Detection of exposure to RIP II toxins
AssigneeThe Israel Institute of Biological Research (Iibr)
InventorsReut Falach, Ofir Israeli, Ohad Shifman, Adi Beth-Din, Tamar Sabo, Chanoch Kronman
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Abstract
The invention concerns methods for detecting exposure to a RIP II family toxin in a biological sample. The method is based on identifying the enzymatic activity of the toxin on 28sRNA and employs sensitive and specific amplification steps that allow detection in clinical samples.
Claims18
A method for detecting exposure to a RIP II family toxin in a biological sample, the method comprising the steps of:
- a. obtaining an isolated RNA preparation comprising 28S rRNA;
- b. performing a reverse transcriptase (RT) reaction using a primer complementary to a sequence of the 28S rRNA which is positioned 3′ to the RIP II family enzyme depurination site, whereby a truncated cDNA transcript is formed if the 28S rRNA was exposed to a RIP II family toxin and was depurinated;
- c. labeling said truncated cDNA transcript obtained in step (b) by ligating a synthetic nucleic acid strand at the truncated 3′ end of the transcript, thereby obtaining a chimeric ligation product;
- and
- d. amplifying the chimeric ligation product using polymerase chain reaction (PCR);
- wherein detection of an amplified chimeric ligation product indicates exposure to a RIP II family toxin.
A method for detecting a RIP II family toxin in a biological sample, the method comprising the steps of:
- a. incubating said biological sample with a composition comprising 28S rRNA;
- b. isolating RNA from said composition comprising 28S rRNA;
- c. performing a reverse transcriptase (RT) reaction using a primer complementary to a sequence of the 28S rRNA which is positioned 3′ to the RIP II family enzyme depurination site, whereby a truncated cDNA transcript is formed if the 28S rRNA was exposed to a RIP II family toxin and was depurinated;
- d. labeling said truncated cDNA transcript obtained in step (c) by ligating a synthetic nucleic acid strand at the truncated 3′ end of the transcript, thereby obtaining a chimeric ligation product;
- and
- e. amplifying the chimeric ligation product using polymerase chain reaction (PCR);
- wherein detection of an amplified chimeric ligation product indicates that said biological sample contains RIP II family toxins.
A method for detecting ribosomal damage induced by exposure to RIP II family toxins in a biological sample, the method comprising the steps of:
- a. isolating RNA from a biological sample, wherein the isolated RNA comprises 28S rRNA;
- b. performing a reverse transcriptase (RT) reaction using a primer complementary to a sequence of the 28S rRNA which is positioned 3′ to the RIP II family enzyme depurination site, whereby a truncated cDNA transcript is formed if the 28S rRNA was exposed to a RIP II family toxin and was depurinated;
- c. labeling said truncated cDNA transcript obtained in step (b) by ligating a synthetic nucleic acid strand at the truncated 3′ end of the transcript, thereby obtaining a chimeric ligation product;
- and
- d. amplifying the chimeric ligation product using PCR;
- wherein detection of an amplified chimeric ligation product indicates the presence of ribosomal damage induced by exposure to RIP II family toxins in said biological sample.
Dependencies are inferred from the claim text.