Multiplex loop-mediated isothermal amplification (multi-LAMP) assay for rapid detection of mcr-1 to mcr-5 in colistin-resistant bacteria.

Purpose: The discovery of the plasmid-mediated colistin resistance genes, mcr, revealed a mechanism of transmission of colistin resistance, which is a major, global public health concern especially among individuals infected with carbapenem-resistant Gram-negative bacteria. To monitor the spread and epidemiology of mcr genes, a convenient and reliable method to detect mcr genes in clinical isolates is needed, especially in the primary care institutions. This study aimed to establish a restriction endonuclease-based multiplex loop-mediated isothermal amplification (multi-LAMP) assay to detect mcr genes (mcr-1 to mcr-5) harbored by colistin-resistant bacteria. Methods: A triple-LAMP assay for mcr-1, mcr-3, and mcr-4 and a double-LAMP assay for mcr-2 and mcr-5 were established. The sensitivity and specificity of the LAMP reactions were determined via electrophoresis and visual detection. Results: The sensitivity of the LAMP assay was 10-fold greater than that of PCR, with high specificity among the screened primers. Specific mcr genes were distinguished in accordance with band numbers and the fragment length of the digested LAMP amplification products. Furthermore, the LAMP assay was confirmed as a rapid and reliable diagnostic technique upon application for clinical samples, and the results were consistent with those of conventional PCR assay. Conclusion: The multi-LAMP assay is a potentially promising method to detect mcr genes and will, if implemented, help prevent infections by drug-resistant bacteria in primary-care hospitals due to rapid and reliable surveillance. To our knowledge, this is the first study to report the application of LAMP to detect mcr-2 to mcr-5 genes and the first time that multi-LAMP has been applied to detect mcr genes.

Folding studies of two hydrostatic pressure sensitive proteins.

High hydrostatic pressure combined with various spectroscopies is a powerful technique to study protein folding. An ideal model system for protein folding studies should have the following characteristics. (1) The protein should be sensitive to pressure, so that the protein can be unfolded under mild pressure. (2) The folding process of the protein should be easily modulated by several chemical or physical factors. (3) The folding process should be easily monitored by some spectroscopic parameters. Here, we summarized the pressure induced folding studies of two proteins isolated from spinach photosystem II, namely the 23-kDa and the 33-kDa protein. They have all the characteristics mention above and might be an ideal model protein system for pressure studies.

An oligonucleotide microarray for microRNA expression analysis based on labeling RNA with quantum dot and nanogold probe.

MicroRNAs (miRNAs) play important regulatory roles in animals and plants by targeting mRNAs for cleavage or translational repression. They have diverse expression patterns and might regulate various developmental and physiological processes. Profiling miRNA expression is very helpful for studying biological functions of miRNAs. We report a novel miRNA profiling microarray, in which miRNAs were directly labeled at the 3' terminus with biotin and hybridized with complementary oligo-DNA probes immobilized on glass slides, and subsequently detected by measuring fluorescence of quantum dots labeled with streptavidin bound to miRNAs through streptavidin-biotin interaction. The detection limit of this microarray for miRNA was approximately 0.4 fmol, and the detection dynamic range spanned about 2 orders of magnitude. We made a model microarray to profile 11 miRNAs from leaf and root of rice (Oryza sativa L. ssp. indica) seedlings. The analysis results of the miRNAs had a good reproducibility and were consistent with the northern blot result. To avoid using high-cost detection equipment, colorimetric detection, a method based on nanogold probe coupled with silver enhancement, was also successfully introduced into miRNA profiling microarray detection.

Pressure equilibrium and jump study on unfolding of 23-kDa protein from spinach photosystem II.

Pressure-induced unfolding of 23-kDa protein from spinach photosystem II has been systematically investigated at various experimental conditions. Thermodynamic equilibrium studies indicate that the protein is very sensitive to pressure. At 20 degrees C and pH 5.5, 23-kDa protein shows a reversible two-state unfolding transition under pressure with a midpoint near 160 MPa, which is much lower than most natural proteins studied to date. The free energy (DeltaG(u)) and volume change (DeltaV(u)) for the unfolding are 5.9 kcal/mol and -160 ml/mol, respectively. It was found that NaCl and sucrose significantly stabilize the protein from unfolding and the stabilization is associated not only with an increase in DeltaG(u) but also with a decrease in DeltaV(u). The pressure-jump studies of 23-kDa protein reveal a negative activation volume for unfolding (-66.2 ml/mol) and a positive activation volume for refolding (84.1 ml/mol), indicating that, in terms of system volume, the protein transition state lies between the folded and unfolded states. Examination of the temperature effect on the unfolding kinetics indicates that the thermal expansibility of the transition state and the unfolded state of 23-kDa protein are closer to each other and they are larger than that of the native state. The diverse pressure-refolding pathways of 23-kDa protein in some conditions were revealed in pressure-jump kinetics.

Colorimetric detection of protein microarrays based on nanogold probe coupled with silver enhancement.

This work presents a method for analyzing protein microarrays using a colorimetric nanogold probe coupled with silver enhancement (gold-silver detection). In this method, the gold nanoparticles were introduced to the microarray by the specific binding of the gold-conjugated antibodies or streptavidins and then coupled with silver enhancement to produce black image of microarray spots, which can be easily detected with a commercial CCD camera. The method showed high detection sensitivity (1 pg of IgG immobilized on slides or 2.75 ng/ml IgG in solution) and a good linear correlation between the signal intensity and the logarithm of the sample concentration. The examination of this method in analyzing a demonstrational ToRCH antigen microarray developed in our lab showed an identical result as in the fluorescent method. These results suggest the colorimetric gold-silver detection method has potential applications in proteomics research and clinical diagnosis.

[Thermodynamic and kinetic analysis of unfolding of P23k protein isolated from spinach photosystem II].

The unfolding of 23kD (P23k) protein isolated from spinach photosystem II particle was studied by high pressure and fluorescence spectroscopy. The thermal equilibrium study indicated that the protein could be totally unfolded by 180 or 160 MPa at 20 degrees C and 3 degrees C, respectively. The standard free energy and standard volume change of the protein for unfolding at 20 degrees C is 23.45 kJ/mol and -150.3 ml/mol, respectively. Kinetics study indicated that at 20 degrees C the activation volume for unfolding, delta V(u)(++), was negative (-66.2 ml/mol), meanwhile the activation volume for folding, deltaV(f)(++), was positive (84.1 ml/mol). The rate constants for folding and unfolding (K(0f), K(0u)) were 1.87 s(-1) and 1.3x10(-4) s(-1), respectively, these results provide some clues to explain why the protein is so sensitive to pressure.

[Application of quantum dot to life science].

As an eminent representation of nanotechnology, quantum dot (semiconductor nanocrystal) has caught great interests of scientists in physics, chemistry, material science, and biology extensively. Although its application to life science has just been explored, valuable progresses have been achieved in both biomacromolecule labeling and coding recently. The progress in quantum dot synthesis, its spectroscopic and photoelectronic properties, and its potential application to life science are reviewed.