Quantification of alpha-amylase mRNA in Bacillus subtilis by nucleic acid sandwich hybridization.

A novel hybridization test for quantification of mRNA from bacterial cells lysed directly in the culture medium was developed and optimized. The method uses two adjacent probes from a DNA fragment of interest in a sandwich hybridization. An unlabeled probe is immobilized on a solid support to capture homologous nucleic acids in the test solution. Hybrid detection is performed with the other, labeled, probe, which can bind to the filter only as a sample-mediated hybrid. We used the Bacillus amyloliquefaciens alpha-amylase gene and its mRNA as models for characterizing gene expression in Bacillus subtilis. To confirm the specificity of mRNA hybridization, nondenatured nucleic acid samples were allowed to react simultaneously with two filters: the mRNA-specific filter containing anti-sense single-stranded DNA and the background filter containing single-stranded DNA of the mRNA sense, respectively. We also developed a rapid enzymatic lysis procedure for B. subtilis, allowing complete degradation of late stationary phase cells grown in rich culture medium, while retaining mRNA molecules of high integrity. The applicability of this rapid lysis procedure and the hybridization method for mRNA analyses in long-term fermentations was demonstrated.