Analysis of hepcidin expression: In situ hybridization and quantitative polymerase chain reaction from paraffin sections

AIM: To establish methods for quantitative polymerase chain reaction (PCR) for hepcidin using RNAs isolated from paraffin-embedded sections and in situ hybridization of hepatocellular carcinoma (HCC). METHODS: Total RNA from paraffin-embedded sections was isolated from 68 paraffin-embedded samples of HCC. Samples came from 54 male and 14 female patients with a mean age of 66.8 ± 7.8 years. Quantitative PCR was performed. Immunohistochemistry and in situ hybridization for hepcidin were also performed. RESULTS: Quantitative PCR for hepcidin using RNAs isolated from paraffin-embedded sections of HCC was performed successfully. The expression level of hepcidin mRNA in cancer tissues was significantly higher than that in non-cancer tissues. A method of in situ hybridization for hepcidin was established successfully, and this demonstrated that hepcidin mRNA was expressed in non-cancerous tissue but absent in cancerous tissue. CONCLUSION: We have established novel methods for quantitative PCR for hepcidin using RNAs isolated from paraffin-embedded sections and in situ hybridization of HCC.

DETECTION OF BREAST CANCER MICROMETASTASES IN BONE MARROW USING REVERSE-TRANSCRIPTASE CHAINREACTION AND SOUTHERN HYBRIDIZATION

Objective: The aim of this study was to detect micrometastases in bone marrow of primary breast cancer patients, and compare with other clinical parameters. Methods: Cytokeratin 19 (CK-19) gene mRNA expression was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot hybridization. Human breast cancer cell line T47D was mixed with bone marrow cells in different proportions. The positive detection rate was compared among RT-PCR, Southern blotting and immunohistochemistry (IHC) methods. Results: Cytokeratin 19 gene was expressed in all 6 positive control samples while the expression was not seen in 8 negative control samples. In all 54 patients 14 cases were CK-19 positive (25.9%) by RT-PCR, another positive signal was obtained in 5/54 (9.3%) of bone marrow samples by Southern blotting. The total positive cases are 19/54 (35.2%). CK-19 IHC+ cells were detected at a dilution of one T47D cell in 5×104 bone marrow cells, while the sensitivity detected by PCR and Southern blot hybridization was at 1∶5×105 and 1∶1×106, respectively. This demonstrates that RT-PCR and Southern blotting was at least 20 times more sensitive than the IHC method. The micrometastases positive rate of the larger tumor size group (>5.0 cm) was significantly (P<0.05) greater than that of the smaller tumor size group (0–2.0 cm). Conclusion: detection of micrometastases in bone marrow by RT-PCR and Southern blotting, using CK-19 as a biological marker, is highly sensitive and it is a method to be used for anticipating the prognosis of breast cancer patients.

基于HCR放大的无标记型荧光传感器的构建及H5N1 DNA检测

对于高致病性H5N1禽流感病毒,构建检测该病毒的高灵敏生物传感器,并与智能包装相结合用于实时监测,这对禽流感的防控具有重要意义。基于杂交链式反应(HCR)信号放大策略,以AgNCs作为荧光信号基团,构建了一种无标记“turn on”型荧光生物传感器用于检测代表H5N1病毒的H5N1基因序列。该传感器以H5N1 DNA作为触发剂引发HCR过程,使AgNCs产生强的荧光信号变化。研究表明,当H5N1 DNA浓度在0.2~800.0 nmol/L内,该传感器具有良好的响应信号,且在0.2~200.0 nmol/L之间的荧光强度与H5N1 DNA浓度呈线性相关,线性方程为y=10.982C+567.435(R^(2)=0.99273),检测限为176 pmol/L。核酸传感体系具有通用性,通过简单调整目标序列,可实现对不同目标物的特异性灵敏检测。该研究有望为高灵敏分析禽流感病毒标志物的通用传感平台设计提供思路。

COMBINED DETECTION OF BREAST CANCER MICROMETASTASES IN THE LYMPH NODES AND BONE MARROW USING REVERSETRANSCRIPTASE CHAIN REACTION AND SOUTHERN HYBRIDIZATION

Objective: The presence of lymph nodes and bone marrow micrometastases of patients with breast carcinoma by immunohistochemistry (IHC) methods has been strongly correlated to early recurrence and shorter overall survival. The aim of this study was to detect micrometastases in matched sample pairs of lymph nodes and the bone marrow of primary breast cancer patients using a more sensitive method, and compare with other clinical parameters. Methods: Cytokeratin 19 (CK-19) gene mRNA expression was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot hybridization. Human breast cancer cell line T47D was mixed with bone marrow cells at different proportions. The positive detection rate was compared among RT-PCR, Southern blotting and IHC methods. Results: Cytokeratin 19 gene was expressed in all 6 positive control samples, while the expression wasn’t seen in 18 negative control samples. CK-19 IHC positive cells were detected at a dilution of one T47D cell in 5×105 bone marrow cells, while the sensitivity detected by PCR and Southern blot hybridization was at 1:5×104 and 1:106, respectively. In the samples from the 35 patients, we found CK-19 positive cells in 2 cases (5.7%) by IHC. CK-19 gene expression signal was detected in 14/35 (40%) by RT-PCR, and 17/35 (48.6%) by southern blotting. Four cases were micrometastases positive both in lymph node and bone marrow (11.4%). There was no correlation between CK-19 detection and other clinical parameters. Conclusion: combined detection of micrometastases in lymph node and bone marrow by RT-PCR and Southern blotting, using CK-19 as a biological marker, is a highly sensitive method for breast cancer.

Hybridization chain reaction-based DNA nanomaterials for biosensing,bioimaging and therapeutics

DNA nanomaterials hold great promise in biomedical fields due to its excellent sequence programmability,molecular recognition ability and biocompatibility.Hybridization chain reaction(HCR)is a simple and efficient isothermal enzyme-free amplification strategy of DNA,generating nicked double helices with repeated units.Through the design of HCR hairpins,multiple nanomaterials with desired functions are assembled by DNA,exhibiting great potential in biomedical applications.Herein,the recent progress of HCR-based DNA nanomaterials for biosensing,bioimaging and therapeutics are summarized.Representative works are exemplified to demonstrate how HCR-based DNA nanomaterials are designed and constructed.The challenges and prospects of the development of HCR-based DNA nanomaterials are discussed.We envision that rationally designing HCR-based DNA nanomaterials will facilitate the development of biomedical applications.

A Bifunctional-Blocker-Aided Hybridization Chain Reaction Lighting-Up Self-calibrating Nanocluster Fluorescence for Reliable Nucleic Acid Detection

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.

Detection of Gene Alteration for Color Vision Defects by Polymerase Chain Reaction

According to the fact that the abnormalities of visual pigment genes were always involved in the changing of the exon 5, two oligonucleotide primers were designed to amplify the exon 5 of red pigment gene and green pigment gene. After electrophoresis of the PCR products digested with Rsal or Sau3A, the DNA fragments from the exon 5 of red pigment gene (RPG) and green pigment gene (GPG) were separated since there are different restriction endonuclease sites. On the other hand, we analyzed the exon 5 rela...

基于适配体-杂交链式反应比色检测生鲜牛乳中四环素类抗生素

)以特异性识别四环素类抗生素(TCs)的广谱型适配体为识别元件,结合杂交链式反应(HCR)信号放大策略,提出了一种TCs多残留比色检测方法,并优化了检测条件和进行了方法学考察。取40 nmol·L^(-1)生物素化检测探针(bio-DP)溶液加入到包被有亲和素的酶标板中,室温孵育后加入牛血清白蛋白(BSA)溶液,封闭反应1 h。加入生鲜牛乳样品稀释液,室温孵育20 min,如果样品中含有TCs,TCs与bio-DP的适配体序列结合,其发夹结构被打开。加入200 nmol·L^(-1)生物素化发夹DNA1(bio-H1)溶液和200 nmol·L^(-1)生物素化发夹DNA2(bio-H2)溶液,室温下进行HCR 40 min,从而形成具有多个重复单元的双链DNA(dsDNA)纳米线。加入辣根过氧化物酶(HRP)标记链霉亲和素(SA-HRP),室温孵育标记dsDNA。加入显色剂[含3,3′,5,5′-四甲基联苯胺(TMB)],HRP催化TMB生成蓝色物质,显色5~8 min后终止反应,在酶标仪中于450 nm测量上述体系的吸光度。结果显示,bio-DP对四环素、土霉素、金霉素和多西环素具有高特异性,和卡那霉素、庆大霉素、氨苄青霉素、泰乐菌素、磺胺嘧啶和恩诺沙星等抗生素无交叉反应。四环素、土霉素、金霉素和多西环素的质量浓度总和在0.8~100μg·L^(-1)内与对应的吸光度总和呈线性关系,检出限(3s/k)为0.18μg·L^(-1)。对生鲜牛乳样品进行加标回收试验,TCs回收率为86.4%~106%,测定值的相对标准偏差(n=3)为2.5%~7.1%。方法应用于生鲜牛乳样品的分析,检测结果与国家标准方法GB 31658.6-2021差异不显著(P>0.05),检出的TCs总量也均未超标(GB 31650-2019)。与文献报道的其他方法相比,上述方法兼具简单、快速、检出限低、准确度高等优点,适用于食品中四环素类抗生素多残留的快速检测。

纳米金核酸适配体HCR放大比色法检测动物源食品中氨苄青霉素残留

为了检测动物原性食品中氨苄青霉素残留,利用氨苄青霉素与核酸适配体的特异性结合能使其脱离纳米金粒子表面的特性,并利用核酸适配体杂交链式反应(HCR)将信号放大,最终通过检测纳米金溶液吸收光谱的变化建立一种氨苄青霉素残留的高灵敏检测方法。首先对各项反应条件进行优化,筛选出HCR放大纳米金比色法的最优检测方法,优化后的检测方法中,Na Cl溶液最优浓度为1mol·L^(-1),HCR的最优终浓度为11.5%(V/V),HCR与纳米金孵育的最优反应条件为37℃、30 min。氨苄青霉素的检测范围是0~1.2μmol·L^(-1),线性方程为y=0.163 6 x+0.025,R2=0.995 3,最低检测限可达到10 nmol·L^(-1),在实际样品中的检测回收率是92.30%~103.27%。建立的氨苄青霉素适配体的HCR放大纳米金比色法具有操作简便、反应快速、灵敏度高等优点,可用于氨苄青霉素残留量的快速检测。

基于杂交链式反应扩增检测奶粉中阪崎肠杆菌的适配体磁珠荧光传感器

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。

Electrochemical analysis of microRNAs with hybridization chain reaction-based triple signal amplification

Selective and sensitive detection of trace microRNA is important for early diagnosis of diseases due to its expression level related to diseases.Herein,a triple signal amplification strategy is developed for trace microRNA-21 (miRNA-21) detection by combining with target-triggered cyclic strand displacement reaction (TCSDR),hybridization chain reaction (HCR) and enzyme catalytic amplification.Four DNA hairpins(H1,H2,H3,H4) are employed to form an ultralong double-strand DNA (dsDNA) structure,which is initiated by target miRNA-21.As H3 and H4 are labeled with horseradish peroxidase (HRP),numerous HRPs are loaded on the long dsDNA,producing significantly enhanced electrocatalytic signals in the hydrogen peroxide (H_(2)O_(2)) and 3,3,5,5-tetramethylbenzidine (TMB) reaction strategy.Compared with single signal amplification,the triple signal amplification strategy shows higher electrochemical response,wider dynamic range and lower detection limit for miRNA-21 detection with excellent selectivity,reproducibility and stability.Taking advantage of the triple signal amplification strategy,the proposed electrochemical biosensor can detect miRNA-21 in 10 He La cell lysates,suggesting that it is a promising method for fruitful assay in clinical diagnosis.

基于金纳米粒子聚集与杂交链式扩增的microRNA传感

MicroRNA(miRNA)是癌症早期诊断的标志物,在生理和病理过程中发挥着关键作用,因此对miRNA的实时准确监测具有重要意义。目前,用于miRNA测定的信号放大/扩增策略多依赖于辅助酶的参与。本文建立了一种基于金纳米粒子(AuNPs)聚集和杂交链式反应(HCR)无酶扩增的高灵敏、特异性miRNA检测方法。为此,设计了一个辅助发夹探针(HP)和两个通用发夹探针(H1/H2),均为单链DNA(ssDNA)且具有粘性末端,可稳定水溶液中的AuNPs而有效防止盐诱导其聚集。靶miRNA与HP环区杂交,启动HCR触发双链DNA(dsDNA)聚合物的形成。dsDNA聚合物无粘性末端,对AuNPs的稳定能力减弱,从而产生盐诱导的AuNPs聚集,导致金胶体溶液由酒红色至蓝色的变化。据此可对miRNA进行光度法检测。该策略无需依赖酶促反应、分离过程及化学修饰,操作简单。通过设计HP环区序列,即可实现对不同靶标的检测,具有通用性。

HPV诊断中应用PCR检验的效果

目的分析高危型人乳头瘤病毒(HPV)诊断中应用实时聚合酶链式反应(PCR)检验的效果。方法以该院2023年1-12月内收治的101例高危型HPV患者为本次研究对象,所有研究对象先使用第二代杂交式捕获法(HC-Ⅱ)检验,再行实时PCR检验,以病理实验检测结果为金标准,统计对比两种检验方式的诊断效能。结果组间对比,HC-Ⅱ与实时PCR对高危型HPV的诊断特异度差异无统计学意义(P>0.05),但实时PCR对高危型HPV的诊断准确率、灵敏度均显著高于HC-Ⅱ,差异有统计学意义(P<0.05)。结论实时PCR对于高危型HPV感染患者具有较高检出率,可为此类患者的早期治疗提供科学依据,倡导临床应用。

Extracellular ATP-activated hybridization chain reaction for accurate and sensitive detection of cancer cells

Accurate and sensitive detection of caner cells is of significant importance for early diagnosis and treat-ment of cancer.Here,we developed an extracellular ATP-dctivated hybridization chain reaction(HCR)amplification strategy to meet this purpose.This strategy relies on three DNA probes,Apt-trigger,H1-AТP aptamer duplex and hairpin H2.The Apt-trigger probe consists of two com sequence for specific recognition of the target cells.and a trigger sequence for the HCR assembly.Theроnents:an aptamer duplex structure of H1-ATP aptamer causes the tochold in hairpin H1 to be hidden,preventing the strand-ent displacement reaction between haipin H1 and Apt-trigger.Upon activation with ATP the ATP aptamer will blnd to ATP to dissoci iate from hairpin H1,thus leading to an Apt-trigger-induced strand-displacement reaction and subsequent HCR with hairpin H2 on the target cell surface.Benefiting from aptamer recogni-tion and ATP-activated HCR amplification,this strategy can not only perform sensitive quantitative anal-ysis with a detection limit of 25 cells in 200 ul.of binding buffer,but also show desirable specificity and accuracy for identifylng target cells from control cells and mixed cell samples,Imporantly,this method retains stable and good perfor mance for target cell detection in 10%fetal bovine ser rum,den onstrating great potential for clinical diagnosis in complex biological matrices.Furthermore,this strategy can be adapted to detect various types of cancer cells by changing the corresponding aptamer sequence.

基于杂交链式(HCR)反应的甲型流感病毒检测技术研究（英文）

甲型流感病毒的现场快速检测对于流感的及时有效防控具有重要意义.本研究基于杂交链式(HCR)反应,利用GO对荧光基团的猝灭作用及共同实现了对甲型流感病毒的快速检测.当目标序列存在时,可引发HCR反应,使短链DNA形成长链,保护FAM荧光基团不被猝灭,从而实现目标物的检测.实验结果表明,该方法在10～40 nmol/L范围内荧光强度与目标检测物浓度表现出了良好的线性关系,检测范围为5～100 nmol/L.这种HCR等温扩增检测技术具有较好的样本检测能力,具有等温、无酶、反应体系简单、操作步骤简便等优点,表现出良好的现场检测应用前景.

Confirmation of Pearl Millet-Napiergrass Hybrids Using EST-Derived Simple Sequence Repeat (SSR) Markers

Prospects for deploying perennial grasses that are currently considered leading candidates for dedicated energy crops over large acreages are debatable because of several limitations, including vegetative propagation or small seed size, low biomass production during the first growing season, and incomplete assessments of crop invasiveness risk. Pearl Millet-Napiergrass hybrids (“PMN”;Pennisetum glaucum [L.] R. Br. × P. purpureum Schumach.), in contrast, are large-seeded, sterile feedstocks capable of high biomass production during establishment year. Novel methods are warranted for confirmation of PMN hybrids, as traditional morphological observations can be inconclusive and chromosome number determination using cytological methods is laborious and time consuming. Six putative PMN lines were produced in this study, and 10 progeny from each line were evaluated using morphological traits, seed fertility, flow cytometry, and expressed sequence tag-simple sequence repeat (EST-SSR) markers. All putative hybrid lines were sterile and failed to produce seed. The PMN hybrids could not be distinguished from either parent using flow cytometry due to highly similar nuclear genome DNA contents. A number of paternal napiergrass-specific EST-SSRs were identified for each PMN line, and four paternal-specific EST-SSRs conserved across all napiergrass accessions were selected to screen the putative PMN hybrids. These EST-SSRs confirmed that all F1 individuals analyzed were PMN hybrids. The use of paternal-specific markers therefore provides a valuable tool in the development of both “Seeded-yet-Sterile” biofuel PMN feedstocks and additional PMN cultivar-and parental species-specific markers.

Detection of mutation in embB gene of Mycobacterium tuberculosis from clinical isolates of tuberculous patients in China by means of reverse-dot blot hybridization

The relationship between embB mutation of Mycobacterium tuberculosis and ethambutol （EMB） resistance of the clinical isolates of tuberculous patients in China was investigated by reversedot blot hybridization （RDBH） in addition to evaluating the clinical value with application of PCR-RDBH technique to detect EMB resistance. In the present study, the genotypes of the 258 bp fragments of embB genes from 196 clinical isolates of M. tuberculosis were analysed with RDBH and DNA sequencing. It was demonstrated that 60 out of 91 phenotypically EMB-resistant isolates （65.9%） showed 5 types of missense mutations at codon 306 of embB gene, resulting in the replacement of the Met residue of the wild type strain with Val, Ile or Leu residues. In these mutations, the GTP mutation （38/91, 41.8% ） and the ATA mutation （16/91, 17.6% ） were the most encountered genotypes. The embB mutation at codon 306 could also be found in 69 isolates of phenotypically EMB-sensitive but resistant to other anti-tuberculous drugs, but no such gene mutation could be found in 36 strains of drug-sensitive isolates. Meanwhile, the concordance with the results of DNA sequencing fcr one wide-type probe and 5 probes for specific mutations was 100%. It was concluded that the EMB-resistance occurring in most M. tuberculosis is due to appearance of embB mutation at codon 306, and the PCR-RDBH assay was proved to be a rapid, simple and reliable method for the detection of gene mutations, which might be a good alternative for the drug-resistance screening.

Optimizing the hybridization chain reaction-fluorescence in situ hybridization(HCR-FISH)protocol for detection of microbes in sediments

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.

基于金纳米和杂交链式反应可视化检测植物病毒RNA

植物病毒病危害严重,严重制约着农业的可持续发展,可造成巨大的经济损失。监测植物健康和及早检测病毒病原对于减少疾病传播至关重要。为实现对植物病毒病的早期田间检测,本文将基于金纳米(AuNPs)的比色法与杂交链式反应(HCR)相结合,设计了一种灵敏、特异与高效的植物病毒RNA可视化检测技术。以烟草花叶病毒(tobacco mosaic virus,TMV)为模型,根据TMV特异性保守片段设计2个具有单链尾的发夹结构H1/H2,TMV可打开发夹结构,使之交替形成长的双直链DNA。HCR反应前后核酸的2种状态与AuNPs之间的结合差异性致使比色信号产生,从而实现对TMV的可视化检测。经过对Tris-HAc浓度、发夹结构浓度、HCR反应时间等进行优化,得到最佳检测条件。在最优条件下,进一步分析了该技术的灵敏性、特异性以及进行了真实样本检测。结果表明,AuNPs的吸光度比值(A 620/A 520)与0~10 nmol/L范围内的目标片段浓度存在线性关系,最低检出限可达412 pmol/L;在实际样本检测中,该技术能从众多病样中准确检出目标病毒,且AuNPs的吸光度比值与0~40ng/μL的TMV也存在良好的线性关系,线性方程为y=0.00827x+0.14606,R 2=0.96405,检出限为4.68 ng/μL,裸眼检出限也可达10 ng/μL。所建立的检测技术具有快速简易、成本低廉、特异性强和灵敏度高等优点,能实现对植物病毒RNA的早期快速可视化检测,具有广阔的应用前景。

基于杂交链反应的新一代RNA-FISH技术检测EV-A71 RNA及其与病毒3D聚合酶的相互作用

RNA荧光原位杂交(RNA-fluorescence in situ hybridization,RNA-FISH)技术利用荧光标记的核苷酸探针,通过互补链杂交,对细胞或组织中特定的RNA序列进行检测和定位。由于RNA-FISH产生的阳性信号较弱,需要结合特异性信号放大,提高信噪比。但传统信号放大技术的背景难以消除,无法定量且分辨率低,是RNA-FISH技术应用的巨大障碍。本文基于第3代杂交链反应(hybridization chain reaction version 3.0,HCR v3.0),利用一对分裂式探针消除非特异杂交背景,并引发荧光信号放大反应,建立了针对肠道病毒A71(enterovirus-A71,EV-A71)RNA的敏感、特异的FISH检测方法,并将该技术与蛋白免疫荧光(immunofluorescence,IF)检测结合,通过高分辨率激光共聚焦成像,成功地在单个细胞水平上检测了EV-A71感染细胞后病毒RNA与其聚合酶3D蛋白的分布变化和相互作用情况,并对细胞中病毒RNA和3D蛋白进行定量。发现相较于传统定量方法,如逆转录定量聚合酶链反应和免疫印迹,新一代RNA-FISH技术在单个细胞水平上病毒RNA和3D聚合酶的表达情况与群体细胞检测的结果在趋势上有明显差异。这说明,基于杂交链反应的新一代RNA-FISH技术,可以克服群体细胞数量增减掩盖病毒组分变化的缺点,从而真实反映病毒在单个细胞中的变化。