5 research outputs found

    Electrochemical DNA Biosensor Based on a Tetrahedral Nanostructure Probe for the Detection of Avian Influenza A (H7N9) Virus

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    A DNA tetrahedral nanostructure-based electrochemical biosensor was developed to detect avian influenza A (H7N9) virus through recognizing a fragment of the hemagglutinin gene sequence. The DNA tetrahedral probe was immobilized onto a gold electrode surface based on self-assembly between three thiolated nucleotide sequences and a longer nucleotide sequence containing complementary DNA to hybridize with the target single-stranded (ss)­DNA. The captured target sequence was hybridized with a biotinylated-ssDNA oligonucleotide as a detection probe, and then avidin-horseradish peroxidase was introduced to produce an amperometric signal through the interaction with 3,3′,5,5′-tetramethylbenzidine substrate. The target ssDNA was obtained by asymmetric polymerase chain reaction (PCR) of the cDNA template, reversely transcribed from the viral lysate of influenza A (H7N9) virus in throat swabs. The results showed that this electrochemical biosensor could specifically recognize the target DNA fragment of influenza A (H7N9) virus from other types of influenza viruses, such as influenza A (H1N1) and (H3N2) viruses, and even from single-base mismatches of oligonucleotides. Its detection limit could reach a magnitude of 100 fM for target nucleotide sequences. Moreover, the cycle number of the asymmetric PCR could be reduced below three with the electrochemical biosensor still distinguishing the target sequence from the negative control. To the best of our knowledge, this is the first report of the detection of target DNA from clinical samples using a tetrahedral DNA probe functionalized electrochemical biosensor. It displays that the DNA tetrahedra has a great potential application as a probe of the electrochemical biosensor to detect avian influenza A (H7N9) virus and other pathogens at the gene level, which will potentially aid the prevention and control of the disease caused by such pathogens

    Mutations detected in the <i>gyrA</i> and <i>parC</i> gene of H<sub>2</sub>S-negative <i>S</i>. Choleraesuis isolates.

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    <p>Ser, serine. Gly, glycine. Ala, alanine. Tyr, tyrosine. Cys, cysteine. Arg, arginine. Pro, proline.</p><p>Mutations detected in the <i>gyrA</i> and <i>parC</i> gene of H<sub>2</sub>S-negative <i>S</i>. Choleraesuis isolates.</p

    Antimicrobial Resistance and Molecular Investigation of H<sub>2</sub>S-Negative <i>Salmonella enterica</i> subsp. <i>enterica</i> serovar Choleraesuis Isolates in China

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    <div><p><i>Salmonella enterica</i> subsp. <i>enterica</i> serovar Choleraesuis is a highly invasive pathogen of swine that frequently causes serious outbreaks, in particular in Asia, and can also cause severe invasive disease in humans. In this study, 21 <i>S</i>. Choleraesuis isolates, detected from 21 patients with diarrhea in China between 2010 and 2011, were found to include 19 H<sub>2</sub>S-negative <i>S</i>. Choleraesuis isolates and two H<sub>2</sub>S-positive isolates. This is the first report of H<sub>2</sub>S-negative <i>S</i>. Choleraesuis isolated from humans. The majority of H<sub>2</sub>S-negative isolates exhibited high resistance to ampicillin, chloramphenicol, gentamicin, tetracycline, ticarcillin, and trimethoprim-sulfamethoxazole, but only six isolates were resistant to norfloxacin. In contrast, all of the isolates were sensitive to cephalosporins. Fifteen isolates were found to be multidrug resistant. In norfloxacin-resistant isolates, we detected mutations in the <i>gyrA</i> and <i>parC</i> genes and identified two new mutations in the <i>parC</i> gene. Pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and clustered regularly interspaced short palindromic repeat (CRISPR) analysis were employed to investigate the genetic relatedness of H<sub>2</sub>S-negative and H<sub>2</sub>S-positive <i>S</i>. Choleraesuis isolates. PFGE revealed two groups, with all 19 H<sub>2</sub>S-negative <i>S</i>. Choleraesuis isolates belonging to Group I and H<sub>2</sub>S-positive isolates belonging to Group II. By MLST analysis, the H<sub>2</sub>S-negative isolates were all found to belong to ST68 and H<sub>2</sub>S-positive isolates belong to ST145. By CRISPR analysis, no significant differences in CRISPR 1 were detected; however, one H<sub>2</sub>S-negative isolate was found to contain three new spacers in CRISPR 2. All 19 H<sub>2</sub>S-negative isolates also possessed a frame-shift mutation at position 760 of <i>phsA</i> gene compared with H<sub>2</sub>S-positive isolates, which may be responsible for the H<sub>2</sub>S-negative phenotype. Moreover, the 19 H<sub>2</sub>S-negative isolates have similar PFGE patterns and same mutation site in the <i>phs</i>A gene, these results indicated that these H<sub>2</sub>S-negative isolates may have been prevalent in China. These findings suggested that surveillance should be increased of H<sub>2</sub>S-negative <i>S</i>. Choleraesuis in China.</p></div