Peptide nucleic acid probes for analysis of pseudomonas (sensu stricto)

Abstract

Disclosed is a PNA probe that includes a nucleobase sequence suitable for the detection, identification and/or quantitation of Pseudomonas (sensu stricto). In one embodiment, the PNA probe is complementary to a target sequence of 23S rRNA or rDNA from all species of the genus Pseudomonas. The invention has a wide range of important uses including detecting Pseudomonas in a sample of interest.

We claim:  
     
         1 . A PNA probe comprising a nucleobase sequence suitable for the detection, identification and/or quantitation of  Pseudomonas  (sensu stricto), said PNA probe being complementary to a target sequence of 23S rRNA or rDNA of all species of the genus  Pseudomonas,  or its complement.  
     
     
         2 . The PNA probe of  claim 1 , wherein at least a portion of the probe is at least about 90% identical to the nucleobase sequence or complement thereof selected from the following sequence: CCT ACC ACC TTA MC (Seq. Id. No. 1).  
     
     
         3 . The PNA probe of  claim 1 , wherein the probe sequence is 8-17 subunits in length.  
     
     
         4 . The PNA probe of  claim 1  for the detection, identification and/or quantification of  Pseudomonas  (sensu stricto) comprising the following probe sequence: CCT ACC ACC TTA MC (Seq. Id. No. 1), the complement and/or variations thereof.  
     
     
         5 . The PNA probe of  claim 1 , wherein the probe is labeled with at least one detectable moiety.  
     
     
         6 . The PNA probe of  claim 5 , wherein the detectable moiety or moieties are selected from the group consisting of: a conjugate, a branched detection system, a chromophore, a fluorophore, a spin label, a radioisotope, an enzyme, a hapten, an acridinium ester and a luminescent compound.  
     
     
         7 . The PNA probe of  claim 5 , wherein the probe is self-reporting.  
     
     
         8 . The PNA probe of claims  7 , wherein the probe comprises a PNA Linear Beacon.  
     
     
         9 . The PNA probe of  claim 1 , wherein the probe is unlabeled.  
     
     
         10 . The PNA probe of  claim 1 , wherein the probe is bound to a support.  
     
     
         11 . The PNA probe of claims  1 , wherein the probe further comprises a spacer or a linker.  
     
     
         12 . The PNA probe of claims  1 , wherein in situ hybridization is used for analysis of  Pseudomonas  (sensu stricto) optionally present in the sample.  
     
     
         13 . A method for the detection, identification and/or quantitation of  Pseudomonas  (sensu stricto) in a sample, said method comprising: a) contacting at least one of the PNA probes of  claim 1  to the sample, 
 b) hybridizing the PNA probe to a target sequence of species of the genus  Pseudomonas  in the sample; and  
 c) detecting the hybridization as being indicative of presence, identity and/or amount of  Pseudomonas  (sensu stricto) in the sample.  
 
     
     
         14 . A method according to  claim 13 , wherein the analysis takes place in situ.  
     
     
         15 . A method according to  claim 12 , wherein the analysis takes place by fluorescence in situ hybridization.  
     
     
         16 . A method according to claims  15 , wherein the analysis does not involve the use of cross-linking reagents or enzymes prior to hybridization.  
     
     
         17 . The method of  claim 12 , wherein the method is used to detect a nucleic acid comprising a target sequence wherein said nucleic acid has been synthesized or amplified in a reaction.  
     
     
         18 . The method of  claim 17 , wherein preferred nucleic acid synthesis or nucleic acid amplification reactions are selected from the group consisting of: Polymerase Chain Reaction (PCR), Ligase Chain Reaction (LCR), Strand Displacement Amplification (SDA), Transcription-Mediated Amplification (TMA), Rolling Circle Amplification (RCA) and Q beta replicase.  
     
     
         19 . The method of  claim 12 , wherein the method further comprises adding at least one blocking probe to reduce or eliminate any hybridization of the PNA probe to non-target sequence.  
     
     
         20 . The method of  claim 12 , wherein the target sequence is immobilized to a surface.  
     
     
         21 . The method of  claim 12 , wherein said PNA probe is immobilized to a surface.  
     
     
         22 . The method of  claim 21 , wherein said PNA probe is one component of an array.  
     
     
         23 . The method of  claim 12 , wherein the sample is a biological sample.  
     
     
         24 . The method of  claim 23 , wherein the biological sample is blood, urine, secretion, sweat, sputum, stool, mucous, or cultures thereof.  
     
     
         25 . A kit adapted to perform an assay for the detection, identification and/or quantitation of  Pseudomonas  (sensu stricto) in a sample, wherein said kit comprises: a) a PNA probe according to  claim 1  and b) other reagents or compositions necessary to perform the assay.  
     
     
         26 . The kit of  claim 25 , wherein  Pseudomonas  (sensu stricto) and at least one other microorganism optionally present in a sample are independently detected, identified and/or quantitated.  
     
     
         27 . The kit of  claim 25 , wherein  Pseudomonas  (sensu stricto) optionally present in a sample is detected, identified and/or quantitated and its susceptibility to antimicrobial agents is determined.  
     
     
         28 . The kit of  claim 25 , wherein the kit is further adapted to perform in an in-situ hybridization assay.  
     
     
         29 . The kit of  claim 25 , wherein the kit is further apdapted to perform a real-time PCR assay.  
     
     
         30 . The kit of  claim 25 , wherein the kit is adapted to examine clinical samples such as clinical specimens or cultures thereof.  
     
     
         31 . The kit of  claim 25 , wherein the kit is adapted to examine food, beverages, water, pharmaceutical products, personal care products, dairy products or environmental samples or cultures thereof.