, aquaculture, fisheries as well as public health. The development of methods for rapid and precise identification and quantification of causative species is essential for the warning and monitoring of blooms, among which the techniques based on taxonomic probes are the most favored. In this study, two harmful algae, i.e., Prorocentrum minimum and Karenia mikimotoi were tak- en into consideration. The partial large subunit rDNA (D1-D2) of both species were firstly PCR-amplified, cloned and sequenced. The obtained sequences were then introduced to carry out alignment analysis for gene specific regions. Three respective candidate probes for each species were designed and used to screen the optimal probe by performing fluorescence in situ hybridization (FISH) tests. The results showed that the probes Pmin0443 and Kmik0602 displayed the best hybridization for P. minimum and K.. mikimotoi, respectively. Both the specific (taxonomic) (Pmin0443 and Kmik0602) and the control probes (UniC0512 and UniR0499) were used for cross-reactivity tests with other microalgae in our laboratory. The probes Pmin0443 and Kmik0602 are specific and could be served as taxonomic probes introduced into the tech- niques targeting rRNA, such as FISH, sandwich hybridization, and DNA-microarray assay of P minimum and K. mikimotoi in the future. Finally, FISH analyses with both probes were performed on the simulated field samples. The probes could hybridize exclusively with the target cells well, and no significant differ- ence (p 〉0.05) was observed in the cell densities of the samples determined by FISH and light microscopy (LM). All suggest that the probes are specific and could be introduced into FISH for the monitoring of both harmful algae.
基金Supported by the National Natural Science Foundation of China(No.41006082)the Postdoctoral Science Foundation(No.20090461272)the National Natural Science Foundation of China for Creative Research Groups(No.40821004)
文摘The dinoflagellate Alexandrium minutum is often associated with harmful algal blooms (HABs). This species consists of many strains that differ in their ability to produce toxins but have similar morphology, making identification difficult. In this study, species-specific rRNA probes were designed for whole-cell fluorescence in situ hybridization (FISH) to distinguish A. minutum from two phylogenetic clades. We acquired the complete SSU to LSU rDNA sequences (GenBank accession numbers JF906989-JF906999) of 11 Alexandrium strains and used these to design rRNA targeted oligonucleotide probes. Three ribotype-specific probes, M-GC-1, M-PC-2, and M-PC-3, were designed. The former is specific for the GC clade ("Global clade") of A. minutum, the majority of which have been found non-toxic, and the latter two are specific for the PSP (paralytic shellfish poisoning)-producing PC clade ("Pacific clade"). The specificity of these three probes was confirmed by FISH. All cells in observed fields of view were fluorescently labeled when probes and target species were incubated under optimized FISH conditions. However, the accessibility of rRNA molecules in ribosomes varied among the probe binding positions. Thus, there was variation in the distribution of positive signals in labeled cells within nucleolus and cytosol (M-GC-1, M-PC-3), or just nucleolus (M-PC-2). Our results provide a methodological basis for studying the biogeography and population dynamics of A. minutum, and providing an early warning of toxic HABs.
文摘HUMAN herpes simplex virus esophagitis (HSVE) was first reported in 1940 by Johnson. ^1HSVE usually occurs in immunocompromised patients,such as those with acquired immunodeficiency syndrome (AIDS), 2-4 malignancies, cutaneous burns, connective tissue diseases, inflammatory bowel disease, those taking immuno-suppressive therapy, and those undergoing organ transplantation,5 etc. In the immunocompetent individuals, HSVE is rare, having been reported in 39 cases and mainly affecting young males^6,7 The aim of this study was to delineate the clinical experience in the diagnosis of HSVE using rapid in situ hybridization and assess the various detection methods.
基金The Fujian Provincial Government of China under contract No 2005YZ1018 the Xiamen Municipal Government of China under contract No 3502Z20041059+4 种基金 the China Postdoctoral Science Foundation under contract No 20060400854the Open Fund of the State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences under contract No 2008FB005 the Specialized Research Fund for the Doctoral Program of Higher Education of China under contract 20070504076 the Open Fund of the Key Laboratory of Freshwater Fish Germplasm and Biotechnology of Ministry of Agriculture, Chinese Academy of Fishery Sciences under contract No LFB20070611the National Natural Science Foundation of China under contract No 40576055
文摘Partial rDNA sequences of Prorocentrum minimum and Takayama pulchella were amplified, cloned and sequenced, and these sequence data were deposited in the GenBank. Eight oligonucleotide probes (DNA probes) were designed based on the sequence analysis. The probes were employed to detect and identify P. minimum and T. pulchella in unialgal and mixed algal samples with a fluorescence in situ hybridization method using flow cytometry. Epifluorescence micrographs showed that these specific probes labeled with fluorescein isothiocyanate entered the algal cells and bound to target sequences, and the fluorescence signal resulting from whole-cell hybridization varied from probe to probe. These DNA probes and the hybridization protocol we developed were specific and effective for P. minimum and T. pulchella, without any specific binding to other algal species. The hybridization efficiency of different probes specific to P. minimum was in the order: PM18S02 PM28S02 〉 PM28S01 〉PM18S01, and that of the probes specific to T. pulcheUa was TP18S02 TP28S01 〉 TP28S02 〉TP18S01. The different hybridization efficiency of the DNA probes could also be shown in the fluorescent signals between the labeled and unlabeled cells demonstrated using flow cytometry. The DNA probes PM18S02, PM28S02, TP18S02 and TP28S01, and the protocol, were also useful for the detection of Mgae in natural samples.
文摘The receptor for autocrine motility factor (AMFR) is known to be involved in the process of AMF-mediated cell migration and metastasis. This paper describes the procedures of non-radioactive in situ hybridization (ISH) detection of AMFR mRNA in both paraffin-embedded surgical sections and cultured cells using either biotinylated oligonucleotide probes or digoxigenin-labeled RNA probes. The results showed that the AMFR mRNA was expressed at an enhanced level in hyperplaJstic and malignant tissues of breast and prostate cancer patient surgical specimens, indicating that the elevated AMFR expression was associated with the tissue malignancy Moreover, AMFR mRNA was detected in both normal and earcinoma cells when cultured at a subconfluent density. However, AMFR expression was inhibited in confluent normal (3T3-A31 murine fibroblast and FHs738BL human bladder) cells while it continued to express in carcinoma (J82 human bladder)and metastatic (3T3-M murine fibroblast) cells irrespective of cell density This suggested a cell-cell contact downregulation of AMFR mRNA expression in normal but not in cancer cells. The ISH data obtained in this study are closely consistent with the AMFR protein expression pattern previously reported, implying that the differential expression of AMFR gene may be regulated and controlled at the transcriptional level.
文摘In this study, in situ hybridization (ISH) was developed to detect avian influenza'virus (AIV) in Madin-Darby canine kidney (MDCK) cells and formalin-fixed, paraffin-embedded chicken tissues. A cDNA probe corresponding to a region of AIV nucleoprotein (NP) gene was synthesized and labeled with digoxigenin. Probe specificity was determined by AIV infected MDCK cells in vitro and the results showed that strong cytoplasmic staining was only detected in AIV-infected cells. Various tissues were collected from 12 h to 35 days post-infection (PI) following inoculation with the H9N2 subtype A1V. AIV was localized in the epithelial cells of the duodenum and cartilage of the throat and trachea at 12 h PI. Tissues from uninfected chickens were negative. The finding of this study indicated ISH was a sensitive and specific technique to detect and localize AIV as well as to study AIV pathogenesis.
文摘We have develoPed a simPle and economical method f0r Chlamydia trachomatisdetecting, called microtiter plate hybridization (PCR-MPH), which may replace stan-dard PCR. This method is similar to that of an ELISA. Brithe, the PCR productslabeled at the 5'termini with biotin were hybridized with probes immobilized on a mi-crotiter well, and the bound PCR products were detected by streptavidin-c0njugatedenzymes followed by color development. Two inprovements have been made in immobi-lizing the probe to the microtiter wells, in terms of increasing both immobility and hy-bridization deciency. One is that singleustranded (ss )DNA, without the complemen-tary strand, is used. The other is that instead of a single copy, a tandem array of theprobe is used for immobilization and hybridization. Using of ssDNA containing abouta 5O-rePeat array of a relevant sequence as an immobilized probe, the sensitivity in-creased 1O-fold over that of a single oligonucleotide unit. We also found that the hy-brldizatlon condltions such as time, temPerature, and solution composition could be simplthed. The advantages of this microtiter plate-hybridization method for routinepathogens detecting are a short time assay, easy processing of large numbers of sansples, and the potential for automation.
基金Supported by Fund of Guangdong Provincial Administration of Traditional Chinese Medicine(20111251)
文摘To screen species-specific DNA probes for identification of Fallopia muhiflora, the genomic DNA (gDNA) suppression subtraction hybridization (SSH) between F. muhiflora and F. muhiflora var. ciliinervis was firstly performed. The obtained differential gDNA fragments by SSH were then hybridized with gDNA ar- rays consisting of multiple whole genomes of several species (adulterants and/or closely related species of F. muhiflora) and four differential fragments were screened uniquely representing F. muhiflora, which could be used as F. muhiflora species-specific probes. The screened DNA probes were tested by reverse dot blot hybridization and the results demonstrated that these probes could be used reliably to identify F, muhiflora. The species-specific DNA probes obtained in this study exhibited broad application prospects in the preparation of gene chips for identifying Chinese traditional medicines and the authentication of germplasm re- sources and crude drugs of F. muhiflora.
文摘Objective To explore if strand breaks of DNA in human early chorionic villus cells in uterus were induced by diagnostic ultrasound and to evaluate the method used for detection of single-stranded breaks and double-stranded breaks in human DNA. Methods 60 normal pregnant women aged 20-30, who underwent artificial abortion during 6-8 weeks of gestation, were randomly divided into 2 experimental groups: All 30 cases were exposed to diagnostic ultrasound in uterus for 10 minutes, and 24 hours later chorionic villi were extracted; the other 30 cases were taken as the control group. Single-stranded DNA and double-stranded DNA in villus cells in all cases were isolated by the alkaline unwinding combined with hydroxylapatite chromatography, and were quantitatively detected using 32 P-labeled Alu probe for dot-blotting hybridization. Results There was no significant difference in quantity and percentage in single-stranded DNA and double-stranded DNA between 2 groups (P>0.05). 32 P-Alu probe could only hybridize with human DNA, and could detect DNA isolated from as few as 2.5×10 3 chorionic villus cells and 0.45ng DNA in human leukocytes. Conclusion The results suggested that there were no DNA strand damages in human chorionic villus cells when the uterus was exposed to diagnostic ultrasound for 10 minutes. The method,^(32)P-Alu probe for dot-blotting hybridization, was even more specific, sensitive and accurate than conventional approaches.
文摘Systematic analysis of factors determining efficiency in discrimination of a point substitution (SNP) within specific DNA sequences was carried out in the context of hybridization approach. There are two types of selectivity that are critical for the rational design of highly specific oligonucleotides probes. The first type is the real selectivity of hybridization (fa) that is the ratio of association degrees of targets with an oligonucleotide probe upon the perfect and imperfect complex formation. This type of selectivity reflects the level of discrimination between matched and mismatched signals, which is determined both by experimental conditions and the thermodynamics of oligonucleotide hybridization. The second parameter characterizing the efficiency of SNP discrimination is the limit selectivity of hybridization, which determines the utmost value of fa at a given temperature. This value can be calculated as the ratio of corresponding equilibrium association constants of perfect and imperfect complex formation determined purely by thermodynamics. We have shown that the fa function is the most reliable characteristic describing the hybridization selectivity. For the analytical system designed to reveal any type of perturbation in DNA (e.g. SNP or modification), there is usually a temperature at which fa has its maximum value. The dependency of the fa maximum on different experimental parameters as well as the structural characteristics of a probe are described in details. The results allowed us to postulate points of principle to rationally design the most selective probes on the basis of oli- gonucleotides or their derivatives.
基金Project supported by the Strategic Priority Research Program of China(Grant No.XDB01010200)the National Natural Science Foundation of China(Grant Nos.61605225,11674337,and 11547035)Natural Science Foundation of Shanghai,China(Grant No.16ZR1448400)
文摘We propose a scheme that can generate tunable double optomechanically induced transparency in a hybrid optomechanical cavity system.In this system, the mechanical resonator of the optomechanical cavity is coupled with an additional mechanical resonator and the additional mechanical resonator can be driven by a weak external coherently mechanical driving field.We show that both the intensity and the phase of the external mechanical driving field can control the propagation of the probe field, including changing the transmission spectrum from double windows to a single-window.Our study also provides an effective way to generate intensity-controllable, narrow-bandwidth transmission spectra, with the probe field modulated from excessive opacity to remarkable amplification.
基金supported by National Natural Science Foundation of China (No. 10635010)Specialized Research Fund for the Doctoral Program of Higher Education of China (No.20090041110026)
文摘A two-dimensional hybrid simulation scheme is proposed to study the characteristics of dual-frequency (DF) capacitively coupled plasma (CCP) discharge based on the geometry of real device. Given the experimental parameters for argon plasma, the output from the fluid module such as ion density, number flux, electron temperature and the Monte-Carlo collision (MCC) results of ion energy distribution function (IEDF) as well as electron energy distribution function (EEDF) are obtained and discussed in detail. A novel complete floating double probe is designed to measure both density and temperature of electron and a quadrupole mass spectrometer is also equipped for IEDF investigations. The measurements on the density of bulk plasma, electron temperature and IEDF agree well, qualitatively, with the simulated results. A comparison with experimental results indicates that, since the structure of real device is taken into account, this model is capable of describing the global dynamic characteristics occurred in DF-CCP and presenting more reliable results than the model with an ideal chamber structure.
文摘To study the technique of D-FISH and its application in the diagnosis of a 49, XXXXY chromosomal abnormality. Methods: Biotin-labeled alpha satellite X chromosome DNA (pBamX7) probe and digoxi-genin-labeled Y chromosome long arm terminal repetitive sequence (pY3.4) probe in situ hybridized with pre-treated slides of peripheral blood chromosome and interphase nucleus. After washing, the slides were treated with avidin-FITC, rhodamine-FITC and anti-avidin, amplified with an additional layer and counter-stained with DAPI in an antifade solution. The hy bridization signals and chromosomal or interphase nucleus settings were observed respectively with WIB, WIG and WU filters under fluorescent microscope (Olympus AX-70) and the number of metaphase chromosome and interphase nucleus in the peripheral blood was counted. Results: The biotin-labeled pBamX7 probe showed 4 green hybridization signal and the digoxigenin-labeled pY3.4 probe showed 1 red hybridization signal. The chromosome or cytoplasm counter-stained with DAPI showed blue. The positive rate of X chromosome hybridization signal for the 350 metaphase chromosomes and interphase nucleus was 91.43 % and 92. 57 %, respectively, while that of the Y chromosome hybridization signal was 99.5 % and 99.8 %, respectively. Conclusion: D-FISH is a valuable technique in diagnosing 49, XXXXY chromosomal abnormality and other sex chromosomal abnormalities. [Reprod Contracep (in Chinese) 2002; 22: 287]
文摘The Plasmodtum falctparum DNA fragment was isolated from a cloned recombinant plasmid pPF14. labelled with Digoxigenin (Dig) and used as a probe (pPF14-F-Dig)to detect 274 blood samples from the eqdemic area population in Hainan Province by dot hybridization.The results showed that out of 274 blood specimens one was positive when using microscopic examination and the positivity rate was 0.36 percent. Fifteen samples showed to be positive (including one positive specimen examined by microscopy) when this probe was applied to detect the 274 samples and its positivity rate was 5.47 percent.The positive coincidence rate between pPF14-F-Dig and microscopic examination was 1/1 and the negative coincidence rate was 94.87 percent. Since a piece of nitrocellulose membrane with the size of 9 by 12 square centimetres can accommodate blots of 96 samples,this probe is fit for large-scale epidemiological surveys.