Method for the specific detection of low abundance rna species in a biological sample

Abstract

The present invention relates to a method and a kit for the detection of low abundance RNA species in a biological sample and to a method and a kit for the detection of a mycoplasma contamination in a biological sample.

1 . A kit for the detection of low abundance target RNA species in a large background of total RNA, comprising at least a reverse transcriptase (RT) and means for the inactivation of the RT. 
     
     
         2 . The kit of  claim 1 , further comprising suitable primers for reverse transcription. 
     
     
         3 . The kit of  claim 1 , further comprising suitable primers for the detection of specific cDNAs. 
     
     
         4 . The kit of  claim 1 , further comprising means for extracting total RNA from a sample. 
     
     
         5 . The kit of  claim 1 , further comprising on or more selected from the group consisting of carrier RNAs, calibrator RNA, and calibrator DNA. 
     
     
         6 . The kit of  claim 1 , further comprising on or more selected from the group consisting of suitable enzymes, buffers, reagents, reaction containers, and consumables. 
     
     
         7 . The kit of  claim 1 , wherein the means for the inactivation of the RT are selected from the group, consisting of proteinase K and a chaotropic agent. 
     
     
         8 . The kit of  claim 1 , wherein the means for the inactivation of the RT is a chaotropic agent and the kit further comprises means for removing the chaotropic agent. 
     
     
         9 . The kit of  claim 1 , wherein the means for the inactivation of the RT is a chaotropic agent selected from the group consisting of urea, lithium perchlorate, and a guanidinium salt. 
     
     
         10 . The kit of  claim 1 , wherein the RT is a hotstart RT. 
     
     
         11 . The kit of  claim 1 , wherein the RT is a  Thermus thermophilus  DNA polymerase (rTth-pol). 
     
     
         12 . The kit of  claim 1 , wherein the RT is a recombinant  Thermus thermophilus  DNA polymerase (rTth-pol). 
     
     
         13 . The kit of  claim 1 , wherein the target RNA is derived from mycoplasma. 
     
     
         14 . The kit of  claim 1 , wherein the target RNA is derived from mycoplasma selected from the group, consisting of  Acholeplasma laidlawii, Mycoplasma gallisepticum, Mycoplasma hyorhinis, Mycoplasma hominis, Mycoplasma fermentans, Mycoplasma synoviae , and  Mycoplasma orate.    
     
     
         15 . The kit of  claim 1 , wherein the target RNA is 16S ribosomal RNA. 
     
     
         16 . The kit of  claim 1 , wherein the kit is a kit for the detection of a mycoplasma contamination in a biological sample, comprising at least a recombinant Thermus thermophilus DNA polymerase (rTth-pol), suitable primers for the reverse transcription of mycoplasma 16S rRNA, suitable primers for the amplification of cDNA derived from mycoplasma 16S ribosomal RNA (16S rRNA), a chaotropic agent for the inactivation of the rTth-pol, means for removing the chaotropic agent from the reaction mixture, and primers for the detection of cDNA derived from mycoplasma 16S rRNA by real-time PCR.