Development of a 16S rRNA-based oligomer probe specific for Listeria monocytogenes

Abstract

Crude rRNA was isolated from Listeria monocytogenes, L. innocua, and L. ivanovii and sequenced by a reverse transcriptase method. Only two sequence regions were found to differ for L. monocytogenes versus L. innocua or L. ivanovii. Two oligonucleotide probes (RL-1 and RL-2) complementary to these two regions of rRNA of L. monocytogenes were synthesized. The RL-1 probe had one base while the RL-2 probe had two bases which differed for L. monocytogenes versus L. innocua and L. ivanovii. Use of a dried gel hybridization in place of Northern (RNA) hybridization or dot blot hybridization indicated that the RL-2 probe hybridized with all 36 strains of L. monocytogenes tested but not with 6 other Listeria species and 11 other bacteria tested. The RL-2 probe is specific for L. monocytogenes, while the RL-1 probe showed some cross-reactions with other Listeria species. An alkaline phosphatase-labeled RL-2 probe could be used in a dot blot hybridization test and gave good results, but a 32P-labeled RL-2 probe was more sensitive than the nonradioactive probe and the 32P-labeled probe was useable for up to 2 months, even though the 32P was highly degenerated.