Recent advances in chemical fluorescent probes sensitive to tumor microenvironments / 药学学报

Cancer seriously threatens human life and health, it is urgent for the development of rapid detection, precise localization and effective treatment of tumors. Chemical fluorescent probes that are sensitive to tumor-specific microenvironments have important significance in tumor theranostics and a variety of such probes have been developed. In this review, we classified chemical fluorescent probes that are sensitive to tumor microenvironments according to biological characteristics and microenvironmental changes while combining spectroscopy or response mechanisms, and systematically introduced the research progress of chemical fluorescent probes with sensitivity to hypoxia, low polarity, high viscosity, abnormal pH values and abundant reactive oxygen species in tumor microenvironments, in order to provide references for the development and applications of these probes.

Research progress of small molecule fluorescent probes for ferrous ion and heme / 药学学报

Small molecule fluorescent probes have gained widespread attention for their advantages of high selectivity, sensitivity, and easy to operate, and have played a critical role in the detection of various species. They have also demonstrated great potential in the field of biomedical research. Iron, as the most abundant transition metal in the human body, plays a vital role in many physiological functions. Due to the influence of the reductive microenvironment of cell, ferrous ion (Fe2+) is the main component of labile iron in living cells. Heme, consisting of Fe2+ and protoporphyrin IX, is one of the main signaling molecules that wrap biological iron in the human body, and also participates in many physiological and pathological processes. Therefore, the development of small molecule fluorescent probes for detecting Fe2+ and heme as effective monitoring tools will help to further understand their pathological and physiological functions, with potential applications in other fields. This review summarizes the research progress of small molecule fluorescent probes for Fe2+ and heme detection in recent years, and provides insights into future directions for their development.

Design and synthesis of peptide-drug conjugates and fluorescent probe based on α-conotoxin ArIBV11L,V16D / 药学学报

italic>α-Conotoxin ArIB[V11L,V16D] is currently the most optimal selective inhibitor of α7 nicotinic acetylcholine receptor (nAChR) known. In order to explore chemical modification methods and enrich its application in targeting nAChR, this study utilized the linker to covalently connect camptothecin and 7-amino-4-methylcoumarin to the [2,4] disulfide bond of ArIB[V11L,V16D]. Therefore, two peptide-drug conjugates (PDCs), ArIB[V11L,V16D]-5 and ArIB[V11L,V16D]-6, and one fluorescent-labeled peptide, ArIB[V11L,V16D]-7 were constructed. Cytotoxicity evaluation showed that the IC50 values against non-small cell lung cancer cell line A549 of the two PDCs were respectively 1.3 and 4.1 times of camptothecin, indicating slight reduction in activity at the cellular level which was related to the linker structure. Fluorescence spectrum scanning revealed that the excitation and emission wavelength of the fluorescent-labeled peptide were 340 nm and 403 nm respectively, and the fluorescence features of 7-amino-4-methylcoumarin as a marker were retained without fluorescence quenching. This modification strategy laid a solid foundation for the further application of α-conotoxin ArIB[V11L,V16D] in PDCs and fluorescent probes.

Screening of rpsL mutations in streptomycin resistance gene of 104 strains of Yersinia pestis strains in south area of Qinghai Province by TaqMan-MGB fluorescent probe / 中国热带医学

@#Abstract: Objective To investigate the current status of streptomycin resistance of Yersinia pestis caused by point mutations of rpsL gene in Qinghai, so as to provide theoretical basis for precise clinical medication and prevention of drug resistance of human plague outbreak in South area of Qinghai Province in the future. Methods A total of 104 representative strains of Yersinia pestis collected from plague patients, vector insects and intermediate hosts in South area of Qinghai Province from 1957 to 2009 were screened, isolated and cultured by Hiss agar plates. The DNA of representative Yersinia pestis was extracted by sodium dodecyl sulfate lysis and phenol-chloroform method. The primers forward primer and reverse primer and TaqMan-MGB probes probe1 [FAM] and probe2 [VIC] were designed for the rpsL gene of streptomycin resistance gene in China. Real-time PCR with TaqMan-MGB fluorescent probe was used to detect the mutations of rpsL gene in streptomycin resistance locus of 104 strains of Yersinia pestis in South area of Qinghai Province. Results The FAM test results of 104 strains in South area of Qinghai Province were positive, corresponding to the detection of rpsL (128 : A ), RFU peak >1 000,negative <200. VIC test results of all tested strains were negative, corresponding to the detection of rpsL (128:G), RFU peak <200, positive >1 000. That is, no strains with rpsL gene mutation related to streptomycin resistance were found in the 104 strains of Yersinia pestis in Qingnan Province. Conclusion This study provides basic data on the distribution of streptomycin resistance of Yersinia pestis in South area of Qinghai Province, and lays a foundation for preventing the occurrence of drug resistance and clinical treatment of Yersinia pestis in South area of Qinghai Province.

Preparation of a polypeptide fluorescent probe based on aggregation induced luminescence and its application in the detection of early dental caries / 国际生物医学工程杂志

Objective:To prepare a peptide fluorescent probe based on aggregation-induced emission and to investigate its application in the detection of early caries.Methods:Eight aspartate-serine-serine (DSS) were combined with aggregation-induced emission material to prepare peptide fluorescent probes, and an artificial demineralization model was established in vitro. The samples were immersed in the peptide fluorescent probe solution for 1 min, and a fluorescence imaging system was applied to examine the tooth samples and collect images and fluorescence data. Scanning electron microscopy was also applied to observe the phenotype of the teeth, and electron microscopy was applied to detect the calcium-phosphorus ratio on the enamel surface of the teeth. Polarized light microscopy was also applied to observe the enamel area of the teeth. Results:The fluorescence intensity of demineralized teeth was clearly observed to be lower than that of normal teeth in the peptide fluorescent probe-treated area, and the difference was statistically significant ( P < 0.05). The results of scanning electron microscopy showed that the enamel surface of the demineralized group had more irregular pores, while the enamel surface of the undemineralized group was flatter with only some irregular accumulation of flakes. The results of polarized light microscopy showed that a clear birefringence could be observed in the enamel region of normal teeth, while a black area or the disappearance of the birefringence effect accompanied by a partial black dark shadow could be observed in the enamel region of demineralized teeth. Conclusions:An aggregation-induced luminescence-based peptide fluorescent probe was successfully prepared, which can precisely localize the enamel and show some application value in early caries detection.

A NIR fluorescent probe for Vanin-1 and its applications in imaging, kidney injury diagnosis, and the development of inhibitor

Vanin-1 is an amidohydrolase that catalyses the conversion of pantetheine into the amino-thiol cysteamine and pantothenic acid (coenzyme A precursor), which plays a vital role in multiple physiological and pathological processes. In this study, an enzyme-activated near-infrared (NIR) fluorescent probe (DDAV) has been constructed for sensitively detecting Vanin-1 activity in complicated biosamples on the basis of its catalytic characteristics. DDAV exhibited a high selectivity and sensitivity toward Vanin-1 and was successfully applied to the early diagnosis of kidney injury in cisplatin-induced kidney injury model. In addition, DDAV could serve as a visual tool for in situ imaging endogenous Vanin-1 in vivo. More importantly, Enterococcus faecalis 20247 which possessed high expression of Vanin-1 was screened out from intestinal bacteria using DDAV, provided useful guidance for the rational use of NSAIDs in clinic. Finally, oleuropein as a potent natural inhibitor for Vanin-1 was discovered from herbal medicines library using a high-throughput screening method using DDAV, which held great promise for clinical therapy of inflammatory bowel disease.

Construction and in vitro evaluation of AIE self-assembled probe based on GSH response covalent cyclization / 国际生物医学工程杂志

Objective:To construct an aggregation induced emission (AIE) self-assembled probe based on glutathione (GSH) response covalent cyclization and evaluate it in vitro.Methods:The peptide sequence containing the 2-cyano-6-aminobenzothiazole-cysteine (CBT-Cys) condensation sequence was synthesized by the solid-phase peptide synthesis method. After coupling with an AIE molecule by click chemical reaction, an AIE self-assembled probe 1 based on GSH response covalent cyclization was constructed, and probe 2 lacking Cys structure was used as the control. The absorption and emission spectra of probes were tested and the specificity of probes to GSH was analyzed. The hydrodynamic diameter and structure of the probes after response were compared. The effects of different pH values, temperatures, probe concentrations, and GSH concentrations on fluorescence intensity were investigated. The toxicity of probes to tumor cells such as HeLa, HepG2 and MDA-MB-231 was evaluated.Results:After GSH response, the fluorescence of probe 1 was enhanced by about 6 times and that of probe 2 was enhanced by about 2 times; probe 1 was converted into a dimer with a hydrodynamic diameter of about 896.1 nm. Probe 2 lacked a cyclization motif and was converted into a monomer with a hydrodynamic diameter of about 427.4 nm. The fluorescence intensity of probe 1 was significantly higher than that of probe 2 at pH=7.0 and 37 ℃, and the toxicity of probes to tumor cells (HeLa, HepG2 and MDA-MB-231) was low.Conclusions:After the disulfide bond of probe 1 was reduced by GSH, the probe molecule lost the hydrophilic sequence, resulting in fluorescence turn-on (the first aggregation), and probe 1 immediately generates an AIE dimer (the second aggregation) because it contains a CBT-Cys cyclization sequence, which realizes the dual AIE effect compared with the single aggregation of probe 2, and significantly enhances the fluorescence emission. Probe 1 has better applicability in physiological environments, which provides an idea for in-situ generation of covalent cycling probes in vivo and is expected to be used in tumor imaging and treatment in the later stages.

Enzyme-responsive fluorescent probes for bacteria imaging / 药学学报

Fluorescent probes are potential fluorophores that display signals based on the changes in tissue microenvironment, interactions with analytes or specific biochemical reactions. Metabolic enzymes are the most important protein involved in bacteria activities. Complex dynamics of biological processes in bacteria are elucidated by these metabolic enzymes-based fluorescent probes with high spatial resolution and sensitivity. Here, we review recent advances in metabolic enzyme-responsive fluorescent probes for bacteria imaging. It was organized according to enzyme classification systems, focused on fluorescence masking strategies, molecular mechanisms of enzyme activation, and bio-related applications.

A Selective Fluorescent Probe for the Determination and Imaging of Fluoride in Living Cells

Abstract Fluoride anions are indispensable trace elements required for sustaining life. To investigate the homeostasis and action of fluoride in the body, a new highly sensitive and selective fluorescence detection method was designed for fluoride in aqueous solutions. A fluorescent probe for fluoride (FP-F) was synthesized for imaging F- in living cells. The design strategy for the probe was based on the specific reaction between fluoride and silica to mediate deprotection of this probe to fluorescein. Upon treatment with F-, FP-F, a closed and weakly fluorescent lactone, was transformed into an open and strongly fluorescent product. Under the optimum conditions, the detection limit for fluoride was 0.526 nM. FP-F could detect micromolar changes in F- concentrations in living cells by confocal microscopy.

Applications of smartphone-based near-infrared (NIR) imaging, measurement, and spectroscopy technologies to point-of-care (POC) diagnostics / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The role of point-of-care (POC) diagnostics is important in public health. With the support of smartphones, POC diagnostic technologies can be greatly improved. This opportunity has arisen from not only the large number and fast spread of cell-phones across the world but also their improved imaging/diagnostic functions. As a tool, the smartphone is regarded as part of a compact, portable, and low-cost system for real-time POC, even in areas with few resources. By combining near-infrared (NIR) imaging, measurement, and spectroscopy techniques, pathogens can be detected with high sensitivity. The whole process is rapid, accurate, and low-cost, and will set the future trend for POC diagnostics. In this review, the development of smartphone-based NIR fluorescent imaging technology was described, and the quality and potential of POC applications were discussed.

Design, synthesis, and activity study of α-conotoxin A10LPnIA fluorescent probe / 药学学报

italic>α7 nicotinic acetylcholine receptor (nAChR) is widely distributed in the central and peripheral nervous systems, and is closely related to a variety of neurological diseases and inflammation response. α-Conotoxin [A10L]PnIA, as an antagonist targeting α7 nAChR, plays an important role in studying the physiological and pathological processes involved in α7 nAChR. [A10L]PnIA was labeled with fluorescein 5-carboxytetramethylrhodamine, and the active peptide ([A10L]PnIA-F) was obtained by a two-step oxidative folding procedure in vitro. The Xenopus oocyte expression system and the two-electrode voltage clamp technique were used to identify the potency of [A10L]PnIA-F fluorescent peptide, and its cytotoxicity was detected by mouse macrophages and CCK8 method. The molecular weight of [A10L]PnIA-F fluorescent peptide was identified by mass spectrometry as 2 077.28 Da, which was consistent with the theoretical value. Electrophysiological determination of its half-maximal inhibitory concentration (IC50) for α7 nAChR is 17.32 nmol·L-1, which is consistent with [A10L]PnIA (IC50, 13.84 nmol·L-1). The cytotoxicity test results showed that within the concentration range of 5 nmol·L-1 to 10 μmol·L-1, there was no significant inhibition on the growth of mouse macrophages. The results showed that the α-conotoxin fluorescent probe [A10L]PnIA could provide pharmacological tools for the research of α7 nAChR-related neurophysiological and pathological mechanisms.

Design and synthesis of tetrazine bioorthogonal fluorogenic probes / 药学学报

Bioorthogonal fluorogenic probes are becoming an ideal tool for live-cell fluorescence imaging. With the tetrazine bioorthogonal fluorogenic probe that displays fluorescence enhancement, the tetrazine plays the dual-role of a bioorthogonal reaction unit and the fluorescence quenching unit. The "off" and "on" states of the fluorescence probe are mainly controlled through inverse electron demand Diels-Alder (IEDDA) bioorthogonal reaction. We designed a series of turn-on tetrazine fluorescent probes with Donor-π-Acceptor (D-π-A) structure to achieve a high signal-to-noise ratio and specificity of fluorescence imaging. This series of probes reacted with the dienophile bicyclononyne, and then generated pyridazine structure in-situ that acted as an electron acceptor, resulting in a new D-π-A effect of fluorescent dyes, turning on the intramolecular charge transfer (ICT) effect. By adjusting the electron-donating groups and the degree of conjugation, tunable fluorescence spectra between 400-647 nm with fluorescence turn-on enhanced up to 500-fold have been achieved. This research lays the foundation for the further optimization of tetrazine bioorthogonal fluorescent probes and their applications in molecular imaging and biomedical fields.

A pyrene-based ratiometric fluorescent probe with a large Stokes shift for selective detection of hydrogen peroxide in living cells / 药物分析学报

Hydrogen peroxide (H2O2) plays a significant role in regulating a variety of biological processes. Dys-regulation of H2O2 can lead to various diseases. Although numerous fluorescent imaging probes for H2O2 have been reported, the development of H2O2 ratiometric fluorescent probe with large Stokes shift re-mains rather limited. Such probes have shown distinct advantages, such as minimized interference from environment and improved signal-to noise ratio. In this work, we reported a new pyrene-based com-pound Py-VPB as H2O2 fluorescent probe in vitro. The probe demonstrated ratiometric detection behavior, large Stokes shift and large emission shift. In addition, the probe showed high sensitivity and selectivity towards H2O2 in vitro. Based on these excellent properties, we successfully applied Py-VPB to the visualization of exogenous and endogenous H2O2 in living cells. Cell imaging study also showed that our probe was localized in the mitochondria. We envision that the probe can provide a useful tool for unmasking the biological roles of mitochondrial H2O2 in living systems.

Application of MAO-A/B subtype activity detection in diagnosis and treatment of neurological diseases / 中国临床药理学与治疗学

The abnormal expression of monoamine oxidases (MAOs), which distributed on the outer mitochondrial membrane can cause the dysfunction of neurotransmitter delivery and related to mental and neurodegenerative diseases. Therefore, the specific detection of MAOs (MAO-A/B) and the regulation of their activities will contribute to the diagnosis and treatment of neurological diseases. In order to distinguish the two subtypes of MAO-A/B, fluorescence detection and imaging techniques with highly specific and sensitive properties have important biological relevance. Herein, our research group based on the concept of "spatial configuration conversion" have designed the two-photon small molecule fluorogenic probes of U1 and F1, which capable of specific fluorescence "Switch-ON" behaviour, and combined with two-photon fluorescence microscopic imaging technology to realize the activity detection of endogenous MAO-B/A in cells and tissues. It is hoped that this works will benefit the understanding of physiological function about MAOs and developing innovative inhibitors, as well as diagnosing and treating in neurological diseases.

Design, synthesis and application of AIE fluorescent probe for lipid raft / 中国药科大学学报

@#Lipid rafts composed of saturated phospholipids,sphingomyelin,and cholesterol are usually defined as liquid ordered microdomains located in the cell membrane. Lipid rafts are involved in many physiological and pathological processes of cells. Based on the difference in composition and distribution between lipid raft and non-raft domains,a lipid raft probe with aggregation-induced emission (AIE),cholesterol-triethylene glycol-tetraphenylethylene (TCHS-TPE),was designed and synthesized for convenient and specific imaging of lipid raft domains on cell membranes in this study. In this paper,TCHS-TPE was successfully synthesized,and the photophysical properties of TCHS-TPE were measured to evaluate its AIE characteristics. And finally the specific imaging of TCHS-TPE on the lipid raft region of B16F10 melanoma cell membrane was studied using confocal laser scanning microscopy. Compared with the existing lipid raft probe cholera toxin B (CTxB),the TCHS-TPE lipid raft probe has the advantages of simple operation and high specificity. The successful synthesis of the fluorescent probe will provide a useful tool for studying the physiological and pathological processes related to lipid raft domains,and offer a theoretical basis for the design of imaging probes for other lipid raft domains.

Establishment of a nucleic acid assay for detection of Echinococcus granulosus based on recombinase-aided isothermal amplification assay / 中国血吸虫病防治杂志

Objective To establish a nucleic acid assay for detection of Echinococcus granulosus based on recombinase-aided isothermal amplification (RAA) assay. Methods The 12S rRNA gene of E. granulosus was selected as the target gene, and the specific primers and fluorescent probes for RAA assay were designed, screened and synthesized to establish a fluorescent RAA assay for detection of E. granulosus. The sensitivity of the fluorescent RAA assay was evaluated using different copy numbers of target gene sequence-contained recombinant plasmids and various concentrations of E. granulosus genomic DNA as templates, and the specificity of the fluorescent RAA assay was evaluated using the genomic DNA from E. granulosus, E. multilocularis, Schistosoma japonicum, S. mansoni, Ancylostoma duodenale, Clonorchis sinensis, Taenia saginata, Spirometra mansoni and Taenia solium as templates. Results A fluorescent RAA assay was successfully established for detection of E. granulosus, which achieved specific amplification of E. granulosus genomic DNA within 20 min at 39 ℃. The lowest detection limit of the fluorescent RAA assay was 10 copies/μL of recombinant plasmids and 0.1 ng/μL E. granulosus genomic DNA, which exhibited a high sensitivity, and the fluorescent RAA assay was all negative for the genomic DNA from E. multilocularis, S. japonicum, S. mansoni, A. duodenale, C. sinensis, T. saginata, Spirometra mansoni and T. solium, which exhibited a high specificity. In addition, this fluorescent RAA assay successfully detected genomic DNA from E. granulosus cysts. Conclusions A rapid, sensitive and specific fluorescent RAA assay is successfully established for nucleic acid detection of E. granulosus.

Establishment of the gene detection method of Schistosoma mansoni based on the recombinase-aided isothermal amplification assay / 中国血吸虫病防治杂志

Objective To establish a recombinase-aided isothermal amplification (RAA) assay for nucleic acid detection of Schistosoma mansoni. Methods The 121 bp highly-repeated sequence of S. mansoni was selected as the target gene fragment to be detected. The primers and fluorescent probes were designed using the Amplfix software, and a fluorescent RAA assay was established and optimized. The fluorescent RAA assay was performed to detect gradient diluent recombinant plasmids containing target gene fragment and different concentrations of S. mansoni genomic DNA to determine the sensitivity, and this assay was applied to detect the genomic DNA of S. japonicum, S. haematobium, Ancylostoma duodenale and Clonorchis sinensis to evaluate the specificity. Results A fluorescent RAA assay was successfully established, which was effective to amplify the specific gene fragments of S. mansoni within 20 min at 39 ℃. The minimum detectable limit of the fluorescent RAA assay was 10 copies/μL using recombinant plasmids as templates and 0.1 fg/μL using S. mansoni genomic DNA samples as templates. The fluorescent RAA assays were all negative for detecting the genomic DNA from S. japonicum, S. haematobium, A. duodenale and C. sinensis. Conclusions A novel fluorescent RAA assay is successfully established, which is simple, rapid, sensitive and specific to detect genomic DNA of S. mansoni.

Lead sulfide quantum dots for the near-infrared IIb window fluorescence imaging of colon cancer / 药学学报

The near-infrared-IIb (NIR-IIb, 1 500-1 700 nm) window fluorescence with long emission wavelength has reduced light scattering and tissue auto-fluorescent background, achieving deep tissue imaging with high spatial resolution. Herein, we prepared an NIR-IIb fluorescent quantum dots (QDs) composed of lead sulfide (PbS). The fluorescence spectrum of PbS QDs were adjusted by controlling the size of the PbS core. Cadmium sulfide (CdS) shell was synthesized by the cation exchange method to form the core/shelled lead sulfide/cadmium sulfide quantum dots (CSQDs). The surface of CSQDs was modified with polyethylene glycol (PEG) to increase their stability in aqueous solution. The resulting PEG-modified CSQDs (PEG-CSQDs) had the emission peak at ~1 550 nm with quantum yield of 7.2%. The animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Fudan University School of Pharmacy. At 2 h postinjection, PEG-CSQDs clearly delineated the tumor region of mice bearing orthotopic CT26-Luc colon cancer model in the NIR-IIb fluorescence imaging. The fluorescent intensity ratio of primary tumor and adjacent normal tissue was 42.3, and that of metastatic tumor and adjacent normal tissue was 22.3, which allowed to detect the primary tumor of 3.4 mm×2.5 mm in dimension and the metastatic tumor of 1.2 mm×0.9 mm in dimension, and accurately guided the excision of tumors. The PEG-CSQDs prepared in this study provided a new approach for the early diagnosis and guidance of surgical resection of colon cancer.

Establishment and evaluation of the detection method of Cryptosporidium specific DNA fragment by recombinase aided isothermal amplification / 中国血吸虫病防治杂志

Objective To establish a recombinase-aided isothermal amplification (RAA) assay for detection of Cryptosporidium. Methods Based on Cryptosporidium-specific 18S rRNA selected as the target gene to be detected, and the primer sequences and fluorescent probes designed using the software Amplfix, and a fluorescent RAA assay was established and optimized. The fluorescent RAA assay was performed to detect 18S RNA target sequence-contained recombinant plasmids at various copies, genomic DNA of Cryptosporidium oocysts at various concentrations, and genomic DNA extracted from various numbers of Cryptosporidium oocysts to assess the sensitivity of the assay, and to detect genomic DNA extracted from Cryptosporidium oocysts, Giardia lamblia cysts, Schistosoma japonicum eggs, Ascaris lumbricoides eggs, Clonorchis sinensis eggs, Salmonella and Shigella to determine the specificity of the assay. Results A fluorescent RAA assay was successfully established, which was effective to amplify the specific 18S RNA gene fragments of Cryptosporidium within 20 min at 39 ℃. The lowest limits of the fluorescent RAA assay were 102 copies/μL for detection of 18S RNA target sequence-contained recombinant plasmids at various copies, 1 pg/μL for detection of genomic DNA of Cryptosporidium oocysts at various concentrations, and one Cryptosporidium oocyst/μL for detection of genomic DNA extracted from various numbers of Cryptosporidium oocysts, and the fluorescent RAA assay was all negative for detection of genomic DNA from G. lamblia cysts, S. japonicum eggs, A. lumbricoides eggs, C. sinensis eggs, Salmonella and Shigella. Conclusion A novel fluorescent RAA assay is successfully established, which is simple, rapid, sensitive and specific to detect genomic DNA of Cryptosporidium oocysts.

Establishment and evaluation of the detection method of Cryptosporidium specific DNA fragment by recombinase aided isothermal amplification / 中国血吸虫病防治杂志

Objective To establish a recombinase-aided isothermal amplification (RAA) assay for detection of Cryptosporidium. Methods Based on Cryptosporidium-specific 18S rRNA selected as the target gene to be detected, and the primer sequences and fluorescent probes designed using the software Amplfix, and a fluorescent RAA assay was established and optimized. The fluorescent RAA assay was performed to detect 18S RNA target sequence-contained recombinant plasmids at various copies, genomic DNA of Cryptosporidium oocysts at various concentrations, and genomic DNA extracted from various numbers of Cryptosporidium oocysts to assess the sensitivity of the assay, and to detect genomic DNA extracted from Cryptosporidium oocysts, Giardia lamblia cysts, Schistosoma japonicum eggs, Ascaris lumbricoides eggs, Clonorchis sinensis eggs, Salmonella and Shigella to determine the specificity of the assay. Results A fluorescent RAA assay was successfully established, which was effective to amplify the specific 18S RNA gene fragments of Cryptosporidium within 20 min at 39 ℃. The lowest limits of the fluorescent RAA assay were 102 copies/μL for detection of 18S RNA target sequence-contained recombinant plasmids at various copies, 1 pg/μL for detection of genomic DNA of Cryptosporidium oocysts at various concentrations, and one Cryptosporidium oocyst/μL for detection of genomic DNA extracted from various numbers of Cryptosporidium oocysts, and the fluorescent RAA assay was all negative for detection of genomic DNA from G. lamblia cysts, S. japonicum eggs, A. lumbricoides eggs, C. sinensis eggs, Salmonella and Shigella. Conclusion A novel fluorescent RAA assay is successfully established, which is simple, rapid, sensitive and specific to detect genomic DNA of Cryptosporidium oocysts.