Evaluation of Dual-Color Fluorescence In Situ Hybridization With Peptide Nucleic Acid Probes for the Detection of Mycobacterium tuberculosis and Non-Tuberculous Mycobacteria in Clinical Specimens

BACKGROUND: Peptide nucleic acid (PNA) probes are artificial DNA analogues with a hydrophobic nature that can penetrate the mycobacterial cell wall. We evaluated a FISH method for simultaneous detection and identification of Mycobacterium tuberculosis (MTB) and non-tuberculous mycobacteria (NTM) in clinical respiratory specimens using differentially labeled PNA probes. METHODS: PNA probes targeting the mycobacterial 16S ribosomal RNA were synthesized. The cross-reactivity of MTB- and NTM-specific probes was examined with reference strains and 10 other frequently isolated bacterial species. A total of 140 sputum specimens were analyzed, comprising 100 MTB-positive specimens, 21 NTM-positive specimens, and 19 MTB/NTM-negative specimens; all of them were previously confirmed by PCR and culture. The PNA FISH test results were graded by using the United States Centers for Disease Control and Prevention-recommended scale and compared with the results from the fluorochrome acid-fast bacterial stain. RESULTS: The MTB- and NTM-specific PNA probes showed no cross-reactivity with other tested bacterial species. The test results demonstrated 82.9% agreement with the culture results with diagnostic sensitivity of 80.2% and diagnostic specificity of 100.0% (kappa=0.52, 95% confidence interval: 0.370-0.676). CONCLUSIONS: Dual-color PNA FISH showed high specificity for detecting and identifying mycobacteria in clinical specimens. However, because of its relatively low sensitivity, this method could be more applicable to culture confirmation. In application to direct specimens, the possibility of false-negative results needs to be considered.

Development of a real-time reverse transcriptase PCR assay for detection of E119V amino acid change in neuraminidase of influenza A (H3N2) using the TaqMan-MGB probe / 中华预防医学杂志

<p><b>OBJECTIVE</b>To develop a rapid duplex Real-time reverse transcription PCR (rRT-PCR) method to detect E119V mutation on neuraminidase (NA) of influenza A(H3N2) subtype with drug resistance to oseltamivir.</p><p><b>METHODS</b>Twenty-six NA genes of influenza A(H3N2) virus between 2000 and 2012 in GenBank database were selected as the target genes, and specific TaqMan-MGB probe was designed to target the E119V amino acid change in neuraminidase protein. rRT-PCR was then performed and evaluated for the sensitivity, specificity and reproducibility using virus with E119V mutation and clinical samples.</p><p><b>RESULTS</b>This study described the validation of a highly sensitive and specific duplex rRT-PCR for detection of substitutions leading to the E119V amino acid change in NA protein of influenza A(H3N2). Fluorescence signals could be detected even when diluted a A (H3N2) virus (HA = 8) into 10(-5) and linear correlation between the logarithm of the viral titer with the Ct values was observed. In addition, the assay was highly specific in that there was no cross-react with other respiratory viruses, nor did two TaqMan-MGB probes. E119V substitution in quasispecies with both sensitive and resistant viruses could be detected as well. The limit of detection was 5% for quasispecies with high concentrations and 50% for quasispecies with low concentrations. The average coefficient of variation (CV) for within-run assays was 2.32% and 0.57% for H3N2-119E and H3N2-119V primer/probe sets separately, 1.77% and 0.97% for average CV of between-run assays, which exhibited good repeatability. Sequence analysis of twenty NA genes verified glutamic acid (E) at amino acid site 119, which was in consistent with the results from our rRT-PCR method.</p><p><b>CONCLUSION</b>The assay developed in this study is highly sensitive and specific, and easy to operate; thereby it could be used for identification of A(H3N2) virus with E119V amino acid change in NA protein.</p>

Preparation and performance assessment of Gamma-peptide nucleic acid gene chip detection system based on surface plasmon resonance / 生物医学工程学杂志

The aim of this study was to build a gene chip system with surface plasmon resonance (SPR) technique, for which Gamma-peptide nucleic acid (Gamma-PNA) functioned as a probe, in order to improve sensitivity and its specificity. With the use of self-assembled monolayer (SAM) technology, surface chemistry of two-dimensional structure was used. Gamma-PNA was designed according to the bioinformatics, and was plated on the SPR chip modified by SAM. Subsequently, relevant parameters of the experiment were ensured and optimized. The results showed that the performances of Gamma-PNA probe was little affected by the ion concentration of buffer, and it had a strong light signal in a stable state. As the ion concentration was 0, there were still good hybrid reactions; pH value had less influence upon Gamma-PNA probe, and acid environment of buffer could be better. Gamma-PNA probe combined with sensor technologies achieved made the probe with dispensable labels and real-time detection. It also improved the efficiency of the hybridization and the stability, providing the foundation for clinical application.

Optimal concentration of c-erbB2 antisense probe labeled with superparamagnetic iron oxide nanoparticles for magnetic resonance imaging in tumor-bearing nude mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To determine the optimal concentration of c-erbB2 antisense probe labeled with superparamagnetic iron oxide (SPIO) nanoparticles for in vivo tumor imaging in mice using magnetic resonance imaging (MRI).</p><p><b>METHODS</b>Thirty BALB/c mice bearing SK-Br-3 tumor were randomized into 5 groups to receive injections of different concentrations of SPIO-labeled c-erbB2 antisense probe (containing 6.0, 9.0, 12.0, 15.0, or 18.0 mg Fe/kg). MRI was performed before and 6 h after the injections, and the signal intensities of the tumor were compared among the groups. The tumor tissues were then dissected for microscopic examination with HE and Prussian blue staining.</p><p><b>RESULTS</b>The tumor-bearing mice all survived after injections of the probe at doses of 6.0, 9.0 and 12.0 mg, but injections at higher doses (15.0 and 18.0 mg) caused death in some mice. Injections of the probe at the doses of 12.0, 15.0 and 18.0 mg resulted in significant signal enhancement of the tumor (P<0.001) to allow visual identification, but the changes showed no significant differences among the 3 groups (P>0.05). Pathological examination revealed irregular structures of the tumor issue containing numerous heterogeneous tumor cells aligned into cancer nests; Prussian blue staining visualized scattered blue iron particles in the tumor issue, which was especially obvious in mice injected with 12.0, 15.0 and 18.0 mg labeled probe.</p><p><b>CONCLUSION</b>Injection of 12.0 mg/kg SPIO-labeled c-erbB2 antisense probe allows optimal tumor imaging in BALB/c mice using MRI.</p>

Connection of magnetic antisense probe with SK-Br-3 oncocyte mRNA nucleotide detected by high resolution atomic force microscope / 生物医学工程学杂志

The present paper is aimed to detect superparamagnetic iron oxide labeled c-erbB2 oncogene antisense oligonucleotide probe (magnetic antisense probe) connected with SK-Br-3 oncocyte mRNA nucleotide by high resolution atomic force microscope (AFM). We transfected SK-Br-3 oncocyte with magnetic antisense probe, then observed the cells by AFM with high resolution and detected protein expression and magnetic resonance imagine (MRI). The high resolution AFM clearly showed the connection of the oligonucleotide remote end of magnetic antisense probe with the mRNA nucleotide of oncocyte. The expression of e-erbB2 protein in SK-Br3 cells were highly inhibited by using magnetic antisense probe. We then obtained the lowest signal to noise ratio (SNR) of SK-Br-3 oncocyte transfected with magnetic antisense probe by MRI (P<0.05). These experiments demonstrated that the high resolution AFM could be used to show the binding of magnetic antisense probe and SK-Br-3 mRNA of tumor cell nuclear.

Malaria diagnosis: a review

Malaria is the most prevalent endemic disease in large parts of the world and is subject to control by health authorities. Today, the goal of malaria control is to prevent mortality and reduce morbidity and socioeconomic losses through the progressive improvement and strengthening of local and national capabilities. The World Health Organization considers early diagnosis as the first basic element of the strategy to control the disease. Traditionally, laboratory diagnosis has been made using the thick blood film, which continues to be the gold standard test. However, this test has disadvantages such as the manner in which the film is prepared, the level of training of the observer, the adequacy of maintenance of materials and equipment and its only fair sensitivity. Thus, many research laboratories have concentrated their efforts on the development of alternative methods for malaria diagnosis. These include methods for the detection of Plasmodia within erythrocytes (fluorescent microscopy, Quantitative Buffy Coat (QBC(), dark field microscopy, nucleic acid probes and immunofluorescence), methods for the detection of plasmodial antigens in body fluids (radioimmunoassay, enzyme immunoassay) and methods for the detection of anti-plasmodial antibodies in serum (indirect immunofluorescence, enzyme immunoassay, Western blotting). Here, we critically review the various methods for malaria diagnosis based on the world's literature and our experience with most of them, with emphasis on recent advances.