Genomic Dna Cloning Of Rickettsia-Like Organisms (Rlo) Of Saint-Jacques Scallop Pecten-Maximus - Evaluation Of Prokaryote Diagnosis By Hybridization With A Non-Isotopically Labeled Probe And By Polymerase Chain-Reaction

Genomic DNA fragments of a rickettsia-like organism (RLO) of Saint-Jacques scallop, Pecten maximus, were extracted from purified RLO and cloned. A DNA sequence of 1500 bp taken from one of the sequenced clones was coupled to horseradish peroxidase and the resulting probe was assayed for the diagnosis of RLO by nucleic acid hybridization. The specificity of the probe for RLO DNA was established from its lack of hybridization with scallop DNA, The sensitivity limit of the probe detected by enhanced chemiluminescence was determined to be around 500 ng of total RLO nucleic acids, equivalent to about 2.5 X 10(6) copies of RLO DNA. A pair of polymerase chain reaction (PCR) primers were synthesized that amplified a 1300 bp segment of the 1500 bp RLO fragment. After 30 cycles of amplification of total RLO nucleic acids, a product of the expected size was easily visible on agarose at all starting template concentrations between 100 ng and 10 pg of RLO total nucleic acids. The lowest level of sensitivity corresponds to about 100 pg of RLO DNA or about 50 genomic molecules. The specificity of the amplified DNA was confirmed by Southern blot analysis using the peroxidase-labelled probe and by the lack of amplified product with scallop DNA as the starting template.