DNA microarrays for visual detection of human pathogenic microorganisms based on tyramine signal amplification coupled with gold label silver stain.

The utility of DNA microarrays is severely limited by their restricted sensitivity. Tyramine signal amplification (TSA) coupled with gold label silver stain (GLSS) was introduced in DNA microarrays for visual detection of human pathogenic microorganisms. First, a TSA system was introduced to the microarrays after the microarrays were prepared and hybridized with biotinylated targets. This procedure leads to large amounts of biotin-conjugated tyramine depositing at the site of enzyme reaction under HRP catalysis. Second, streptavidin-nanogold was introduced and accumulated by specific binding of biotin and streptavidin. Finally, silver staining was performed. The images of the spots were scanned with a visible light scanner and quantified with ArrayVision 7.0 software. Detection conditions were systematically optimized. Then the sensitivity among TSA coupled with GLSS, GLSS, and TSA coupled with Cy3 was compared. The optimized conditions were: streptavidin-HRP (1 mg mL(-1)) dilution 1:1500, biotin-tyramine dilution 1:200 (+0.5% H(2)O(2)), streptavidin-nanogold dilution 1:100 (all diluted in 1 × PBS + 1% BSA) and silver stain time of 10 min. The sensitivity of TSA coupled with GLSS was 100-fold higher than that of GLSS, and was identical with that of TSA coupled with Cy3. Meanwhile, the specificity of the microarrays were not affected. This implied that TSA coupled with GLSS was a sensitive visual detection method and would be an ideal alternative to fluorescence-based detection for DNA microarrays.

[Different expressions of inflammatory cytokines in two types of cardiac hypertrophy in rats].

OBJECTIVE: To compare the different expressions of cardiac inflammatory cytokines including tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta(IL-1beta), interleukin-6 (IL-6) in two types of cardiac hypertrophy rat models induced by volume overload and pressure overload. METHODS: Volume overload-induced cardiac hypertrophy was established by abdominal arteriovenous fistula (ACF) and pressure overload-induced cardiac hypertrophy was developed by constriction of aorta (CA). Heart weight measurement and histological examination were performed 1 week or 2 weeks after the operation respectively. The cytokine expression was measured by enzyme linked immunosorbent assay. RESULTS: All the operated groups developed cardiac hypertrophy. The left ventricular fractional shortening of each operated group had no significant difference with the sham-operated groups respectively. As far as the total amount of each cytokine in left ventricular myocardium was concerned, compared to the sham-operated groups, IL-6 and IL-1beta both increased significantly in CA groups [IL-6(23 722+/-8 671)pg vs (17 693+/-5 705)pg,P<0.05 ;IL-1beta(335+/-95)pg vs (159+/-99) pg,P<0.01].There was no difference of TNF-alpha between operated and sham-operated groups in ACF or CA groups . CONCLUSION: The contents of IL-6 and IL-1beta in myocardium increased in pressure overload-induced cardiac hypertrophy.

[In vitro selection and affinity function of the aptamers to Bacillus anthracis spores by SELEX].

To obtain oligonucleotide aptamers, specifically binding to Bacillus anthracis spores, and to find the relationship between the structures and the affinities, and to determine whether the aptamers can be used as a novel molecule for spore detection, a synthetic 35 mer random DNA library was subjected to 18 rounds of selection by using SELEX (systematic evolution of ligands by exponential enrichment) protocol against spores of Bacillus anthracis vaccine strain A. 16R. The selected aptamers were cloned and sequenced. Software packages CLUSTALX (1.8) and DNASIS v2.5 were employed to analyze the conserved sequences and second structures of the aptamers, respectively. Affinities of aptamers to the spores were visualized by biotin streptavidin horseradish peroxidase system. DAB was used to visualize signals, as an assay method. A membrane-based hybrid sandwich assay was developed for detecting Bacillus anthracic spores by using a 5'-biotinylated ssDNA aptamers and anti-spore antibodies. PCR amplification band pattern of the first round selection was different from that of the ninth round. The binding assay demonstrated that the affinity of the eighteenth round pool increased thirty-seven folds more than that of the first round pool. The affinities of the aptamers were different: the highest A at 450 nm was 1.20, and the lowest was 0.20. The secondary structure analysis revealed possible stem-loop and hairpin structures for binding to the spores. The colorimetry on the immuno-membrane got the best signal with a ratio of 16 microgram aptamer to 4x10(7) spores. A set of aptamers with considerable binding affinity to Bacillus anthracis spores was successfully selected from the initial random ssDNA pool. The stem-loop and hairpin at 5' end of the aptamers worked as the main motif in the interaction between oligonucleotides and spores, while the neighbor bases of the triple structure might affect the stability. Therefore ssDNA aptamers seem to be a type of potential diagnostic molecule.

Analysis on the clinical features of 507 HDV-infected patients.

The objective of this study was to analyze the clinical feature of hepatitis delta virus (HDV)-infected patients and to discuss the pathological mechanism of hepatitis D. A total of 507 patients with hepatitis due to the infection of HDV were included. The incidence rates of various hepatitis subtypes, the sequelae, the clinical manifestation, the hepatic function, and the hepatic virus makers for all the 507 patients were analyzed statistically. A cohort of 213 patients with hepatitis B, who were also HDV free, served as the control. HDV infection significantly contributed to the increased incidence rate and mortality of severe hepatitis (SH) and cirrhosis (P < 0.01). HDV was also associated with higher incidence rates of hemorrhage in the gastrointestinal tract, abdominal ascites and hepatic encephalopathy, repetitive augmentation of alanine transaminase, and its enhanced magnitude (P < 0.01 or 0.05). The major liver function changes in patients with SH or chronic serious hepatitis was significant compared to the control (P < 0.01). Within the HDV(+) category, HBeAg(-) expression was significantly higher in the HBV DNA(-) group than the HBV DNA(+) group (P < 0.01). The expression of HDAg(+) HBeAg(-) in acute hepatitis, SH, and cirrhosis was significantly higher than that of HDAg(+) HBeAg(+) (P < 0.01 or P < 0.05). The HDV infection was closely associated with the development and prognosis of chronic serious hepatitis, SH, and cirrhosis. HDV infection could inhibit the HBV DNA replication or the HBcAg expression. The direct cytotoxicity of HDV might be the leading pathological factor in AH. HDV might play a major role in the deterioration and chronicization of HDV-co-infected hepatitis B and was responsible for the increased mortality of HBV/HDV patients.

Screening of specific antigens for SARS clinical diagnosis using a protein microarray.

In this study several SARS-CoV structural proteins and fragments were expressed in E. coli as GST or TRX fusion proteins. They were fabricated on a microarray and tested with sera from SARS patients. Antigenic screening indicated that recombinant GST-N2 fusion protein, the carboxy-terminus 213aa-423aa of N protein, was strongest positive and weakest non-specific compared with others. An indirect antibody ELISA method was developed and clinical positive and negative sera for their antibodies against GST-N2 fusion protein were assayed. 311 out of the 442 sera from clinical SARS inpatients, as well as 229 out of 302 sera from convalescent patients gave positive reactivities; positive rates were 70.4% and 75.8% respectively. Sera from a total of 2726 non-SARS patients and healthy individuals were tested and the false positive rate was only 0.07%. When the sensitivity control sample was diluted 1 : 64, it yielded OD values above the cutoff value. Reported data showed that this was a relatively high degree of sensitivity and specificity for SARS-CoV antibody testing. The data indicate that GST-N2 fusion protein, which was screened by protein microarray, may be a valuable diagnostic antigen for the development of serological assays for SARS. In addition, protein microarray assay presents a higher positive rate and sensitivity (86.1% and 1 : 200) compared with the traditional ELISA screening method, and could provide a rapid, parallel and high-throughput antigen screening platform.