Temperature-mediated heteroduplex analysis for the detection of drug-resistant gene mutations in clinical isolates of Mycobacterium tuberculosis by denaturing HPLC, SURVEYOR nuclease.

Denaturing high-performance liquid chromatography (DHPLC) is a relatively new technique, which utilizes heteroduplex formation between wild-type and mutated DNA strands to identify point mutations. Heteroduplex molecules are separated from homoduplex molecules by ion-pair, reverse-phase liquid chromatography on a special column matrix with partial heat denaturation of the DNA strands. In order to investigate the application of this method for point mutation detection in drug-resistant genes of Mycobacterium tuberculosis, katG, rpoB, embB, gyrA, pncA and rpsL genes, which are responsible for isoniazid, rifampicin, ethambutol, fluoroquinolone, pyrazinamide and streptomycin resistance, respectively, were detected by temperature-mediated DHPLC in 10 multidrug-resistant and 10 drug-susceptible clinical isolates. The DHPLC data were compared with those from a conventional MIC test. The results show that DHPLC is cost-effective with high capacity and accuracy, and is potentially useful for genotypic screening for mutations associated with anti-tuberculosis drug resistance.

Imaging of pulmonary granulomas using a photon imager.

To clarify the location of pulmonary granulomas in vivo, we prepared a Mycobacterium tuberculosis H37Rv mutant in which the gene for a green fluorescent protein (GFP) (GFP-H37Rv) was introduced. Five weeks after aerosol infection with GFP-H37Rv, the infected lungs from guinea pigs and mice were subjected to imaging using a photon imager. Pulmonary granulomas more than 1 mm in diameter were localized clearly by the photon imager. Therefore, if a method for binding a dye (GFP, fluorescein isothiocyanate [FITC], etc.) specifically to M. tuberculosis can be developed, it will be possible to visualize granulomas using a photon imager.

Antimicrobial activity of cationic gemini surfactant containing an oxycarbonyl group in the lipophilic portion against gram-positive and gram-negative microorganisms.

We evaluated the antimicrobial activities of a cationic Gemini surfactant, trans-1,4-bis[2-(alkanoyloxy)ethyldimethylammonio]-2-butene dichloride [II-m-2(t-butene)] and its derivatives against Gram-positive and Gram-negative microorganisms. The II-m-2(t-butene) compound was previously shown to have good surface activity and biodegradability. A dodecanoyloxy derivative (m = 12) of II-m-2(t-butene) showed excellent antimicrobial activity against Gram-positive Streptococcus aureus [minimum inhibitory concentration (MIC): 7.8 µg/mL] and Gram-negative Escherichia coli (MIC: 31.2 µg/mL).

SNP genotyping in the beta(2)-adrenergic receptor by electronic microchip assay, DHPLC, and direct sequencing.

The beta(2)-adrenergic receptor (beta2AR) is the key target for the beta(2)-agonist drugs used for bronchodilation in asthma and chronic obstructive pulmonary disease. To detect four SNPs with amino acid variations at positions -47T/C (CysBUP19Arg), 46A/G (Gly16Arg), 79C/G (Gln27Glu), and 491C/T (Thr164Ile) in the beta 2AR gene, we used the electronic microchip assay, denaturing high-performance liquid chromatography (DHPLC), and direct sequencing. Genomic DNA samples were obtained from the blood of 84 Japanese healthy volunteers. The agreement rates of the first data set with the final data (allele calls) were 99.7% (332/333), 99.2% (246/248), and 96.7% (329/340). The percentages of no allele designation (ND) were 2.06% (7/340), 2.75% (7/255), and 0.00% (0/340) for the electronic microchip assay, DHPLC, and direct sequencing, respectively. Furthermore, we found three samples that had a novel haplotype.