BACKGROUND Quantitative fluorescent polymerase chain reaction(QF-PCR)is a rapid prenatal diagnostic method for abnormalities on chromosomes 21,18,and 13 and sex chromosomal aneuploidy.However,the value of QF-PCR in di...BACKGROUND Quantitative fluorescent polymerase chain reaction(QF-PCR)is a rapid prenatal diagnostic method for abnormalities on chromosomes 21,18,and 13 and sex chromosomal aneuploidy.However,the value of QF-PCR in diagnosing chromosomal structural abnormalities is limited.In this article,we report a confusing QF-PCR finding in a pregnant woman who underwent amniocentesis.CASE SUMMARY The short tandem repeat marker AMXY(Xp22.2/Yp11.2)located on the sex chromosome exhibited a trisomic biallelic pattern,indicating that the karyotype of the fetus might be 47,XYY.Chromosome analysis performed on cultured amniocytes showed a normal male karyotype of the fetus.Copy number variation sequencing confirmed a 500 kb duplication at Yp11.2-Yp11.2(chrY:6610001_7110000)and a 250 kb duplication at Yp11.2-Yp11.2(chrY:7110001_7360000).CONCLUSION In conclusion,the comprehensive application of different methods could achieve a higher detection rate and accuracy for the prenatal diagnosis of chromosomal disorders through chromosomal testing.展开更多
Burkholderia glumae causing seedling rot and grain rot of rice was listed as a plant quarantine disease of China in 2007. It＇s quite necessary to set up effective detection methods for the pathogen to manage further ...Burkholderia glumae causing seedling rot and grain rot of rice was listed as a plant quarantine disease of China in 2007. It＇s quite necessary to set up effective detection methods for the pathogen to manage further dispersal of this disease. The present study combined the real-time PCR method with classical PCR to increase the detecting efficiency, and to develop an accurate, rapid and sensitive method to detect the pathogen in the seed quarantine for effective management of the disease. The results showed that all the tested strains of B. glumae produced about 139 bp specific fragments by the real-time PCR and the general PCR methods, while others showed negative PCR result. The bacteria could be detected at the concentrations of 1×10^4 CFU/mL by general PCR method and at the concentrations below 100 CFU/mL by real-time fluorescence PCR method. B. glumae could be detected when the inoculated and healthy seeds were mixed with a proportion of 1：100.展开更多
Objective: To develop a new high sensitivity, rapid and simple mycobacterium tuberculosis DNA detection method using fluorescence polarization technology. Methods: In our asymmetric PCR protocol, 100 times mole of TB-...Objective: To develop a new high sensitivity, rapid and simple mycobacterium tuberculosis DNA detection method using fluorescence polarization technology. Methods: In our asymmetric PCR protocol, 100 times mole of TB-R primer was added than in the usual symmetric PCR to get enough single strands PCR product. The probe TB-5′-TAMRA and PCR products were mixed in a tube and incubate for 5-15 min at 46 ℃.The polarization (mp) was measured using victor2 Multilabel Plate Reader. Results: Asymmetric and symmetric PCR products were analyzed by the FP method. Asymmetric PCR products are detected more sensitively than symmetric ones. The polarization values of probe associated with asymmetric products were much higher than with symmetric products. Conclusion: This fluorescence polarization assay in conjunction with asymmetric PCR is a powerful and widely applicable method for the rapid and sensitive detection of micro-organisms in clinical laboratories.展开更多
Objective:The aim of this study was to detect the expression level of Metadherin (MTDH) in peripheral blood of the breast cancer patients by real-time fluorescence quantitative polymerase chain reaction (PCR),and to e...Objective:The aim of this study was to detect the expression level of Metadherin (MTDH) in peripheral blood of the breast cancer patients by real-time fluorescence quantitative polymerase chain reaction (PCR),and to explore the relationship between expression of Metadherin gene in the patients peripheral blood and the clinic-pathological features in breast cancer. Methods:Real-time fluorescence quantitative polymerase chain reaction was employed to determine the expression level of Metadherin gene in 80 peripheral blood samples of breast cancer patients and healthy donors. Results:The expression of Metadherin gene in breast cancer patients peripheral blood were positive,in which 34 breast cancer patients were highly expressed,accounting for 55.7%,while the expression of Metadherin gene in normal females peripheral blood were negative,there was statistical significance (Ratio = 2.02±0.81,P < 0.05); Ratio of the Metadherin expression in breast cancer patients peripheral blood and the glyceraldehyde-3-phosphate dehydrogenase expression was 1.15 ± 0.36. REST software analysis showed that the expression of Metadherin gene was significantly up-regulated in breast cancer. Conclusion:The SYBR Green I quantitative real-time polymerase chain reaction method can successfully detect the expression level of Metadherin gene. Expression level of Metadherin gene in breast cancer patients peripheral blood is closely related to survival,and it maybe involved in the development of breast cancer and used as an indicator of prognosis.展开更多
In this work,we proposed a ratiometric silver nanoclusters(AgNCs)fluorescent assay by designing a bifunctional-blockeraided hybridization chain reaction(HCR).Hairpin probe 1(HP1)containing two special DNA fragments(5...In this work,we proposed a ratiometric silver nanoclusters(AgNCs)fluorescent assay by designing a bifunctional-blockeraided hybridization chain reaction(HCR).Hairpin probe 1(HP1)containing two special DNA fragments(5′-CAC CGC T-3′and 5′-ATT TGC CTT TTG GGG ACG GATA-3′)at two terminals creates a red-emitting AgNC nucleation sequence(rNS,5′-CAC CGC TAT TTG CCT TTT GGG GAC GGATA-3′).We found that the presence of a toehold fragment(5′-TGCCC-3′)in HP1 could silence the rNS.Upon the addition of a target nucleic acid,HCR of HP1 and hairpin probe 2(HP2)could be initiated,resulting in the formation of long chain of DNA duplexes with multibranched rNS.As the toehold fragment in HP1participated in generating duplexes,a strong emission of rNS-templated AgNCs was observed at 670 nm.More significantly,a bifunctional blocker was introduced not only to reduce the background red-emitting fluorescence but also to play as an internal green-emitting AgNCs nucleation sequence.On the one hand,the blocker could increase the signal-to-noise-ratio of the constructed biosensor,and on the other hand,the blocker also helped to prepare ratiometric HCR-AgNCs assay with self-calibrating ability to strengthen its reproducibility.Compared with the traditional HCR-AgNCs sensors,the developed ratiometric assay based on the bifunctional-blocker-aided HCR has higher reliability,which is important for the fabrication of biosensors in various fields for practical biosensing applications.展开更多
Background Enamel demineralization occurs frequently during orthodontic treatment. In this study, we evaluated the changes of the density of mutans streptococcus （MS） in plaque after bracket bonding and using fluori...Background Enamel demineralization occurs frequently during orthodontic treatment. In this study, we evaluated the changes of the density of mutans streptococcus （MS） in plaque after bracket bonding and using fluoride adhesive on maxillary incisors by real time fluorescence-quantitative polymerase chain reaction （RT-FQ PCR）.Methods The study was designed as a self-paired test. Brackets were bonded with fluoride adhesive on the left side, while non-fluoride adhesive on the right side for each patient. Plaque samples were taken from the surfaces around the brackets of four maxillary incisors before brackets bonding and after the bonding 4 weeks later. The amount of MS was measured by RT-FQ PCR. The data obtained were analyzed statistically using the SPSS 11.5 version and the alpha level was set at 0. 05 （ 2-tailed）.Results The amount of MS in plaque increased significantly after bracket bonding （ P 〈 0.01 ）, whereas no significant differences were observed among four maxillary incisors both before and after brackets bonding （P 〉 0. 05 ）, and among the incisors using and not using fluoride adhesive （ P 〉 0. 05 ）.Conclusions The increase of the density of MS in plaque after bracket bonding is one of the etiological factors for enamel demineralization in orthodontic patients. The result of this study did not support what we observed clinically that the incidence of enamel demineralization for lateral incisors was higher than that for central incisors. Using fluoride adhesive for bonding did not affect the amount of MS in plaque in our study. Further study is needed.展开更多
Fluorescence in situ hybridization(FISH)is a canonical tool commonly used in environmental microbiology research to visualize targeted cells.However,the problems of low signal intensity and false-positive signals impe...Fluorescence in situ hybridization(FISH)is a canonical tool commonly used in environmental microbiology research to visualize targeted cells.However,the problems of low signal intensity and false-positive signals impede its widespread application.Alternatively,the signal intensity can be amplified by incorporating Hybridization Chain Reaction(HCR)with FISH,while the specificity can be improved through protocol modification and proper counterstaining.Here we optimized the HCR-FISH protocol for studying microbes in environmental samples,particularly marine sediments.Firstly,five sets of HCR initiator/amplifier pairs were tested on the laboratory-cultured bacterium Escherichia coli and the archaeon Methano-coccoides methylutens,and two sets displayed high hybridization efficiency and specificity.Secondly,we tried to find the best combination of sample pretreatment methods and HCR-FISH protocol for environmental sample analysis with the aim of producing less false positive signals.Various detachment methods,extraction methods and formulas of hybridization buffer were tested using sediment samples.Thirdly,an image processing method was developed to enhance the DAPI signal of microbial cells against that of abiotic particles,providing a reliable reference for FISH imaging.In summary,our optimized HCR-FISH protocol showed promise to serve as an addendum to traditional FISH for research on environmental microbes.展开更多
构建一种基于杂交链式反应(hybridization chain reaction,HCR)扩增的适配体磁珠荧光传感器。巧妙设计序列HP和发卡序列H1、H2,其中HP是由适配体序列与触发序列结合而成的,并且序列互补形成稳定的二级结构。然后采用戊二醇反应和亲和素...构建一种基于杂交链式反应(hybridization chain reaction,HCR)扩增的适配体磁珠荧光传感器。巧妙设计序列HP和发卡序列H1、H2,其中HP是由适配体序列与触发序列结合而成的,并且序列互补形成稳定的二级结构。然后采用戊二醇反应和亲和素-生物素反应进行适配体功能化磁珠的制备。将阪崎肠杆菌与适配体磁珠一起孵育,HP中的适配体序列识别靶标,引起HP构象变化,露出触发序列,通过HCR触发H1和H2的链状组装,产生长双链DNA。荧光指示剂SYBR Green I以插层和小槽结合的方式与HCR产物的长双链结合。最后加入氧化石墨烯(graphene oxide,GO)后,游离的H1、H2和SYBR Green I将通过π-π堆积紧密吸附在GO表面,荧光信号被猝灭。HCR产物不能被吸附在GO表面,因此与HCR产物结合的SYBR Green I发出依赖于靶浓度的强荧光信号,从而实现阪崎肠杆菌的定量检测。本方法在纯培养条件下的检出限为2CFU/mL,对奶粉的检出限为8CFU/g,对奶粉样品的检测结果与传统微生物培养法具有良好的一致性。该方法具有无需DNA提取,快速、稳定性高、高特异性和高灵敏度等优点,因此为阪崎肠杆菌的现场快速检测提供了一种很有潜力的方法。展开更多
文摘BACKGROUND Quantitative fluorescent polymerase chain reaction(QF-PCR)is a rapid prenatal diagnostic method for abnormalities on chromosomes 21,18,and 13 and sex chromosomal aneuploidy.However,the value of QF-PCR in diagnosing chromosomal structural abnormalities is limited.In this article,we report a confusing QF-PCR finding in a pregnant woman who underwent amniocentesis.CASE SUMMARY The short tandem repeat marker AMXY(Xp22.2/Yp11.2)located on the sex chromosome exhibited a trisomic biallelic pattern,indicating that the karyotype of the fetus might be 47,XYY.Chromosome analysis performed on cultured amniocytes showed a normal male karyotype of the fetus.Copy number variation sequencing confirmed a 500 kb duplication at Yp11.2-Yp11.2(chrY:6610001_7110000)and a 250 kb duplication at Yp11.2-Yp11.2(chrY:7110001_7360000).CONCLUSION In conclusion,the comprehensive application of different methods could achieve a higher detection rate and accuracy for the prenatal diagnosis of chromosomal disorders through chromosomal testing.
基金supported by National Natural Science Foundation of China(Grant No.30671397 and No.30871655)the Public Beneficial Research Project of Agricultural Ministry,China(Grant No.nyhyzx07-056)
文摘Burkholderia glumae causing seedling rot and grain rot of rice was listed as a plant quarantine disease of China in 2007. It＇s quite necessary to set up effective detection methods for the pathogen to manage further dispersal of this disease. The present study combined the real-time PCR method with classical PCR to increase the detecting efficiency, and to develop an accurate, rapid and sensitive method to detect the pathogen in the seed quarantine for effective management of the disease. The results showed that all the tested strains of B. glumae produced about 139 bp specific fragments by the real-time PCR and the general PCR methods, while others showed negative PCR result. The bacteria could be detected at the concentrations of 1×10^4 CFU/mL by general PCR method and at the concentrations below 100 CFU/mL by real-time fluorescence PCR method. B. glumae could be detected when the inoculated and healthy seeds were mixed with a proportion of 1：100.
文摘Objective: To develop a new high sensitivity, rapid and simple mycobacterium tuberculosis DNA detection method using fluorescence polarization technology. Methods: In our asymmetric PCR protocol, 100 times mole of TB-R primer was added than in the usual symmetric PCR to get enough single strands PCR product. The probe TB-5′-TAMRA and PCR products were mixed in a tube and incubate for 5-15 min at 46 ℃.The polarization (mp) was measured using victor2 Multilabel Plate Reader. Results: Asymmetric and symmetric PCR products were analyzed by the FP method. Asymmetric PCR products are detected more sensitively than symmetric ones. The polarization values of probe associated with asymmetric products were much higher than with symmetric products. Conclusion: This fluorescence polarization assay in conjunction with asymmetric PCR is a powerful and widely applicable method for the rapid and sensitive detection of micro-organisms in clinical laboratories.
文摘Objective:The aim of this study was to detect the expression level of Metadherin (MTDH) in peripheral blood of the breast cancer patients by real-time fluorescence quantitative polymerase chain reaction (PCR),and to explore the relationship between expression of Metadherin gene in the patients peripheral blood and the clinic-pathological features in breast cancer. Methods:Real-time fluorescence quantitative polymerase chain reaction was employed to determine the expression level of Metadherin gene in 80 peripheral blood samples of breast cancer patients and healthy donors. Results:The expression of Metadherin gene in breast cancer patients peripheral blood were positive,in which 34 breast cancer patients were highly expressed,accounting for 55.7%,while the expression of Metadherin gene in normal females peripheral blood were negative,there was statistical significance (Ratio = 2.02±0.81,P < 0.05); Ratio of the Metadherin expression in breast cancer patients peripheral blood and the glyceraldehyde-3-phosphate dehydrogenase expression was 1.15 ± 0.36. REST software analysis showed that the expression of Metadherin gene was significantly up-regulated in breast cancer. Conclusion:The SYBR Green I quantitative real-time polymerase chain reaction method can successfully detect the expression level of Metadherin gene. Expression level of Metadherin gene in breast cancer patients peripheral blood is closely related to survival,and it maybe involved in the development of breast cancer and used as an indicator of prognosis.
基金supported by the National Natural Science Foundation of China(22304062)the Zhejiang Provincial Natural Science Foundation of China(LTGY24B050002)+2 种基金the Program for Science and Technology of Jiaxing(2023AY40028)the Baiqing Foundation of Jiaxing University(CD70621010)Springer Nature or its licensor(e.g.a society or other partner)holds exclusive rights to this article under a publishing agreement with the author(s)or other rightsholder(s)author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law。
文摘In this work,we proposed a ratiometric silver nanoclusters(AgNCs)fluorescent assay by designing a bifunctional-blockeraided hybridization chain reaction(HCR).Hairpin probe 1(HP1)containing two special DNA fragments(5′-CAC CGC T-3′and 5′-ATT TGC CTT TTG GGG ACG GATA-3′)at two terminals creates a red-emitting AgNC nucleation sequence(rNS,5′-CAC CGC TAT TTG CCT TTT GGG GAC GGATA-3′).We found that the presence of a toehold fragment(5′-TGCCC-3′)in HP1 could silence the rNS.Upon the addition of a target nucleic acid,HCR of HP1 and hairpin probe 2(HP2)could be initiated,resulting in the formation of long chain of DNA duplexes with multibranched rNS.As the toehold fragment in HP1participated in generating duplexes,a strong emission of rNS-templated AgNCs was observed at 670 nm.More significantly,a bifunctional blocker was introduced not only to reduce the background red-emitting fluorescence but also to play as an internal green-emitting AgNCs nucleation sequence.On the one hand,the blocker could increase the signal-to-noise-ratio of the constructed biosensor,and on the other hand,the blocker also helped to prepare ratiometric HCR-AgNCs assay with self-calibrating ability to strengthen its reproducibility.Compared with the traditional HCR-AgNCs sensors,the developed ratiometric assay based on the bifunctional-blocker-aided HCR has higher reliability,which is important for the fabrication of biosensors in various fields for practical biosensing applications.
文摘Background Enamel demineralization occurs frequently during orthodontic treatment. In this study, we evaluated the changes of the density of mutans streptococcus （MS） in plaque after bracket bonding and using fluoride adhesive on maxillary incisors by real time fluorescence-quantitative polymerase chain reaction （RT-FQ PCR）.Methods The study was designed as a self-paired test. Brackets were bonded with fluoride adhesive on the left side, while non-fluoride adhesive on the right side for each patient. Plaque samples were taken from the surfaces around the brackets of four maxillary incisors before brackets bonding and after the bonding 4 weeks later. The amount of MS was measured by RT-FQ PCR. The data obtained were analyzed statistically using the SPSS 11.5 version and the alpha level was set at 0. 05 （ 2-tailed）.Results The amount of MS in plaque increased significantly after bracket bonding （ P 〈 0.01 ）, whereas no significant differences were observed among four maxillary incisors both before and after brackets bonding （P 〉 0. 05 ）, and among the incisors using and not using fluoride adhesive （ P 〉 0. 05 ）.Conclusions The increase of the density of MS in plaque after bracket bonding is one of the etiological factors for enamel demineralization in orthodontic patients. The result of this study did not support what we observed clinically that the incidence of enamel demineralization for lateral incisors was higher than that for central incisors. Using fluoride adhesive for bonding did not affect the amount of MS in plaque in our study. Further study is needed.
基金We thank for the funding:National Key R&D Program of China(grant numbers 2018 YFC0310800,2018YFC0310803)COMRA Project DY135-B2-12+3 种基金the National Natural Science Foundation of China(grant numbers 41525011,91751205,11774225)the Recruitment Program of Global Experts(Program for Young Professionals),and the Natural Science Foundation of Shanghai(grant no.18ZR1419800)This is also a contribution to the Center for Ocean Mega-Science,Chinese Academy of Sciences,the Senior User Project of RV KEXUE(KEXUE2019GZ06)the International Center for Deep-Life Investigation(IC-DLI).We thank Gunter Wegener for providing the ANME enrichment sample.
文摘Fluorescence in situ hybridization(FISH)is a canonical tool commonly used in environmental microbiology research to visualize targeted cells.However,the problems of low signal intensity and false-positive signals impede its widespread application.Alternatively,the signal intensity can be amplified by incorporating Hybridization Chain Reaction(HCR)with FISH,while the specificity can be improved through protocol modification and proper counterstaining.Here we optimized the HCR-FISH protocol for studying microbes in environmental samples,particularly marine sediments.Firstly,five sets of HCR initiator/amplifier pairs were tested on the laboratory-cultured bacterium Escherichia coli and the archaeon Methano-coccoides methylutens,and two sets displayed high hybridization efficiency and specificity.Secondly,we tried to find the best combination of sample pretreatment methods and HCR-FISH protocol for environmental sample analysis with the aim of producing less false positive signals.Various detachment methods,extraction methods and formulas of hybridization buffer were tested using sediment samples.Thirdly,an image processing method was developed to enhance the DAPI signal of microbial cells against that of abiotic particles,providing a reliable reference for FISH imaging.In summary,our optimized HCR-FISH protocol showed promise to serve as an addendum to traditional FISH for research on environmental microbes.
文摘构建一种基于杂交链式反应(hybridization chain reaction,HCR)扩增的适配体磁珠荧光传感器。巧妙设计序列HP和发卡序列H1、H2,其中HP是由适配体序列与触发序列结合而成的,并且序列互补形成稳定的二级结构。然后采用戊二醇反应和亲和素-生物素反应进行适配体功能化磁珠的制备。将阪崎肠杆菌与适配体磁珠一起孵育,HP中的适配体序列识别靶标,引起HP构象变化,露出触发序列,通过HCR触发H1和H2的链状组装,产生长双链DNA。荧光指示剂SYBR Green I以插层和小槽结合的方式与HCR产物的长双链结合。最后加入氧化石墨烯(graphene oxide,GO)后,游离的H1、H2和SYBR Green I将通过π-π堆积紧密吸附在GO表面,荧光信号被猝灭。HCR产物不能被吸附在GO表面,因此与HCR产物结合的SYBR Green I发出依赖于靶浓度的强荧光信号,从而实现阪崎肠杆菌的定量检测。本方法在纯培养条件下的检出限为2CFU/mL,对奶粉的检出限为8CFU/g,对奶粉样品的检测结果与传统微生物培养法具有良好的一致性。该方法具有无需DNA提取,快速、稳定性高、高特异性和高灵敏度等优点,因此为阪崎肠杆菌的现场快速检测提供了一种很有潜力的方法。
