PNA Probes, Mixtures, Methods And Kits Pertaining To The Determination Of Mycoplasma and related Mollicutes

Abstract

This invention is related to PNA probes, probe sets, mixtures, methods and kits pertaining to the determination of Mycoplasma and related Mollicutes.

1 . A PNA probe, of 12-20 nucleobase containing subunits, comprising a probing nucleobase sequence for detecting, identifying and/or quantifying one or more  Mollicutes  in a sample. 
     
     
         2 . The PNA probe of  claim 1 , wherein the one or more  Mollicutes  are  Mycoplasma, Acholeplasma  or  Ureaplasma.    
     
     
         3 . The PNA probe of  claim 1 , wherein the probe comprises a probing nucleobase sequence selected from the group consisting of:
 SEQ. ID. NO: 6, SEQ. ID. NO: 7, SEQ. ID. NO: 8, SEQ. ID. NO: 9, the complement to SEQ. ID. NO: 6, the complement to SEQ. ID. NO: 7, the complement to SEQ. ID. NO: 8 and the complement to SEQ. ID. NO: 9.   
     
     
         4 . The PNA probe of  claim 1 , wherein the probing nucleobase sequence of the PNA probe is SEQ. ID. NO: 6, SEQ. ID. NO: 7, SEQ. ID. NO: 8, SEQ. ID. NO: 9, the complement to SEQ. ID. NO: 6, the complement to SEQ. ID. NO: 7, the complement to SEQ. ID. NO: 8 or the complement to SEQ. ID. NO: 9. 
     
     
         5 . The PNA probe of  claim 1 , wherein the probe is unlabeled. 
     
     
         6 . The PNA probe of  claim 1 , wherein the probe is labeled with at least one detectable moiety. 
     
     
         7 . The PNA probe of  claim 6 , wherein the detectable moiety or moieties are selected from the group consisting of: a dextran conjugate, a branched nucleic acid detection system, a chromophore, a fluorophore, a spin label, a radioisotope, an enzyme, a hapten, an acridinium ester and a chemiluminescent compound. 
     
     
         8 . The PNA probe of  claim 1 , wherein the probe is support bound. 
     
     
         9 . The PNA probe of  claim 1 , wherein the probe is self-indicating. 
     
     
         10 . A PNA probe set suitable for detecting, identifying and/or quantifying one or more  Mollicutes  in a sample. 
     
     
         11 . The probe set of  claim 10 , wherein the one or more  Mollicutes  are  Mycoplasma, Acholeplasma  or  Ureaplasma.    
     
     
         12 . The probe set of  claim 10 , wherein each PNA probe comprises 12-20 nucleobase containing subunits. 
     
     
         13 . The probe set of  claim 10 , wherein at least one PNA probe of the set comprises a probing nucleobase sequence selected from the group consisting of:
 SEQ. ID. NO: 6, SEQ. ID. NO: 7, SEQ. ID. NO: 8, SEQ. ID. NO: 9, the complement to SEQ. ID. NO: 6, the complement to SEQ. ID. NO: 7, the complement to SEQ. ID. NO: 8 and the complement to SEQ. ID. NO: 9.   
     
     
         14 . The probe set of  claim 10 , wherein the set comprises a PNA probe comprising SEQ. ID. NO: 6 as the probing nucleobase sequence, a PNA probe comprising SEQ. ID. NO: 7 as the probing nucleobase sequence, a PNA probe comprising SEQ. ID. NO: 8 as the probing nucleobase sequence and/or a PNA probe comprising SEQ. ID. NO: 9 as the probing nucleobase sequence. 
     
     
         15 . The probe set of  claim 10 , wherein the set comprises a PNA probe of SEQ. ID. NO: 6 as the probing nucleobase sequence, a PNA probe of SEQ. ID. NO: 7 as the probing nucleobase sequence, a PNA probe of SEQ. ID. NO: 8 as the probing nucleobase sequence and/or a PNA probe of SEQ. ID. NO: 9 as the probing nucleobase sequence. 
     
     
         16 . The probe set of  claim 10 , wherein all probes of the set are labeled with one or more detectable moieties. 
     
     
         17 . The probe set of  claim 10 , wherein at least one probe of the set is self-indicating. 
     
     
         18 . The probe set of  claim 10 , wherein at least one probe of the set is support bound. 
     
     
         19 . A mixture comprising:
 a) at least two nucleic acid primers, of 15-30 nucleotides in length, each capable of hybridizing to a primer sequence within the nucleic acid of one or more  Mollicutes;  and   b) at least one PNA probe, of 12-20 nucleobase containing subunits, capable of hybridizing to a target sequence within the nucleic acid of one or more  Mollicutes.      
     
     
         20 . The mixture of  claim 19 , wherein the one or more  Mollicutes  are  Mycoplasma, Acholeplasma  or  Ureaplasma.    
     
     
         21 . The mixture of  claim 19 , further comprising:
 c) other reagents selected to perform a PCR amplification reaction.   
     
     
         22 . The mixture of  claim 21 , further comprising an internal positive control. 
     
     
         23 . The mixture of  claim 19 , further comprising:
 c) other reagents selected to perform an in-situ hybridization assay.   
     
     
         24 . The mixture of  claim 19 , wherein the at least two nucleic acid primers are each independently SEQ ID. NO: 1, SEQ ID. NO: 2, SEQ ID. NO: 3, SEQ ID. NO: 4 or SEQ ID. NO: 5. 
     
     
         25 . The mixture of  claim 19 , wherein the PNA probe or probes each comprise a probing nucleobase sequence independently selected from the group consisting of:
 SEQ. ID. NO: 6, SEQ. ID. NO: 7, SEQ. ID. NO: 8, SEQ. ID. NO: 9, the complement to SEQ. ID. NO: 6, the complement to SEQ. ID. NO: 7, the complement to SEQ. ID. NO: 8 and the complement to SEQ. ID. NO: 9.   
     
     
         26 . The mixture of  claim 19 , wherein the probing nucleobase sequence of each probe is independently:
 SEQ. ID. NO: 6, SEQ. ID. NO: 7, SEQ. ID. NO: 8, SEQ. ID. NO: 9, the complement to SEQ. ID. NO: 6, the complement to SEQ. ID. NO: 7, the complement to SEQ. ID. NO: 8 or the complement to SEQ. ID. NO: 9.   
     
     
         27 . The mixture of  claim 19 , wherein the PNA probe or probes are self-indicating. 
     
     
         28 . The mixture of  claim 19 , comprising:
 a) a primer of SEQ ID NO: 1; a primer of SEQ ID NO: 2; a primer of SEQ ID NO: 3; a primer of SEQ ID NO: 4; and/or a primer of SEQ ID NO: 5; and   b) a PNA probe consisting of SEQ. ID. NO: 6 as the probing nucleobase sequence; a PNA probe consisting of SEQ. ID. NO: 7 as the probing nucleobase sequence; a PNA probe consisting of SEQ. ID. NO: 8 as the probing nucleobase sequence; and/or a PNA probe consisting of SEQ. ID. NO: 9 as the probing nucleobase sequence.   
     
     
         29 . A method comprising:
 a) contacting a sample with one or more PNA probes of 12-20 nucleobase containing subunits, wherein each of the one or more PNA probes comprises a probing nucleobase sequence independently selected from the group consisting of:
 SEQ. ID. NO: 6, SEQ. ID. NO: 7, SEQ. ID. NO: 8, SEQ. ID. NO: 9, the complement to SEQ. ID. NO: 6, the complement to SEQ. ID. NO: 7, the complement to SEQ. ID. NO: 8 and the complement to SEQ. ID. NO: 9; and 
   b) determining hybridization of the probing nucleobase sequence of the PNA probe or probes to at least one target sequence in the sample, under suitable hybridization conditions or suitable in-situ hybridization conditions, and correlating the result with the presence, absence and/or quantity of one or more  Mollicutes  in the sample.   
     
     
         30 . The method of  claim 29 , wherein, the one or more  Mollicutes  are  Mycoplasma, Acholeplasma  or  Ureaplasma.    
     
     
         31 . The method of  claim 29 , further comprising:
 c) contacting the sample with at least two nucleic acid primers, of 15-30 nucleotides in length, and other reagents selected to perform a PCR amplification reaction; and   d) performing a PCR amplification reaction to thereby produce one or more amplicons comprising the target sequence or target sequences.   
     
     
         32 . The method of  claim 31 , wherein the PCR assay is an end-point PCR assay or a real-time PCR assay. 
     
     
         33 . The method of  claim 29 , wherein the PNA probe or probes are self-indicating. 
     
     
         34 . The method of  claim 32 , further comprising:
 e) contacting a sample with at least one internal positive control.   
     
     
         35 . A kit comprising:
 a) at least two nucleic acid primers, of 15-30 nucleotides in length, each capable of hybridizing to a primer sequence within the nucleic acid of one or more  Mollicutes ; and   b) at least one PNA probe, of 12-20 nucleobase containing subunits, capable of hybridizing to a target sequence within the nucleic acid of one or more  Mollicutes.      
     
     
         36 . The kit of  claim 35 , wherein the one or more  Mollicutes  are  Mycoplasma, Acholeplasma  or  Ureaplasma.    
     
     
         37 . The kit of  claim 35 , further comprising:
 c) other reagents selected to perform a PCR amplification reaction.   
     
     
         38 . The kit of  claim 35 , further comprising:
 c) at least one internal positive control.   
     
     
         39 . The kit of  claim 38 , wherein the at least two nucleic acid primers are each independently:
 SEQ ID. NO: 1, SEQ ID. NO: 2, SEQ ID. NO: 3, SEQ ID. NO: 4 or SEQ ID. NO: 5.   
     
     
         40 . The kit of  claim 38 , wherein the PNA probe or probes each comprise a probing nucleobase sequence independently selected from the group consisting of:
 SEQ. ID. NO: 6, SEQ. ID. NO: 7, SEQ. ID. NO: 8, SEQ. ID. NO: 9, the complement to SEQ. ID. NO: 6, the complement to SEQ. ID. NO: 7, the complement to SEQ. ID. NO: 8 and the complement to SEQ. ID. NO: 9.   
     
     
         41 . The kit of  claim 35 , wherein the PNA probe or probes are self-indicating.