Insitu hybridization method for detecting target nucleic acid

Abstract

The present invention provides a method for detecting a target nucleic acid fragment directly from a specimen obtained from a patient by in situ hybridization. The method is comprised of several steps which are performed in the listed order. A sample of the specimen is deposited onto a slide. The sample is fixed onto the slide with fixative, the fixative comprising either methanol-acetic acid at a ratio of from 99:1 to 80:20, or formalin-acetic acid at a ratio of from 99:1 to 80:20. The nucleic acids of the fixed sample are contacted with a probe complex specific for the target nucleic acid fragment, under conditions appropriate for hybridization. Non-hybridized probe complex is rinsed from the sample. The rinsed sample is stained with Evans Blue. The hybridized probe complex is visually detected by microscopy, with the presence of the probe complex being an indication of the presence of the target nucleic acid fragment. The method can be performed with different hybridization buffers, several of which are disclosed. The method of the present invention is useful for detecting pathogens in a specimen. Specific probe complexes are disclosed which are useful for detecting pathogens of the species Babesia. The method is useful in detecting nucleic acids from a wide variety of specimens, including serum, plasma, sputum, urine, cerebral spinal fluids, tissue, breast milk, and insects such as ticks.