Probes and methods for detection of pathogens and antibiotic resistance

Abstract

Described are probes and methods for detecting pathogens and antibiotic resistance of a specimen. The method comprises contacting the specimen with a growth medium; and lysing the specimen to release nucleic acid molecules from the specimen. The lysate of the specimen is contacted with a capture probe immobilized on a substrate, wherein the capture probe comprises an oligonucleotide that specifically hybridizes with a first target nucleic acid sequence region of ribosomal RNA. The lysate is in contact with a detector probe that comprises a detectably labeled oligonucleotide that specifically hybridizes with a second target nucleic acid sequence region of ribosomal RNA. The presence or absence of labeled oligonucleotide complexed with the substrate is determined. Detection of labeled oligonucleotide complexed with the substrate is indicative of the presence of pathogen. Performing the method in the presence and absence of an antibiotic permits detection of antibiotic resistance.

1 . A kit comprising oligonucleotide probes, each of which is 10-35 bases in length, and that specifically hybridize under highly stringent conditions to target regions of bacterial ribosomal RNA (rRNA), wherein the target regions are, or are fully complementary to, nucleic acid molecules substantially corresponding to:
 (a) SEQ ID NO: 181 of  E. coli,  and   (b) SEQ ID NO: 179 of  K. pneumoniae,      wherein the highly stringent conditions comprise hybridization and washes at 20° C. to 39° C. in 1M phosphate buffer at native pH, and wherein the probes share at least 80% identity or complementarity to SEQ ID NO: 181 and 179, respectively, over the length of the probe.   
     
     
         2 . The kit of  claim 1 , wherein the target regions comprise:
 (a) at least one of the bases at positions 432-439 of  Escherichia coli;  and   (b) the cytosine at position 440 of  Klebsiella pneumoniae.      
     
     
         3 . The kit of  claim 1 , wherein the probes are labeled with a detectable marker. 
     
     
         4 . The kit of  claim 1 , wherein each of the probes is 10-15 bases in length. 
     
     
         5 . The kit of  claim 1 , wherein each of the probes is 15-25 bases in length. 
     
     
         6 . The kit of  claim 1 , wherein the probes share at least 90% identity or complementarity to SEQ ID NO: 181 and 179, respectively, over the length of the probe. 
     
     
         7 . The kit of  claim 1 , wherein the probes share 100% identity or complementarity to SEQ ID NO: 181 and 179, respectively, over the length of the probe. 
     
     
         8 . The kit of  claim 1 , wherein the target regions include:
 (a)  Escherichia coli,  SEQ ID NO: 28 or 29; and   (b)  Klebsiella pneumoniae,  SEQ ID NO: 26 or 27.   
     
     
         9 . The kit of  claim 1 , wherein the probes comprise:
 (a) the  Escherichia coli  probe of SEQ ID NO: 44 or 45; and   (b) the  Klebsiella pneumoniae  probe of SEQ ID NO: 48 or 49.   
     
     
         10 . The kit of  claim 1  that further comprises a probe that specifically hybridizes under highly stringent conditions to a target region of bacterial ribosomal RNA (rRNA), wherein the target region is, or is fully complementary to, nucleic acid molecules substantially corresponding to:
 (c) SEQ ID NO: 176 of  E. faecalis,  wherein the highly stringent conditions comprise hybridization and washes at 20° C. to 39° C. in 1M phosphate buffer at native pH, and wherein the probe shares at least 80% identity or complementarity to SEQ ID NO: 176, respectively, over the length of the probe. 
 
     
     
         11 . The kit of  claim 10 , wherein the target regions include:
 (c)  Enterococcus faecalis  SEQ ID NO: 24 or 25.   
     
     
         12 . The kit of  claim 1  that further comprises a substrate to which the probes are immobilized, wherein the substrate comprises a sensor array. 
     
     
         13 . The kit of  claim 1 , wherein the kit comprises at least four probes, the four probes comprising one  E. coli  capture probe and one  E. coli  detector probe that specifically hybridize under highly stringent conditions to the target region of (a) and one  K. pneumoniae  capture probe and one  K. pneumoniae  detector probe that specifically hybridize under highly stringent conditions to the target region of (b). 
     
     
         14 . The kit of  claim 13  that further comprises one  E. faecalis  capture probe and one  E. faecalis  detector probe that specifically hybridize under highly stringent conditions to the target region of (c) SEQ ID NO: 176. 
     
     
         15 . A method for detecting the presence of bacterial pathogens in a specimen, the method comprising:
 (a) contacting a lysate of the specimen with a kit of  claim 1  under conditions that permit hybridization of the probes of the kit with target nucleic acid sequences of the specimen; and   (b) determining the presence of the probe hybridization, whereby detection of probe hybridization is indicative of the presence of bacterial pathogens in the specimen.   
     
     
         16 . The method of  claim 15 , wherein the conditions that permit hybridization are a temperature of 20° C. to 39° C. and a buffered saline solution. 
     
     
         17 . The method of  claim 15 , wherein the conditions that permit hybridization are a temperature of 20° C. to 25° C. and a buffered saline solution. 
     
     
         18 . The method of  claim 15 , wherein the lysate is prepared by contacting the specimen with a first lysis buffer comprising a non-denaturing detergent and lysozyme. 
     
     
         19 . The method of  claim 18 , wherein the lysate is further prepared by contacting the specimen with a second lysis buffer comprising NaOH. 
     
     
         20 . The method of  claim 19 , wherein the contacting of the specimen with the second lysis buffer occurs prior to the contacting of the specimen with the first lysis buffer. 
     
     
         21 . The method of  claim 19 , wherein the contacting of the specimen with second lysis buffer occurs after the contacting of the specimen with the first lysis buffer. 
     
     
         22 . The method of  claim 19 , wherein the contacting of the specimen with the first and/or second lysis buffers occurs at 20° C. to 39° C. 
     
     
         23 . The method of  claim 19 , wherein the contacting of the specimen with the first and/or second lysis buffers occurs for about 5 minutes per lysis buffer. 
     
     
         24 . The method of  claim 15 , wherein the oligonucleotide probes are 10-15 bases in length. 
     
     
         25 . The method of  claim 15 , wherein at least one probe is immobilized onto an electrochemical sensor assay and the determining comprises measuring current output. 
     
     
         26 . The method of  claim 25 , wherein the determining comprises comparing current output at 15 minutes after contacting the specimen with the growth medium. 
     
     
         27 . The method of  claim 15 , wherein the specimen is a bodily fluid. 
     
     
         28 . The method of  claim 27 , wherein the specimen is urine. 
     
     
         29 . The method of  claim 27 , wherein the specimen is blood.