Highly conserved genes and their use to generate probes and primers for detection of microorganisms

Abstract

Compositions and methods for the detection of vancomycin-resistant pathogens using primers and/or probes to the vanA and vanB genes.

1 . A composition for the detection of a vancomycin resistant pathogen in a sample using a nucleic acid amplification assay, wherein said composition comprises a primer pair for the amplification of vanA sequences, said primer pair comprising a forward and a reverse oligonucleotides, wherein said forward and reverse oligonucleotides are between 13 and 30 nucleotides, and wherein said forward and reverse oligonucleotides each comprises a binding region that is complementary to primer binding sites present on opposite strands of the pathogen's DNA, wherein said primer pair is adapted to amplify a target region of a vancomycin resistance gene of said pathogen's DNA between and including said primer binding sites to produce a detectable vanA vancomycin amplification product, wherein at least one of said oligonucleotides comprises, or is fully complementary to, at least 11 consecutive nucleotides of SEQ ID NO: 1090 and SEQ ID NO: 1091, or the complements thereof. 
     
     
         2 . (canceled) 
     
     
         3 . (canceled) 
     
     
         4 . The composition of  claim 1 , further comprising at least one internal hybridization probe, wherein said internal hybridization probe can hybridize under stringent conditions to said vanA vancomycin amplification product. 
     
     
         5 . The composition of  claim 4 , wherein said internal hybridization probe is a molecular beacon. 
     
     
         6 . The composition of  claim 4 , wherein said internal hybridization probe comprises at least 10 consecutive nucleotides of SEQ ID NO: 2299. 
     
     
         7 . (canceled) 
     
     
         8 . The composition of  claim 4 , further comprising:
 an internal control DNA; and   an internal control probe,   wherein said internal control DNA is amplified using the said forward and reverse oligonucleotides to produce a control amplification product in said nucleic acid amplification assay,   and wherein said internal control probe hybridizes to said internal control DNA under the same conditions as said internal hybridization probe hybridizes to said vancomycin amplification product.   
     
     
         9 . The composition of  claim 8 , wherein said internal control probe comprises at least 10 consecutive nucleotides of the sequence of SEQ ID NO: 2301. 
     
     
         10 . A kit comprising the composition of  claim 1 . 
     
     
         11 . A method to detect the presence of vancomycin-resistant organisms in a sample comprising nucleic acids, comprising:
 annealing the nucleic acids of said sample with the composition of  claim 4 ;   amplifying said sample nucleic acids with said annealed primer pair; and   determining the amount of internal hybridization probe that is annealed to the vanA vancomycin amplification product and the target region.   
     
     
         12 . The method of  claim 11 , wherein said primer pair consists of SEQ ID NO's 1090 and 1091, or the complements thereof. 
     
     
         13 . The method of  claim 11 , wherein said primer pair and said internal hybridization probe are placed in the same physical enclosure. 
     
     
         14 . (canceled) 
     
     
         15 . The method of  claim 11  wherein amplification step comprises a method selected from the group consisting of:
 (a) polymerase chain reaction (PCR),   (b) ligase chain reaction,   (c) nucleic acid sequence-based amplification,   (d) self-sustained sequence replication,   (e) strand displacement amplification,   (f) branched DNA signal amplification,   (g) nested PCR, and   (h) multiplex PCR.   
     
     
         16 . The method of  claim 15 , wherein said amplification step comprises PCR. 
     
     
         17 . (canceled) 
     
     
         18 . The method of  claim 11 , wherein said internal hybridization probe comprises at least 10 consecutive nucleotides of the sequence of SEQ ID NO: 2299. 
     
     
         19 . The composition of  claim 1 , wherein said forward or said reverse oligonucleotide consists of SEQ ID NO: 1090. 
     
     
         20 . The composition of  claim 1 , wherein said forward or said reverse oligonucleotide consists of SEQ ID NO: 1091. 
     
     
         21 . The composition of  claim 1 , wherein said forward and reverse oligonucleotides consist of SEQ ID NO: 1090 and SEQ ID NO: 1091, respectively. 
     
     
         22 . The composition of  claim 21 , further comprising an internal hybridization probe, wherein said internal hybridization probe can hybridize under stringent conditions to said vancomycin amplification product. 
     
     
         23 . The composition of  claim 22 , wherein said internal hybridization probe is a molecular beacon. 
     
     
         24 . The composition of  claim 22 , wherein said internal hybridization probe comprises at least 10 consecutive nucleotides of the sequence of SEQ ID NO: 2299. 
     
     
         25 . The composition of  claim 22 , further comprising:
 an internal control DNA; and   an internal control probe,   wherein said internal control DNA is amplified under the same conditions and using said forward and reverse primers to produce a control amplification product, and wherein said internal control probe hybridizes to said internal control DNA under the same conditions as said internal hybridization probe hybridizes to said vanA vancomycin amplification product.   
     
     
         26 . The composition of  claim 25 , wherein said internal control DNA is SEQ ID NO: 2302. 
     
     
         27 . The composition of  claim 25 , wherein said internal control probe comprises at least 10 consecutive nucleotides of the sequence of SEQ ID NO: 2301. 
     
     
         28 . The composition of  claim 8 , wherein said internal control DNA is SEQ ID NO: 2302. 
     
     
         29 . The composition of  claim 1 , further comprising a second amplification primer pair for the amplification of vanB sequences, said second primer pair comprising a second forward and a second reverse oligonucleotide, wherein said second forward and second reverse oligonucleotides are each between 13 and 30 nucleotides and wherein each of said forward and reverse oligonucleotides comprises a binding region that is complementary to primer binding sites present on opposite strands of the pathogen's DNA, wherein said primer pair is adapted to amplify a vancomycin resistance gene of said pathogen's DNA between and including said primer binding sites to produce a detectable vanB vancomycin amplification product, wherein at least one of said oligonucleotides of said second primer pair is selected from the group consisting of: SEQ ID NO: 1095 and SEQ ID NO: 1096, or the complements thereof. 
     
     
         30 . The composition of  claim 29 , wherein said second forward and second reverse oligonucleotides of said second primer pair consist of SEQ ID NO: 1095 and SEQ ID NO: 1096, respectively. 
     
     
         31 . The composition of  claim 29  further comprising an internal hybridization probe, wherein said internal hybridization probe can hybridize under stringent conditions to said vanA vancomycin amplification product or said vanB vancomycin amplification product. 
     
     
         32 . The composition of  claim 31 , wherein said internal hybridization probe is a molecular beacon. 
     
     
         33 . The composition of  claim 31 , wherein said internal hybridization probe comprises at least 10 consecutive nucleotides of the sequence of SEQ ID NO: 2299. 
     
     
         34 . The composition of  claim 31 , wherein said internal hybridization probe comprises at least 10 consecutive nucleotides of the sequence of SEQ ID NO: 2300. 
     
     
         35 . The composition of  claim 29 , further comprising:
 an internal control DNA; and   an internal control probe,   wherein said internal control DNA is amplified under the same conditions and using the same forward and reverse oligonucleotides to produce a control amplification product, and wherein said internal control probe hybridizes to said internal control DNA under the same conditions as said internal hybridization probe hybridizes to said vanA or vanB vancomycin amplification product.   
     
     
         36 . The composition of  claim 36 , wherein said internal control DNA comprises SEQ ID NO: 2302. 
     
     
         37 . The composition of  claim 36 , wherein said internal control probe comprises at least 10 consecutive nucleotides of the sequence of SEQ ID NO: 2301. 
     
     
         38 . The method of  claim 11 , further comprising annealing the sample nucleic acids with:
 an internal control DNA; and   an internal control probe,   wherein said internal control DNA is amplified under the same conditions and using the same forward and reverse oligonucleotides as to produce a control amplification product, and wherein said internal control probe hybridizes to said internal control DNA under the same conditions as said internal hybridization probe hybridizes to said vanA vancomycin amplification product.   
     
     
         39 . The method of  claim 38 , wherein said internal control probe comprises at least 10 consecutive nucleotides of the sequence of SEQ ID NO: 2301. 
     
     
         40 . The method of  claim 38 , wherein said internal control DNA comprises of SEQ ID NO: 2302.