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Yuan J.,Jiangnan University | Yu Y.,Zhangjiagang Entry Exit Inspection and Quarantine Bureau | Li C.,Jiangnan University | Ma X.,Jiangnan University | And 3 more authors.
Microchimica Acta | Year: 2014

We have developed a specific method for the visual detection of Staphylococcus aureus based on aptamer recognition coupled to tyramine signal amplification technology. A biotinylated aptamer specific for S. aureus was immobilized on the surface of the wells of a microplate via biotin-avidin binding. Then, the target bacteria (S. aureus), the biotinylated-aptamer-streptavidin-HRP conjugates, biotinylated tyramine, hydrogen peroxide and streptavidin-HRP were successively placed in the wells of the microplate. After adding TMB reagent and stop solution, the intensity of the yellow reaction product can be visually inspected or measured with a plate reader. Under optimized conditions, there is a linear relationship between absorbance at 450 nm and the concentration of S. aureus in the 10 to 107 cfu mL-1 concentration range (with an R2 of 0.9976). The limit of detection is 8 cfu mL-1. [Figure not available: see fulltext.] © 2013 Springer-Verlag Wien.


Yuan J.,Jiangnan University | Tao Z.,Jiangnan University | Yu Y.,Zhangjiagang Entry Exit Inspection and Quarantine Bureau | Ma X.,Jiangnan University | And 3 more authors.
Food Control | Year: 2014

A rapid, specific and visible detection method for Salmonella Typhimurium was developed based on the recognition of aptamers coupled with nanogold labeling and silver signal amplification. Briefly, biotinylated aptamer 1, which was specific to Salmonella Typhimurium, was immobilized onto the surface of microtiter plate-wells modified with streptavidin through the binding of biotin and streptavidin. Then, the target bacteria, Salmonella Typhimurium, and the aptamer 2-gold nanoparticle (aptamer 2-AuNPs) conjugates were incubated in the wells to form sandwich-type aptamer/bacteria/aptamer-AuNPs complexes. Under optimal conditions, the correlation between the concentration of Salmonella Typhimurium and the intensity of the signals was observed to be linear within the range of 10-106cfumL-1 (R2=0.9913), and the detection limit of the proposed method was observed to be 7cfumL-1 This developed method offers the potential for rapid, sensitive and visible detection of Salmonella Typhimurium in samples. © 2013 Elsevier Ltd.


Duan N.,Jiangnan University | Wu S.,Jiangnan University | Yu Y.,Zhangjiagang Entry Exit Inspection and Quarantine Bureau | Ma X.,Jiangnan University | And 4 more authors.
Analytica Chimica Acta | Year: 2013

A sensitive, specific method for the collection and detection of pathogenic bacteria was demonstrated using quantum dots (QDs) as a fluorescence marker coupled with aptamers as the molecular recognition element by flow cytometry. The aptamer sequences were selected using a bacterium-based SELEX strategy in our laboratory for Vibrio parahaemolyticus and Salmonella typhimurium that, when applied in this method, allows for the specific recognition of the bacteria from complex mixtures including shrimp samples. Aptamer-modified QDs (QD-apt) were employed to selectively capture and simultaneously detect the target bacteria with high sensitivity using the fluorescence of the labeled QDs. The signal intensity is amplified due to the high photostability of QDs nanoparticles, resulting in improved sensitivity over methods using individual dye-labeled probes. This proposed method is promising for the sensitive detection of other pathogenic bacteria in food stuff if suitable aptamers are chosen. The method may also provide another potential platform for the application of aptamer-conjugated QDs in flow cytometry. © 2013 Elsevier B.V.


Xie Y.,Jiangnan University | Xu L.,Jiangnan University | Wang Y.,Zhangjiagang Entry Exit Inspection and Quarantine Bureau | Shao J.,Zhangjiagang Entry Exit Inspection and Quarantine Bureau | And 4 more authors.
Analytical Methods | Year: 2013

Label-free and rapid assessment of foodborne pathogenic contamination, which can have significant implications for food safety, is critically important. In this study, seven foodborne bacteria (Salmonella typhimurium ATCC 50013, Salmonella O7HZ10, Shigella boydii CMCC51514, Shigella sonnei CMCC51529, Shigella dysenteriae CMCC51252, Citrobacter freundii ATCC43864, and Enterobacter sakazakii 154) have been examined, which are classified as Enterobacteriaceae according to biological taxonomy. They often have high rates of outbreak. A rapid and sensitive measurement method has been developed based on surface-enhanced Raman spectroscopy (SERS) coupled with gold nanoparticles as SERS substrates. The respective SERS spectra of the pathogens and their reproducibility have been compared. The principal component analysis (PCA) multivariate statistical technique has been employed to analyze the data, and a dendrogram cluster was constructed by hierarchical cluster analysis (HCA). Our results have shown that a rapid, sensitive, label-free identification of the foodborne pathogenic bacteria could be obtained by using gold nanoparticles as effective and stable SERS substrates. © 2013 The Royal Society of Chemistry.


Ma X.,Jiangnan University | Jiang Y.,Jiangnan University | Jia F.,Jiangnan University | Yu Y.,Zhangjiagang Entry Exit Inspection and Quarantine Bureau | And 2 more authors.
Journal of Microbiological Methods | Year: 2014

Salmonella is one of the most common causes of food-associated disease. An electrochemical biosensor was developed for Salmonella detection using a Salmonella-specific recognition aptamer. The biosensor was based on a glassy carbon electrode modified with graphene oxide and gold nanoparticles. Then, the aptamer ssDNA sequence could be linked to the electrode. Each assembly step was accompanied by changes to the electrochemical parameters. After incubation of the modified electrode with Salmonella, the electrochemical properties between the electrode and the electrolyte changed accordingly. The electrochemical impedance spectrum was measured to quantify the Salmonella. The results revealed that, when more Salmonella were added to the reaction system, the current between the electrode and electrolyte decreased; in other words, the impendence gradually increased. A detection limit as low as 3. cfu/mL was obtained. This novel method is specific and fast, and it has the potential for real sample detection. © 2014 Elsevier B.V.


Jiang H.,Jiangnan University | Jiang D.,Jiangnan University | Jiang D.,Yangzhou University | Shao J.,Zhangjiagang Entry Exit Inspection And Quarantine Bureau | Sun X.,Jiangnan University
Biosensors and Bioelectronics | Year: 2016

We have developed a novel and economical electrochemical sensor to measure Gram-negative bacterial quorum signaling molecules (AHLs) using magnetic nanoparticles and molecularly imprinted polymer (MIP) technology. Magnetic molecularly imprinted polymers (MMIPs) capable of selectively absorbing AHLs were successfully synthesized by surface polymerization. The particles were deposited onto a magnetic carbon paste electrode (MGCE) surface, and characterized by electrochemical measurements. Differential Pulse Voltammetry (DPV) was utilized to record the oxidative current signal that is characteristic of AHL. The detection limit of this assay was determined to be 8×10-10molL-1 with a linear detection range of 2.5×10-9molL-1 to 1.0×10-7molL-1. This Fe3O4@SiO2-MIP-based electrochemical sensor is a valuable new tool that allows quantitative measurement of Gram-negative bacterial quorum signaling molecules. It has potential applications in the fields of clinical diagnosis or food analysis with real-time detection capability, high specificity, excellent reproducibility, and good stability. © 2015 Elsevier B.V.


PubMed | Bright Dairy & Food Co., Zhangjiagang Entry Exit Inspection and Quarantine Bureau and Jiangnan University
Type: | Journal: Biosensors & bioelectronics | Year: 2016

In this study, a sensitive and simple electrochemical murine macrophage (Ana-1) cell sensor has been developed for early detection of lipopolysaccharides (LPS) to evaluate the toxicity of pathogenic bacteria. Magnetic glassy carbon electrode (MGCE), which possesses excellent reproducibility and regeneration qualities, was modified with a nanocomposite to improve electrochemical signals and enhance the sensitivity. The synthesized magnetic nanoparticles (MNPs) were internalized into murine macrophages, which completed the immobilization of macrophages onto the modified electrode for evaluating the cytotoxicity of LPS by electrochemical impedance spectroscopy (EIS). The MNPs facilitated reusability of the proposed sensor by allowing removal of the magnetic core from the electrode. Our results indicated that LPS caused a marked decrease in electrochemical impedance in a dose-dependent manner in range of 1-5g/mL. By SEM, we found that microvilli on the plasma membrane became scarce and the membrane became smooth on cells incubated with LPS, which lessens the absorption of cells to reduce the impedance. And biological assay indicated that EIS patterns were correlated with the calcium concentration in cells, and suggested that [Ca(2+)]i production increased in cells incubated with LPS and its mobilization altered electrochemical signals. Compared with conventional methods, this electrochemical test is inexpensive, highly sensitive, and has a quick response, and thus provides a new avenue for evaluating the cytotoxicity of pathogens.


PubMed | Yangzhou University, Zhangjiagang Entry Exit Inspection And Quarantine Bureau and Jiangnan University
Type: | Journal: Scientific reports | Year: 2016

Due to the high toxicity of bacterial lipopolysaccharide (LPS), resulting in sepsis and septic shock, two major causes of death worldwide, significant effort is directed toward the development of specific trace-level LPS detection systems. Here, we report sensitive, user-friendly, high-throughput LPS detection in a 96-well microplate using a transcriptional biosensor system, based on 293/hTLR4A-MD2-CD14 cells that are transformed by a red fluorescent protein (mCherry) gene under the transcriptional control of an NF-B response element. The recognition of LPS activates the biosensor cell, TLR4, and the co-receptor-induced NF-B signaling pathway, which results in the expression of mCherry fluorescent protein. The novel cell-based biosensor detects LPS with specificity at low concentration. The cell-based biosensor was evaluated by testing LPS isolated from 14 bacteria. Of the tested bacteria, 13 isolated Enterobacteraceous LPSs with hexa-acylated structures were found to increase red fluorescence and one penta-acylated LPS from Pseudomonadaceae appeared less potent. The proposed biosensor has potential for use in the LPS detection in foodstuff and biological products, as well as bacteria identification, assisting the control of foodborne diseases.


PubMed | Zhangjiagang Entry Exit Inspection And Quarantine Bureau and Jiangnan University
Type: | Journal: Biosensors & bioelectronics | Year: 2015

We have developed a novel and economical electrochemical sensor to measure Gram-negative bacterial quorum signaling molecules (AHLs) using magnetic nanoparticles and molecularly imprinted polymer (MIP) technology. Magnetic molecularly imprinted polymers (MMIPs) capable of selectively absorbing AHLs were successfully synthesized by surface polymerization. The particles were deposited onto a magnetic carbon paste electrode (MGCE) surface, and characterized by electrochemical measurements. Differential Pulse Voltammetry (DPV) was utilized to record the oxidative current signal that is characteristic of AHL. The detection limit of this assay was determined to be 810(-10)molL(-1) with a linear detection range of 2.510(-9)molL(-1) to 1.010(-7)molL(-1). This Fe3O4@SiO2-MIP-based electrochemical sensor is a valuable new tool that allows quantitative measurement of Gram-negative bacterial quorum signaling molecules. It has potential applications in the fields of clinical diagnosis or food analysis with real-time detection capability, high specificity, excellent reproducibility, and good stability.


Shao J.D.,Zhangjiagang Entry Exit Inspection and Quarantine Bureau
Zhongguo ji sheng chong xue yu ji sheng chong bing za zhi = Chinese journal of parasitology & parasitic diseases | Year: 2012

The special DnaJ-like protein gene of Cryptosporidium parvum was amplified through designing special primers and TaqMan probes within the conserved and specific regions for this gene. method of real-time PCR assay for the detection of C. parvum was established. The specificity and sensitivity of PCR were also analyzed. By adding standard culture fluid in blank fecal sample, the sensitivity of the method was evaluated. The results showed that the detection limit of pure culture with real-time PCR assay was 26 oocysts/ml. The detection limit for C. parvum in artificially contaminated fecal sample was 2 600 oocysts/ml. The specificity of the method was verified with no amplification on DNA from other enteric parasites and bacteria. These results indicated that the real-time PCR method for C. parvum detection in fecal sample is simple, rapid, with high specificity and sensitivity.

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