In situ mRNA hybridization(ISH)is a powerful tool for examining the spatiotemporal expression of genes in shoot apical meristems and flower buds of cucumber.The most common ISH protocol uses paraffin wax;however,embed...In situ mRNA hybridization(ISH)is a powerful tool for examining the spatiotemporal expression of genes in shoot apical meristems and flower buds of cucumber.The most common ISH protocol uses paraffin wax;however,embedding tissue in paraffin wax can take a long time and might result in RNA degradation and decreased signals.Here,we developed an optimized protocol to simplify the process and improve RNA sensitivity.We combined embedding tissue in low melting-point Steedman’s wax with processing tissue sections in solution,as in the whole-mount ISH method in the optimized protocol.Using the optimized protocol,we examined the expression patterns of the CLAVATA3(CLV3)and WUSCHEL(WUS)genes in shoot apical meristems and floral meristems of Cucumis sativus(cucumber)and Arabidopsis thaliana(Arabidopsis).The optimized protocol saved 4–5 days of experimental period compared with the standard ISH protocol using paraffin wax.Moreover,the optimized protocol achieved high signal sensitivity.The optimized protocol was successful for both cucumber and Arabidopsis,which indicates it might have general applicability to most plants.展开更多
Objective:To evaluate the diagnostic value of fluorescence in situ hybridization(FISH)in bladder cancer.Methods:We enrolled healthy volunteers and patients who were clinically suspected to have bladder cancer and cond...Objective:To evaluate the diagnostic value of fluorescence in situ hybridization(FISH)in bladder cancer.Methods:We enrolled healthy volunteers and patients who were clinically suspected to have bladder cancer and conducted FISH tests and cytology examinations from August 2007 to December 2008.Receiver operating characteristic(ROC)curve analysis was performed and the area under curve(AUC)values were calculated for both the FISH and urine cytology tests.Results:A cohort of 988 healthy volunteers was enrolled to establish a reference range for the normal population.A total of 4807 patients with hematuria were prospectively,randomly enrolled for the simultaneous analysis of urine cytology,FISH testing,and a final diagnosis as determined by the pathologic findings of a biopsy or a surgically-excised specimen.Overall,the sensitivity of FISH in detecting transitional-cell carcinoma was 82.7%,while that of cytology was 33.4%(p<0.001).The sensitivity values of FISH for non-muscle invasive and muscle invasive bladder transitional-cell carcinoma were 81.7%and 89.6%,respectively(p=0.004).The sensitivity values of FISH for low and high grade bladder cancer were 82.6%and 90.1%,respectively(p=0.002).Conclusion:FISH is significantly more sensitive than voided urine cytology for detecting bladder cancer in patients evaluated for gross hematuria at all cancer grades and stages.Higher sensitivity using FISH was obtained in high grade and muscle invasive tumors.展开更多
A microbial community structure of granules harvested from an anaerobic sludge blanket reactor treating phenolic wastewater was investigated using fluorescence in situ hybridization(FISH)and clone library construction...A microbial community structure of granules harvested from an anaerobic sludge blanket reactor treating phenolic wastewater was investigated using fluorescence in situ hybridization(FISH)and clone library construction.Clones of Syntrophorhabdaceae and Cryptanaerobacter were observed to be responsible for phenol degradation.For accurate taxonomic assignment of Cryptanaerobacter clones,phylogenetic analysis using nearly full-length 16S ribosomal RNA(rRNA)gene sequences was necessary.Three oligonucleotide probes were designed to detect the following three taxonomic groups:Syntrophorhabdaceae,Cryptanaerobacter,and Syntrophus.FISH analysis of thin sections of anaerobic granules showed a random distribution of bacteria and archaea.However,a well-defined distribution of Syntrophorhabdaceae,Cryptanaerobacter,and Syntrophus was observed.Cryptanaerobacter and Syntrophus were found on the outer layer of the granules and were closely associated with each other,while Syntrophorhabdaceae was located in the deeper part of the granules.Such specific distribution of the bacteria is most likely due to their metabolic association and affinity for the substrate.Phenol degradation in the granular sludge was observed to be carried out in the following way.First,Cryptanaerobacter converts phenol to benzoate,which is then degraded by Syntrophus into acetate.This syntrophic degradation of phenol occurs near the surface of the granule,where the phenol concen-tration is high.In the deeper part of the granule,where the phenol concentration is lower,Syntrophorhabdaceae degrades phenol into acetate.We observed that Syntrophorhabdaceae is less likely to produce benzoate as an intermediate to feed the neighboring organisms,which contradicts the theo-ries presented by previous studies.展开更多
For sludge samples from EBPR reactor fed with municipal wastewater,fluorescent in situ hybridization (FISH) operation process including moisture chamber,pretreatment,treatment with lysozyme and Proteinase K and washin...For sludge samples from EBPR reactor fed with municipal wastewater,fluorescent in situ hybridization (FISH) operation process including moisture chamber,pretreatment,treatment with lysozyme and Proteinase K and washing time was optimized and improved.Preserving box was chosen to be moisture chamber due to its bigger depth /radii ratio,good sealability and big volume contrast with Petri dish.3-5 mm diameter glass balls could disperse samples without destroying microorganism cells and community structure.Impurities and ECPs could be removed easily and sludge samples became thinner after dispersing which benefit the observation.Permeabilized cells with lysozyme and Proteinase K could enhance probe penetration before hybridization.Experiments of different treatment time with lysozyme and Proteinase K were carried out.Best results were observed when sludge samples treated with lysozyme 10 min/Proteinase K 20 min or lysozyme 20 min/Proteinase K 10 min.Slides were washed at 48 ℃ for 10,20,30,40 and 60 min in parallel.The best washing time was 20 min when washing temperature was 48 ℃.Fluorescent dye could residue when washing time was 10 min and washing out happened when washed for 30 min or more.展开更多
In order to explore researches about the chromosome karyotype analysis and fluorescence in situ hybridization(FISH) technology in China,using the bibliometric method,taking " fluorescence in situ hybridization(FI...In order to explore researches about the chromosome karyotype analysis and fluorescence in situ hybridization(FISH) technology in China,using the bibliometric method,taking " fluorescence in situ hybridization(FISH) " and " chromosome" as key words,this paper made a statistical analysis on the literature published in China National Knowledge Infrastructure(CNKI) during 2002-2016.The results indicated that the number of papers published in 2002 was the smallest(37),while the number of papers published in 2012 was the largest(125).In terms of the distribution of organizations of authors,in 1201 papers,11 organizations published papers ≥15,accounting for 21.65%.In terms of distribution of papers published by different periodicals,11 periodicals published papers ≥10,accounting for 17.65%.In terms of the papers supported by foundation projects,in all papers searched,377 papers were supported by foundation projects,accounting for 31.39%.In terms of the distribution of doctoral and master's dissertations,259 papers were master's dissertations,accounting for 21.57%;92 papers were doctoral dissertations,accounting for 7.66%.展开更多
It has been reported that there was a linkage of 5S rRNA gene to 18S-5.8S-25S rRNA gene in a few of species in Ochrophyta.In regard to the usual two positions of linked 5S rDNA to the 3′end of 25S rDNA,two pairs of p...It has been reported that there was a linkage of 5S rRNA gene to 18S-5.8S-25S rRNA gene in a few of species in Ochrophyta.In regard to the usual two positions of linked 5S rDNA to the 3′end of 25S rDNA,two pairs of primers were designed for amplification to verify this linkage of two genes in a kelp cultivar of Saccharina japonica,one of species in Ochrophyta.This result supplemented the previous report that 5S rDNA was unlinked to 25S rDNA in this kelp.In order to simultaneously visualize this unlinkage of two genes,dual-color fluorescence in situ hybridization(FISH)technique was applied to the cytogenetics of S.japonica.Dual-color FISH images showed that two and four hybridization signals were present in the kelp gametophyte and sporophyte,respectively,metaphase nuclei hybridized simultaneously with the labeled probes of 18S rDNA and 5S rDNA.Both haploid and diploid karyotypes in decreasing length of chromosomes showed that 18S-5.8S-25S rDNA was localized at the interstitial region of Chromosome 23,whereas 5S rDNA resided at the sub-telomeric region of Chromosome 27.These karyotypes suggested that the kelp nuclear genome had only one locus of each rRNA gene,and their loci on different chromosomes indicated the physical unlinkage of 5S rDNA to 18S-5.8S-25S rDNA in this kelp.Therefore,dual-color FISH seems to be a powerful technique for the discrimination and pairing of chromosomes featured in both small size and nearly identical shape in S.japonica.展开更多
BACKGROUND Although acute graft-vs-host disease(aGvHD)is a rare complication of liver transplantation,it is poorly understood and has an extremely high mortality rate.No standardized diagnostic criteria or treatment r...BACKGROUND Although acute graft-vs-host disease(aGvHD)is a rare complication of liver transplantation,it is poorly understood and has an extremely high mortality rate.No standardized diagnostic criteria or treatment regimens currently exist.CASE SUMMARY The present study investigated the etiology,diagnosis,and treatment of aGvHD following liver transplantation.Presentation,diagnosis,disease course,histology,and treatment of an aGvHD case are reported,and associated literature is reviewed.A 64-year-old female required LTx due to primary biliary cirrhosis.The donor was a 12-year-old male.Three weeks following liver transplantation,the recipient developed pyrexia,diarrhea,rashes,and antibiotic-unresponsive pancytopenia.Clinical symptoms together with laboratory investigations suggested a diagnosis of aGvHD,which was confirmed via peripheral blood fluorescent in situ hybridization.Donor XY chromosome fluorescent in situ hybridization indicating early chimerism achieved 93%sensitivity in the detection of GvHD.Existing immunosuppressants were discontinued,and high-dose intravenous methylprednisolone was initiated along with antibiotics.While diarrhea resolved,the patient’s general condition continued to deteriorate until demise due to multi-system organ failure at 37 d post-liver transplantation.This case illustrates the life-threatening nature of aGvHD.CONCLUSION Herein,we have summarized a post-LTx aGvHD case and reviewed associated literature in order to increase awareness and provide potentially risk-mitigating recommendations.展开更多
A total of 80 piglets (7.9 ± 1.0 kg) were used in a feeding experiment with dried oregano. The diets differed in their oregano content: 0 g, 2 g, 4 g and 8 g oregano/kg feed, corresponding to 0, 23.5, 46.9 and 93...A total of 80 piglets (7.9 ± 1.0 kg) were used in a feeding experiment with dried oregano. The diets differed in their oregano content: 0 g, 2 g, 4 g and 8 g oregano/kg feed, corresponding to 0, 23.5, 46.9 and 93.9 mg carvacrol/kg DM. After the experimental period of 5 weeks, 20 piglets of both extreme feeding groups were slaughtered: 10 animals of the control group and 10 animals of the group that received 8 g oregano/kg. Ingesta samples of jejunum, caecum and colon were collected and analyzed by FISH and PCR RFLP to compare the diversity of microbiota. The results showed no significant changes in microbiota in response to oregano. The patterns of the PCR-RFLP showed a similarity of 61.8% - 91.8% in both feeding groups. In conclusion, an effect of oregano on the in- testinal microbiota could not be shown under the methods used.展开更多
Fluorescence in situ hybridization (FISH) has become an important tool both for defining initial chromosomal abnormalities within a disease process, and for monitoring response to therapy as well as minimal residual d...Fluorescence in situ hybridization (FISH) has become an important tool both for defining initial chromosomal abnormalities within a disease process, and for monitoring response to therapy as well as minimal residual disease. We report the results of interphase FISH (iFISH) analysis of 92 patients. We have used five different FISH probes to detect common cytogenetic rearrangements associated with hematological malignancies. A total of 83 patients were screened for BCR/ABL gene rearrangements. Displayed iFISH patterns of BCR/ABL gene rearrangements in 37.3% of patients (31/83) ranged between 10% to 98%. In addition, while 3 patients and one patient with AML showed t(15;17) (12.5%) and inv(16;16) (8.3%) respectively, t(8;21) was not found. Furthermore, secondary chromosomal aberrations (6.5% of all cases) were clearly non random in the present study. The diagnosis of BCR/ABL gene rearrangements are likely become an important tool for the monitoring of therapies in patients with CML. Atypical patterns also may have clinical prognostic implications. Further studies in larger groups of patients are needed in order to elucidate the role of AML1/ETO, PML/RARA, CBFB and p53, and to identify the specific chromosomal regions and interacting genes involved in this process.展开更多
BACKGROUND: Molecular cytogenetics of oncogene HER-2 amplification in primary hepatocellular carcinoma (HCC) is still unknown. The aim of this study was to in vestigate the frequency of HER-2 oncogene amplification in...BACKGROUND: Molecular cytogenetics of oncogene HER-2 amplification in primary hepatocellular carcinoma (HCC) is still unknown. The aim of this study was to in vestigate the frequency of HER-2 oncogene amplification in primary HCC and its relations to clinicopathological pa rameters and prognosis. METHODS: Forty-two surgical samples from patients with primary HCC were detected for their HER-2 oncogene am plification. The number of chromosome 17 and their ratio were tested by dual fluorescence in situ hybridization (FISH) technique, and then the correlations between HER-2 amplification, clinicopathological characteristics and prog nosis were analyzed statistically. RESULTS: HER-2 oncogene amplification was detected in 9 (21.4%) of the 42 primary HCCs, including 4 patient with high copy (HC) (9.5%) and 5 patients with low copy (LC) (11.9%). HER-2 amplification was associated signifi cantly with tumor size and postoperative survival time o HCC patients (P<0.05), and the presence of HER-2 gene amplification was correlated with postoperative relapse (P— 0.257), but not related to sex, age, AFP level, HBV infec tion, histopathological grading and clinical staging of HCC patients (P>0.05). The HER-2 oncogene copy was exa mined in 31 (73.8%) of the 42 primary HCCs, consisting of 9 patients with HER-2 amplification (21.4%) and 22 pa tients with aneuploidy (52.4%). No significant relation were observed between the HER-2 oncogene copy, patien sex, tumor size, histopathological grading, clinical stag ing, postoperative relapse and survival time (P >0.05); bu the HER-2 oncogene copy was correlated significantly to age, AFP level and HBV infection (P <0.05). CONCLUSIONS: There are a lower frequency of HER-2 oncogene amplification and a higher frequency of chromo- some 17 aneuploidy in primary HCC. HER-2 oncogene amplification may be involved in the development and pro- gression of large HCC in some patients, and seems to be a valuably independent prognostic factor predicting the re- currence and poor survival in patients with large HCC.展开更多
White Spot Syndrome Virus (WSSV) was purified from hemolymph of infected shrimp. After nucleic acid extraction from the purified virus particles, EcoR I digested fragments of the WSSV genome were cloned; three of thes...White Spot Syndrome Virus (WSSV) was purified from hemolymph of infected shrimp. After nucleic acid extraction from the purified virus particles, EcoR I digested fragments of the WSSV genome were cloned; three of these fragments were used as non radioactive probes labeled with DIG 11 dUTP. The probes hybridized in situ, with sections located in the nuclei of all WSSV infected tissues. The virus was detected in the gill, stomach, epidermis, and connective tissue and so on, but not detected in healthy shrimp tissues and epithelial cells of hepatopancreatic tubules of diseased shrimp.展开更多
H pylori is etiologically associated with gastritis, gas-tric and duodenal ulcers, gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. Eradicating H pylori may convert rapidly the outcome of ...H pylori is etiologically associated with gastritis, gas-tric and duodenal ulcers, gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. Eradicating H pylori may convert rapidly the outcome of related diseases with the use of more accurate diagnostic molecular tests. Indeed some of the tests cannot give the evidence of current infection; H pylori can be detected by noninvasive and invasive methods, the latter requiring an endoscopy. Eradication failure is a big problem in H pylori infection. Recently, clarithromycin resistance in H pylori strains is increasing and eradicati-on therapy of this bacterium is becoming more difficult. Molecular methods have frequently been applied besides phenotypic methods for susceptibility testing to detect clarithromycin resistance due to mutations in the 2143 and 2144 positions of 23S rRNA gene. Fluorescence in situ hybridization (FISH) method on paraffin embedded tissue is a rapid, accurate and cost-effective method for the detection of H pylori infection and to determine clarithromycin resistance within three hours according to the gold standards as a non-culture method. This method can also be applied to fresh biopsy samples and the isolated colonies from a culture of H pylori, detecting both the culturable bacillary forms and the coccoid forms of H pylori, besides the paraffin embedded tissue secti-ons. This technique is helpful for determining the bac-terial density and the results of treatment where clarith-romycin has been widely used in populations to increase the efficacy of the treatment and to clarify the treatment failure in vitro.展开更多
Longiflorum and Asiatic lilies of the genus Lilium of the family Liliaceae are two important groups of modern lily cultivars.One of the main trends of lily breeding is to realize introgression between these groups.Wit...Longiflorum and Asiatic lilies of the genus Lilium of the family Liliaceae are two important groups of modern lily cultivars.One of the main trends of lily breeding is to realize introgression between these groups.With cut style pollination and embryo rescue,distant hybrids between the two groups have been obtained.However,the F1 hybrids are highly sterile or some of them could produce a small number of 2n gametes,and their BC1 progenies are usually triploids.Dutch lily breeders have selected many cultivars from these BC1 progenies based on their variation.It is presumably suggested that such variation could be caused by intergenomic recombination and abnormal meiosis during gamete formation in F1 hybrids of Longiflorum × Asiatic(LA) hybrids in Lilium.Therefore,the meiotic process of ten F1 LA hybrids was cytologically investigated using genomic in situ hybridization and traditional cytological methods in the present research.The results showed that:at metaphase I,the homoeologous chromosome pairing among different F1 hybrids ranged from 2.0 to 11.4 bivalents formed by homoeologous chromosomes per pollen mother cell(PMC),and very few multivalents,and even very few bivalents were formed by two chromosomes within one genome rather than homoeologous chromosomes in some PMCs;at anaphase I,all bivalents were disjoined and most univalents were divided.Both the disjoined bivalents(half-bivalents) and the divided univalents(sister chromatids) moved to the opposite poles,and then formed two groups of chromosomes;because the two resulting half-bivalents retained their axes in the cell undisturbed,many crossover types,including single crossovers,three strand double crossovers,four strand double crossovers,four strand triple crossovers,and four strand multiple crossovers between the non-sister chromatids in the tetrads of bivalents,were clearly inferred by analyzing the breakpoints on the disjoined bivalents.The present investigation not only explained the reason for sterility of the F1 LA hybrids and the variation of their BC1 progenies,but also provided a new method to analyze crossover types in other F1 interspecific hybrids as well.展开更多
Chromosome aberrations are distinctive features of human malignant tumors. Analysis of chromosomal changes can illuminate the molecular mechanisms underlying the development and progression of cancer. To establish the...Chromosome aberrations are distinctive features of human malignant tumors. Analysis of chromosomal changes can illuminate the molecular mechanisms underlying the development and progression of cancer. To establish the technique of multicolor fluorescence in situ hybridization (M-FISH) for identifying chromosome aberrations in esophageal carcinoma cell line KYSE 410-4, four pools of 6-color whole-chromosome painting probes have been designed and hybridized on the same metaphase spread by four rounds of repetitive FISH. Repetitive 6-color M-FISH was successfully established and the cytogenetic abnormalities in KYSE 410-4 cells were characterized. Chromosome gains occurred at 2q, 3, 8, 17p, and X. An isochromosome 3q was visualized in the cell line, which might be one intermedi- ate mechanism leading to 3p losses and/or 3q gains. Furthermore, 16 structural arrangements were detected, including four derivative chromosomes. The rearrangement of the centromeric regions accounted for approximately 44% of all rearrangements. The results added a more complete and accurate information of the genetic alterations to the classical cytogenetic description of KYSE 410-4 and provided a detailed cytogenetic background data for appropriate use of the cell line. The established 6-color M-FISH was useful for analyzing chro- mosomes in the whole genome of human tumors.展开更多
The accurate assessment of a proto-oncogene, human epidermal growth factor receptor-2 gene (HER-2), is extremely important for the therapy and prognosis of breast cancer. Currently, im-munohistochemistry (IHC) is the ...The accurate assessment of a proto-oncogene, human epidermal growth factor receptor-2 gene (HER-2), is extremely important for the therapy and prognosis of breast cancer. Currently, im-munohistochemistry (IHC) is the method widely used for the detection of HER-2 protein. Fluores-cence in situ hybridization (FISH) has been suggested to be a golden standard assay for HER-2 am-plification. This study examined the expression and amplification of HER-2 in paraffin-embedded sections of breast cancer tissues, and compared the two methods on the measurement of HER-2 status. HER-2 gene and protein were determined in breast cancer samples from 52 Chinese women by FISH and IHC respectively. The findings indicated that the HER-2 gene amplification was found in 18 cases (34.6%) by FISH and the HER-2 protein over-expression (score 3+) in 15 cases (28.8%) by IHC. Immunohistochemically, 28.6% of the cases scored as 2+ and 93.3% of the cases scored as 3+ were HER-2-positive by FISH. There was a significant correlation between the HER-2 gene amplifi-cation and HER-2 protein over-expression in breast cancer (P<0.005). No correlation was noted be-tween the HER-2 gene amplification and any of the clinicopathological parameters examined, in-cluding age, menopausal status, menarche age, tumor size, histological tumor type, histological grade, lymph node status, and the expression of ER and PR. It was concluded that the detection of HER-2 gene amplification in breast cancer by FISH is valuable and can compare with HER-2 protein detec-tion by IHC.展开更多
AIM: To investigate the presence of H. pylori DNA withingastric epithelial cells in patients with H. pylori infection andits possible carcinogenic mechanism.METHODS: Total 112 patients, with pathologically confirmedch...AIM: To investigate the presence of H. pylori DNA withingastric epithelial cells in patients with H. pylori infection andits possible carcinogenic mechanism.METHODS: Total 112 patients, with pathologically confirmedchronic superficial gastritis, chronic atrophic gastritis,intestinal metaplasia, atypical hyperplasia or gastrio cancerwere studied .Among them, 28 were H. pylori negative and84 H. pylori positive. H. pylori DNA in gastric epithelialcells was detected by GenPoint catalyzed signalamplification system for in situ hybridization.RESULTS: In the H. pylori positive group, zero out of 24chronic superficial gastritis (0. 0 %), four out of 25precancerous changes (16.0 %) and thirteen out of 35gastric cancers (37. 1 %) showed H. pylori DNA in thenucleus of gastric epithelial cells, the positive rates of H.pylori DNA in the nucleus of gastric epithelial cells wereprogressively inoreased in chronic superficial gastritis,precancerous changes and gastric cancer groups (χ2 = 12.56, P = 0. 002); One out of 24 ohronic superficial gastritis(4.2 %), eleven out of 25 precancerous ohangas (44.0 %)and thirteen out of 35 gastric cancers (37. 1 %) showed H.pylori DNA in the cytoplasm of gastric epithelial cells (χ2 =10.86, P = 0.004). In the H. pylorinegative group, only onepatient with gastric cancer was found H. pylori DNA in thenucleus of gastric epithelial cells; Only two patients, onepatient with precancerous changes and another with gastriccancer, showed H. pylori DNA in the cytoplasm of gastricepithelial calls. Furthermore, H. pylori DNA must have been inthe ayteplasm as long as it existed in the nucleus of gastricepithelial cells.CONCLUSION: H. pyloriDNA exists both in the nucleus andthe cytoplasm of gastric epithelial cells in patients with H.pylori infections. The pathological progression from chronicsuperficial gastritis, precancerous changes to gastric canceris associated with higher positive rates of H. pylori DNApresence in the nucleus of gastric epithelial cells.展开更多
Objective: To establish a method to improve the detection of disseminated tumor cells in bone marrow and peripheral blood samples of neuroblastoma patients and analysis of cytogenetic aberration. Methods: Immunofluore...Objective: To establish a method to improve the detection of disseminated tumor cells in bone marrow and peripheral blood samples of neuroblastoma patients and analysis of cytogenetic aberration. Methods: Immunofluorescent staining was performed using a cocktail of primary monoclonal neuroblastoma antibodies (14.G2a, 5.1H11). Fluorescence in situ hybridization was applied with fluorescent probes specific for Nmyc genes afterwards. A novel computer assisted scanning system for automatic search, image analysis and repositioning of these positive cells was developed. Fifty-six bone marrow and peripheral blood samples from 7 patients were evaluated by this method. Results: Fluorescence in situ hybridization can be combined with immunofluorescent staining in detecting Nmyc amplification in neuroblastoma patients. Fluorescence in situ hybridization results correlated well with data obtained by conventional cytogenetic procedures. Conclusion: The technique described allows search of tumor cells in the bone marrow as well as detection of Nmyc amplification in interphase nuclei.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
基金supported by the National Natural Science Foundation of China(32002036)。
文摘In situ mRNA hybridization(ISH)is a powerful tool for examining the spatiotemporal expression of genes in shoot apical meristems and flower buds of cucumber.The most common ISH protocol uses paraffin wax;however,embedding tissue in paraffin wax can take a long time and might result in RNA degradation and decreased signals.Here,we developed an optimized protocol to simplify the process and improve RNA sensitivity.We combined embedding tissue in low melting-point Steedman’s wax with processing tissue sections in solution,as in the whole-mount ISH method in the optimized protocol.Using the optimized protocol,we examined the expression patterns of the CLAVATA3(CLV3)and WUSCHEL(WUS)genes in shoot apical meristems and floral meristems of Cucumis sativus(cucumber)and Arabidopsis thaliana(Arabidopsis).The optimized protocol saved 4–5 days of experimental period compared with the standard ISH protocol using paraffin wax.Moreover,the optimized protocol achieved high signal sensitivity.The optimized protocol was successful for both cucumber and Arabidopsis,which indicates it might have general applicability to most plants.
基金This clinical research was funded by the Ministry of Health People’s Republic of China(No.WKJ2007-3-001).
文摘Objective:To evaluate the diagnostic value of fluorescence in situ hybridization(FISH)in bladder cancer.Methods:We enrolled healthy volunteers and patients who were clinically suspected to have bladder cancer and conducted FISH tests and cytology examinations from August 2007 to December 2008.Receiver operating characteristic(ROC)curve analysis was performed and the area under curve(AUC)values were calculated for both the FISH and urine cytology tests.Results:A cohort of 988 healthy volunteers was enrolled to establish a reference range for the normal population.A total of 4807 patients with hematuria were prospectively,randomly enrolled for the simultaneous analysis of urine cytology,FISH testing,and a final diagnosis as determined by the pathologic findings of a biopsy or a surgically-excised specimen.Overall,the sensitivity of FISH in detecting transitional-cell carcinoma was 82.7%,while that of cytology was 33.4%(p<0.001).The sensitivity values of FISH for non-muscle invasive and muscle invasive bladder transitional-cell carcinoma were 81.7%and 89.6%,respectively(p=0.004).The sensitivity values of FISH for low and high grade bladder cancer were 82.6%and 90.1%,respectively(p=0.002).Conclusion:FISH is significantly more sensitive than voided urine cytology for detecting bladder cancer in patients evaluated for gross hematuria at all cancer grades and stages.Higher sensitivity using FISH was obtained in high grade and muscle invasive tumors.
基金supported by Grant-in-Aids for Scientific Research(B)(JP18H01564)from the Japan Society for the Promotion of Science.
文摘A microbial community structure of granules harvested from an anaerobic sludge blanket reactor treating phenolic wastewater was investigated using fluorescence in situ hybridization(FISH)and clone library construction.Clones of Syntrophorhabdaceae and Cryptanaerobacter were observed to be responsible for phenol degradation.For accurate taxonomic assignment of Cryptanaerobacter clones,phylogenetic analysis using nearly full-length 16S ribosomal RNA(rRNA)gene sequences was necessary.Three oligonucleotide probes were designed to detect the following three taxonomic groups:Syntrophorhabdaceae,Cryptanaerobacter,and Syntrophus.FISH analysis of thin sections of anaerobic granules showed a random distribution of bacteria and archaea.However,a well-defined distribution of Syntrophorhabdaceae,Cryptanaerobacter,and Syntrophus was observed.Cryptanaerobacter and Syntrophus were found on the outer layer of the granules and were closely associated with each other,while Syntrophorhabdaceae was located in the deeper part of the granules.Such specific distribution of the bacteria is most likely due to their metabolic association and affinity for the substrate.Phenol degradation in the granular sludge was observed to be carried out in the following way.First,Cryptanaerobacter converts phenol to benzoate,which is then degraded by Syntrophus into acetate.This syntrophic degradation of phenol occurs near the surface of the granule,where the phenol concen-tration is high.In the deeper part of the granule,where the phenol concentration is lower,Syntrophorhabdaceae degrades phenol into acetate.We observed that Syntrophorhabdaceae is less likely to produce benzoate as an intermediate to feed the neighboring organisms,which contradicts the theo-ries presented by previous studies.
基金Sponsored by the National Natural Science Foundation of China (Grant No. 50638020)
文摘For sludge samples from EBPR reactor fed with municipal wastewater,fluorescent in situ hybridization (FISH) operation process including moisture chamber,pretreatment,treatment with lysozyme and Proteinase K and washing time was optimized and improved.Preserving box was chosen to be moisture chamber due to its bigger depth /radii ratio,good sealability and big volume contrast with Petri dish.3-5 mm diameter glass balls could disperse samples without destroying microorganism cells and community structure.Impurities and ECPs could be removed easily and sludge samples became thinner after dispersing which benefit the observation.Permeabilized cells with lysozyme and Proteinase K could enhance probe penetration before hybridization.Experiments of different treatment time with lysozyme and Proteinase K were carried out.Best results were observed when sludge samples treated with lysozyme 10 min/Proteinase K 20 min or lysozyme 20 min/Proteinase K 10 min.Slides were washed at 48 ℃ for 10,20,30,40 and 60 min in parallel.The best washing time was 20 min when washing temperature was 48 ℃.Fluorescent dye could residue when washing time was 10 min and washing out happened when washed for 30 min or more.
基金Supported by Project of National Natural Science Foundation(31160292)Crop Discipline Construction Project of Tibet Agricultural and Animal Husbandry College(2015ZWXKJS&2016ZWXKJS)
文摘In order to explore researches about the chromosome karyotype analysis and fluorescence in situ hybridization(FISH) technology in China,using the bibliometric method,taking " fluorescence in situ hybridization(FISH) " and " chromosome" as key words,this paper made a statistical analysis on the literature published in China National Knowledge Infrastructure(CNKI) during 2002-2016.The results indicated that the number of papers published in 2002 was the smallest(37),while the number of papers published in 2012 was the largest(125).In terms of the distribution of organizations of authors,in 1201 papers,11 organizations published papers ≥15,accounting for 21.65%.In terms of distribution of papers published by different periodicals,11 periodicals published papers ≥10,accounting for 17.65%.In terms of the papers supported by foundation projects,in all papers searched,377 papers were supported by foundation projects,accounting for 31.39%.In terms of the distribution of doctoral and master's dissertations,259 papers were master's dissertations,accounting for 21.57%;92 papers were doctoral dissertations,accounting for 7.66%.
基金Supported by the National Natural Science Foundation of China(Nos.41376136,31201992)the Double First-Class Discipline of Fisheries Science in China。
文摘It has been reported that there was a linkage of 5S rRNA gene to 18S-5.8S-25S rRNA gene in a few of species in Ochrophyta.In regard to the usual two positions of linked 5S rDNA to the 3′end of 25S rDNA,two pairs of primers were designed for amplification to verify this linkage of two genes in a kelp cultivar of Saccharina japonica,one of species in Ochrophyta.This result supplemented the previous report that 5S rDNA was unlinked to 25S rDNA in this kelp.In order to simultaneously visualize this unlinkage of two genes,dual-color fluorescence in situ hybridization(FISH)technique was applied to the cytogenetics of S.japonica.Dual-color FISH images showed that two and four hybridization signals were present in the kelp gametophyte and sporophyte,respectively,metaphase nuclei hybridized simultaneously with the labeled probes of 18S rDNA and 5S rDNA.Both haploid and diploid karyotypes in decreasing length of chromosomes showed that 18S-5.8S-25S rDNA was localized at the interstitial region of Chromosome 23,whereas 5S rDNA resided at the sub-telomeric region of Chromosome 27.These karyotypes suggested that the kelp nuclear genome had only one locus of each rRNA gene,and their loci on different chromosomes indicated the physical unlinkage of 5S rDNA to 18S-5.8S-25S rDNA in this kelp.Therefore,dual-color FISH seems to be a powerful technique for the discrimination and pairing of chromosomes featured in both small size and nearly identical shape in S.japonica.
文摘BACKGROUND Although acute graft-vs-host disease(aGvHD)is a rare complication of liver transplantation,it is poorly understood and has an extremely high mortality rate.No standardized diagnostic criteria or treatment regimens currently exist.CASE SUMMARY The present study investigated the etiology,diagnosis,and treatment of aGvHD following liver transplantation.Presentation,diagnosis,disease course,histology,and treatment of an aGvHD case are reported,and associated literature is reviewed.A 64-year-old female required LTx due to primary biliary cirrhosis.The donor was a 12-year-old male.Three weeks following liver transplantation,the recipient developed pyrexia,diarrhea,rashes,and antibiotic-unresponsive pancytopenia.Clinical symptoms together with laboratory investigations suggested a diagnosis of aGvHD,which was confirmed via peripheral blood fluorescent in situ hybridization.Donor XY chromosome fluorescent in situ hybridization indicating early chimerism achieved 93%sensitivity in the detection of GvHD.Existing immunosuppressants were discontinued,and high-dose intravenous methylprednisolone was initiated along with antibiotics.While diarrhea resolved,the patient’s general condition continued to deteriorate until demise due to multi-system organ failure at 37 d post-liver transplantation.This case illustrates the life-threatening nature of aGvHD.CONCLUSION Herein,we have summarized a post-LTx aGvHD case and reviewed associated literature in order to increase awareness and provide potentially risk-mitigating recommendations.
文摘A total of 80 piglets (7.9 ± 1.0 kg) were used in a feeding experiment with dried oregano. The diets differed in their oregano content: 0 g, 2 g, 4 g and 8 g oregano/kg feed, corresponding to 0, 23.5, 46.9 and 93.9 mg carvacrol/kg DM. After the experimental period of 5 weeks, 20 piglets of both extreme feeding groups were slaughtered: 10 animals of the control group and 10 animals of the group that received 8 g oregano/kg. Ingesta samples of jejunum, caecum and colon were collected and analyzed by FISH and PCR RFLP to compare the diversity of microbiota. The results showed no significant changes in microbiota in response to oregano. The patterns of the PCR-RFLP showed a similarity of 61.8% - 91.8% in both feeding groups. In conclusion, an effect of oregano on the in- testinal microbiota could not be shown under the methods used.
文摘Fluorescence in situ hybridization (FISH) has become an important tool both for defining initial chromosomal abnormalities within a disease process, and for monitoring response to therapy as well as minimal residual disease. We report the results of interphase FISH (iFISH) analysis of 92 patients. We have used five different FISH probes to detect common cytogenetic rearrangements associated with hematological malignancies. A total of 83 patients were screened for BCR/ABL gene rearrangements. Displayed iFISH patterns of BCR/ABL gene rearrangements in 37.3% of patients (31/83) ranged between 10% to 98%. In addition, while 3 patients and one patient with AML showed t(15;17) (12.5%) and inv(16;16) (8.3%) respectively, t(8;21) was not found. Furthermore, secondary chromosomal aberrations (6.5% of all cases) were clearly non random in the present study. The diagnosis of BCR/ABL gene rearrangements are likely become an important tool for the monitoring of therapies in patients with CML. Atypical patterns also may have clinical prognostic implications. Further studies in larger groups of patients are needed in order to elucidate the role of AML1/ETO, PML/RARA, CBFB and p53, and to identify the specific chromosomal regions and interacting genes involved in this process.
基金This study was supported by grants from the National Outstanding YouthFoundation of China (type B, No. 3982511 ) and the Provincial NaturalScience Foundation of Guangdong, China (No. 980107)
文摘BACKGROUND: Molecular cytogenetics of oncogene HER-2 amplification in primary hepatocellular carcinoma (HCC) is still unknown. The aim of this study was to in vestigate the frequency of HER-2 oncogene amplification in primary HCC and its relations to clinicopathological pa rameters and prognosis. METHODS: Forty-two surgical samples from patients with primary HCC were detected for their HER-2 oncogene am plification. The number of chromosome 17 and their ratio were tested by dual fluorescence in situ hybridization (FISH) technique, and then the correlations between HER-2 amplification, clinicopathological characteristics and prog nosis were analyzed statistically. RESULTS: HER-2 oncogene amplification was detected in 9 (21.4%) of the 42 primary HCCs, including 4 patient with high copy (HC) (9.5%) and 5 patients with low copy (LC) (11.9%). HER-2 amplification was associated signifi cantly with tumor size and postoperative survival time o HCC patients (P<0.05), and the presence of HER-2 gene amplification was correlated with postoperative relapse (P— 0.257), but not related to sex, age, AFP level, HBV infec tion, histopathological grading and clinical staging of HCC patients (P>0.05). The HER-2 oncogene copy was exa mined in 31 (73.8%) of the 42 primary HCCs, consisting of 9 patients with HER-2 amplification (21.4%) and 22 pa tients with aneuploidy (52.4%). No significant relation were observed between the HER-2 oncogene copy, patien sex, tumor size, histopathological grading, clinical stag ing, postoperative relapse and survival time (P >0.05); bu the HER-2 oncogene copy was correlated significantly to age, AFP level and HBV infection (P <0.05). CONCLUSIONS: There are a lower frequency of HER-2 oncogene amplification and a higher frequency of chromo- some 17 aneuploidy in primary HCC. HER-2 oncogene amplification may be involved in the development and pro- gression of large HCC in some patients, and seems to be a valuably independent prognostic factor predicting the re- currence and poor survival in patients with large HCC.
文摘White Spot Syndrome Virus (WSSV) was purified from hemolymph of infected shrimp. After nucleic acid extraction from the purified virus particles, EcoR I digested fragments of the WSSV genome were cloned; three of these fragments were used as non radioactive probes labeled with DIG 11 dUTP. The probes hybridized in situ, with sections located in the nuclei of all WSSV infected tissues. The virus was detected in the gill, stomach, epidermis, and connective tissue and so on, but not detected in healthy shrimp tissues and epithelial cells of hepatopancreatic tubules of diseased shrimp.
文摘H pylori is etiologically associated with gastritis, gas-tric and duodenal ulcers, gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. Eradicating H pylori may convert rapidly the outcome of related diseases with the use of more accurate diagnostic molecular tests. Indeed some of the tests cannot give the evidence of current infection; H pylori can be detected by noninvasive and invasive methods, the latter requiring an endoscopy. Eradication failure is a big problem in H pylori infection. Recently, clarithromycin resistance in H pylori strains is increasing and eradicati-on therapy of this bacterium is becoming more difficult. Molecular methods have frequently been applied besides phenotypic methods for susceptibility testing to detect clarithromycin resistance due to mutations in the 2143 and 2144 positions of 23S rRNA gene. Fluorescence in situ hybridization (FISH) method on paraffin embedded tissue is a rapid, accurate and cost-effective method for the detection of H pylori infection and to determine clarithromycin resistance within three hours according to the gold standards as a non-culture method. This method can also be applied to fresh biopsy samples and the isolated colonies from a culture of H pylori, detecting both the culturable bacillary forms and the coccoid forms of H pylori, besides the paraffin embedded tissue secti-ons. This technique is helpful for determining the bac-terial density and the results of treatment where clarith-romycin has been widely used in populations to increase the efficacy of the treatment and to clarify the treatment failure in vitro.
基金the National Natural Science Foundation of China（No.30471222）
文摘Longiflorum and Asiatic lilies of the genus Lilium of the family Liliaceae are two important groups of modern lily cultivars.One of the main trends of lily breeding is to realize introgression between these groups.With cut style pollination and embryo rescue,distant hybrids between the two groups have been obtained.However,the F1 hybrids are highly sterile or some of them could produce a small number of 2n gametes,and their BC1 progenies are usually triploids.Dutch lily breeders have selected many cultivars from these BC1 progenies based on their variation.It is presumably suggested that such variation could be caused by intergenomic recombination and abnormal meiosis during gamete formation in F1 hybrids of Longiflorum × Asiatic(LA) hybrids in Lilium.Therefore,the meiotic process of ten F1 LA hybrids was cytologically investigated using genomic in situ hybridization and traditional cytological methods in the present research.The results showed that:at metaphase I,the homoeologous chromosome pairing among different F1 hybrids ranged from 2.0 to 11.4 bivalents formed by homoeologous chromosomes per pollen mother cell(PMC),and very few multivalents,and even very few bivalents were formed by two chromosomes within one genome rather than homoeologous chromosomes in some PMCs;at anaphase I,all bivalents were disjoined and most univalents were divided.Both the disjoined bivalents(half-bivalents) and the divided univalents(sister chromatids) moved to the opposite poles,and then formed two groups of chromosomes;because the two resulting half-bivalents retained their axes in the cell undisturbed,many crossover types,including single crossovers,three strand double crossovers,four strand double crossovers,four strand triple crossovers,and four strand multiple crossovers between the non-sister chromatids in the tetrads of bivalents,were clearly inferred by analyzing the breakpoints on the disjoined bivalents.The present investigation not only explained the reason for sterility of the F1 LA hybrids and the variation of their BC1 progenies,but also provided a new method to analyze crossover types in other F1 interspecific hybrids as well.
基金Acknowledgements This study was supported by the National Science Foundation （No. 30630067）；the State Key Basic Research Grant of China （No. 2004CB518705）； the Program for Changjiang Scholars and Innovative Research Team in University （No. IRT0416）.
文摘Chromosome aberrations are distinctive features of human malignant tumors. Analysis of chromosomal changes can illuminate the molecular mechanisms underlying the development and progression of cancer. To establish the technique of multicolor fluorescence in situ hybridization (M-FISH) for identifying chromosome aberrations in esophageal carcinoma cell line KYSE 410-4, four pools of 6-color whole-chromosome painting probes have been designed and hybridized on the same metaphase spread by four rounds of repetitive FISH. Repetitive 6-color M-FISH was successfully established and the cytogenetic abnormalities in KYSE 410-4 cells were characterized. Chromosome gains occurred at 2q, 3, 8, 17p, and X. An isochromosome 3q was visualized in the cell line, which might be one intermedi- ate mechanism leading to 3p losses and/or 3q gains. Furthermore, 16 structural arrangements were detected, including four derivative chromosomes. The rearrangement of the centromeric regions accounted for approximately 44% of all rearrangements. The results added a more complete and accurate information of the genetic alterations to the classical cytogenetic description of KYSE 410-4 and provided a detailed cytogenetic background data for appropriate use of the cell line. The established 6-color M-FISH was useful for analyzing chro- mosomes in the whole genome of human tumors.
基金supported by a grant from Ministry of Public Health of China (No. WKJ2007-3-001)
文摘The accurate assessment of a proto-oncogene, human epidermal growth factor receptor-2 gene (HER-2), is extremely important for the therapy and prognosis of breast cancer. Currently, im-munohistochemistry (IHC) is the method widely used for the detection of HER-2 protein. Fluores-cence in situ hybridization (FISH) has been suggested to be a golden standard assay for HER-2 am-plification. This study examined the expression and amplification of HER-2 in paraffin-embedded sections of breast cancer tissues, and compared the two methods on the measurement of HER-2 status. HER-2 gene and protein were determined in breast cancer samples from 52 Chinese women by FISH and IHC respectively. The findings indicated that the HER-2 gene amplification was found in 18 cases (34.6%) by FISH and the HER-2 protein over-expression (score 3+) in 15 cases (28.8%) by IHC. Immunohistochemically, 28.6% of the cases scored as 2+ and 93.3% of the cases scored as 3+ were HER-2-positive by FISH. There was a significant correlation between the HER-2 gene amplifi-cation and HER-2 protein over-expression in breast cancer (P<0.005). No correlation was noted be-tween the HER-2 gene amplification and any of the clinicopathological parameters examined, in-cluding age, menopausal status, menarche age, tumor size, histological tumor type, histological grade, lymph node status, and the expression of ER and PR. It was concluded that the detection of HER-2 gene amplification in breast cancer by FISH is valuable and can compare with HER-2 protein detec-tion by IHC.
文摘AIM: To investigate the presence of H. pylori DNA withingastric epithelial cells in patients with H. pylori infection andits possible carcinogenic mechanism.METHODS: Total 112 patients, with pathologically confirmedchronic superficial gastritis, chronic atrophic gastritis,intestinal metaplasia, atypical hyperplasia or gastrio cancerwere studied .Among them, 28 were H. pylori negative and84 H. pylori positive. H. pylori DNA in gastric epithelialcells was detected by GenPoint catalyzed signalamplification system for in situ hybridization.RESULTS: In the H. pylori positive group, zero out of 24chronic superficial gastritis (0. 0 %), four out of 25precancerous changes (16.0 %) and thirteen out of 35gastric cancers (37. 1 %) showed H. pylori DNA in thenucleus of gastric epithelial cells, the positive rates of H.pylori DNA in the nucleus of gastric epithelial cells wereprogressively inoreased in chronic superficial gastritis,precancerous changes and gastric cancer groups (χ2 = 12.56, P = 0. 002); One out of 24 ohronic superficial gastritis(4.2 %), eleven out of 25 precancerous ohangas (44.0 %)and thirteen out of 35 gastric cancers (37. 1 %) showed H.pylori DNA in the cytoplasm of gastric epithelial cells (χ2 =10.86, P = 0.004). In the H. pylorinegative group, only onepatient with gastric cancer was found H. pylori DNA in thenucleus of gastric epithelial cells; Only two patients, onepatient with precancerous changes and another with gastriccancer, showed H. pylori DNA in the cytoplasm of gastricepithelial calls. Furthermore, H. pylori DNA must have been inthe ayteplasm as long as it existed in the nucleus of gastricepithelial cells.CONCLUSION: H. pyloriDNA exists both in the nucleus andthe cytoplasm of gastric epithelial cells in patients with H.pylori infections. The pathological progression from chronicsuperficial gastritis, precancerous changes to gastric canceris associated with higher positive rates of H. pylori DNApresence in the nucleus of gastric epithelial cells.
基金This work was supported by a grant fromthe CSC scholarship of the Ministry of Education ofChina(No. 99837010)
文摘Objective: To establish a method to improve the detection of disseminated tumor cells in bone marrow and peripheral blood samples of neuroblastoma patients and analysis of cytogenetic aberration. Methods: Immunofluorescent staining was performed using a cocktail of primary monoclonal neuroblastoma antibodies (14.G2a, 5.1H11). Fluorescence in situ hybridization was applied with fluorescent probes specific for Nmyc genes afterwards. A novel computer assisted scanning system for automatic search, image analysis and repositioning of these positive cells was developed. Fifty-six bone marrow and peripheral blood samples from 7 patients were evaluated by this method. Results: Fluorescence in situ hybridization can be combined with immunofluorescent staining in detecting Nmyc amplification in neuroblastoma patients. Fluorescence in situ hybridization results correlated well with data obtained by conventional cytogenetic procedures. Conclusion: The technique described allows search of tumor cells in the bone marrow as well as detection of Nmyc amplification in interphase nuclei.
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.
