Diagnostic and therapeutic system for Crohn's disease and Colitia ulcerosa

A method for the analysis of Crohn's Disease and/or ulcerative colitis which method comprises the steps of:

• (a) obtaining a sample of bowel tissue containing granulomas or lymphoid follicles from a patient, and securing the same in an enclosed reaction container;
• (b) adding a reagent comprising a PCR buffer, MgCl2 and DNTP random hexamers diluted aqueously to a desired dilution factor;
• (c) subsequently closing the enclosed reaction container, adding M-MLV-RT and subjecting to heat cycling for at least one cycle;
• (d) treating with a washing buffer;
• (e) adding a buffered Taq DNA polymerase, MgCl2, DNTP and a base primer or probe at a predetermined dilution and then heat cycling for at least 25 cycles to produce a labeled product;
• (f) subsequently repeating step (d) and viewing for the labeled product to indicate the presence of measles virus RNA;
• and
• (g) determining whether the patient suffers from Crohn's Disease or ulcerative colitis based upon the presence or absence of the measles virus RNA.

A method for the in vitro analysis of a sample of bowel tissue containing granulomas or lymphoid follicles from a patient with Crohn's Disease and/or ulcerative colitis which method comprises abstracting measles virus RNA from bowel tissue containing granulomas or lymphoid follicles and:

• (a) adding thereto a buffer further comprising DTT, DNTPs and RNase inhibitor and oligo (Dt) 12-18;
• (b) adding thereto M-MLV-RT and subjecting to incubation to provide a cDNA product;
• (c) purifying the product and adding thereto a PCR buffer along with MgCl2, dNTPs and an outer primer or probe;
• (d) subsequently adding Taq DNA polymerase and heating for at least 15 cycles, whereby a first reaction product is formed;
• (e) recovering an aliquot of the first reaction product and adding buffered PCR with inner primers or probes and further Taq DNA polymerase and recycling for at least 15 cycles, whereby a second reaction product is formed;
• (f) removing the second reaction product and adding a loading dye to form a dyed product and subjecting the dyed product to electrophoresis to identify a selected product band;
• and
• (g) sequencing the amplified products or hybridizing the amplified products with a homologous or heterologous specific probe to distinguish the measles virus to abstract the measles virus RNA from the tissue.

A method for the in vitro analysis of a sample of bowel tissue containing granulomas or lymphoid follicles from a patient with Crohn's Disease and/or ulcerative colitis which method comprises abstracting measles virus RNA from bowel tissue containing granulomas or lymphoid follicles and:

• (a) adding thereto a room temperature buffer further comprising DNTP and room temperature primer or probe mixes to produce a master mix;
• (b) aliquoting master mix into a reaction mixture comprising the template RNA to produce a resulting mixture, heating the resulting mixture for 5 minutes at 65° C., then at 41° C. for 5 minutes;
• (c) subsequently adding the enzyme mix further comprising a RNA polymerase;
• (d) incubating at 41° C. for 90 minutes in a water bath to produce a reaction product;
• (e) detecting the reaction product by Northern blotting or an enzyme-linked gel assay;
• and
• (f) sequencing of the amplified products or hybridizing the amplified products with a homologous or heterologous specific probe to distinguish the measles virus.