Patent Application: US-201314370752-A

Abstract:
a method for performing a diagnostic assay for the detection of the presence or amount of a microorganism within a sample matrix containing active dna polymerase , is disclosed . the method utilizes the measurement of dna polymerase extension activity , wherein the assay comprises the steps of incubating dna polymerase in the sample matrix with a selected suitable substrate , and performing pcr cycling and detection via the use of a selected suitable nucleic acid probe , thereby to detect endogenous dna polymerase extension activity in the sample matrix as an indication of the presence or amount of said microorganism .

Description:
during the past fifty years , in vitro measurement of dna polymerase activity has become an essential molecular biology tool . traditional methods used to measure dna polymerase activity in vitro are undesirable due to the usage of radionucleotides . fluorescence - based dna polymerase assays have been developed ; however , they also suffer from various limitations . herein is disclosed a rapid , highly sensitive and quantitative assay capable of measuring dna polymerase extension activity from purified enzymes or directly from microbial lysates . when tested with purified dna polymerase , the assay has been found to detect as little as 2 × 10 − 11 u of enzyme (≈ 50 molecules ), while demonstrating excellent linearity ( r 2 = 0 . 992 ). the assay was also able to detect endogenous dna polymerase extension activity down to at least 10 colony forming units of input gram - positive or gram - negative bacteria when coupled to bead mill lysis while maintaining an r 2 = 0 . 999 . furthermore , experimental evidence presented herein suggests that dna polymerase extension activity is an indicator of bacterial viability , as demonstrated by the reproducibly strong concordance between assay signal and bacterial colony formation . together , the novel methodology of the invention described herein represents a significant advancement toward sensitive detection of potentially any microorganism containing active dna polymerase within a given sample matrix . to further illustrate the foregoing concepts and advantages of the invention , the following examples are provided as illustrative of this invention , but are in no way to be construed as limitative thereof . the sequence of the dna substrate ( and qpcr primers presented below ) was adapted from dna oligos previously used to measure bacterial - derived atp via t4 dna ligase ( 18 ). briefly , oligo 1 and oligo 2 ( see fig1 ) were pre - annealed and diluted to a working concentration of 0 . 01 μm . dna pol i ( neb cat # m0209l ), klenow ( neb cat # m0210s ) and klenow exo (−) ( neb cat # m0212s ) were diluted to the indicated u / μl stock in tris edta ( t . e .) ph 8 . 0 . to begin , 2 μl of dna polymerase stock at each concentration were placed into a 50 μl polymerase assay mixture containing the following components : 50 μm dntp , 20 mm tris ph 8 . 0 , 10 mm ammonium sulfate , 10 mm potassium chloride , 2 mm magnesium sulfate , 1 % bsa , 0 . 1 % triton , 0 . 1 % tween , and 0 . 001 μm pre - annealed dna substrate . reactions were vortexed briefly and placed at 37 ° c . for 20 minutes . after 20 minutes , 3 μl of each reaction were immediately placed into a quantitative pcr ( qpcr ) reaction . the qpcr reaction master mix was prepared using the following components : lightcycler 480 master mix ( roche cat # 04707494001 ), 333 nm of each primer , 166 nm target probe ( fam ), 166 nm internal control probe ( txred ), and 1 . 2 u of udg ( bioline cat # bio - 20744 ). as a tool to monitor pcr inhibition , each qpcr reaction also included 40 copies of competitive internal control dna . for each qpcr reaction , 3 μl of dna polymerase reaction were added to 27 μl of master mix and a two - step qpcr was run on a smartcycler ( cepheid , sunnyvale calif .) as follows : initial incubation of 40 ° c . for 10 minutes and 50 ° c . for 10 minutes and at 95 ° c . for 5 minutes ( to activate taq ), followed by 45 cycles of 5 s denaturation at 95 ° c . and 20 s annealing / extension at 65 ° c . cycle threshold ( ct ) values were generated automatically by the smartcycler software using 2 nd derivative analysis of the emerging qpcr curves . staphylococcus aureus ( atcc 25923 ) and escherichia coli ( atcc 25922 ) were used in this study . cultures were grown in / on brain - heart infusion liquid media / agar ( teknova .) the atcc reference numbers and growth media for the additional 17 microorganisms tested are listed in fig5 . s . aureus and e . coli cultures were grown to an od 600 of 1 . 0 ± 0 . 2 ( approximately 1 × 10 9 cfu / ml .) for each organism , 1 ml of culture was pelleted and washed three times in t . e . bacterial suspensions were serially diluted in t . e ., and 5 μl of each stock were added to bead lysis - reactions containing 50 μl of lysis - reaction buffer . a titration curve of 1 × 10 5 to 1 × 10 0 cfu / reaction was performed in triplicate for each organism , including triplicate reactions without bacterial suspension ( no input control ). after the addition of 5 μl bacterial stock ( or no input control ), lysis / reaction tubes were bead milled for 6 min . at 2800 rpm , followed by incubation at 37 ° c . for 20 min . after a 20 minute incubation , samples were heated to 95 ° c . for 5 min . and removed to cool at room temperature . samples were then spun at 12 k × g for 30 seconds and 3 μl of each reaction were placed into qpcr . five micro - liters of each bacterial stock was plated to obtain more accurate cfu input levels . organism - specific pcr was also performed on the same lysates used for dna polymerase detection . primer and probe sequences for s . aureus and e . coli gene specific pcr are listed in fig2 . dna polymerase assay reactions were prepared as described above with a dntp mix containing either 50 μm dctp or 50 μm ddctp ( affymetrix # 77332 .) reactions containing either dntp mix were spiked with 2 × 10 − 9 u of dna polymerase i ( new england biolabs # m0209 ). reactions were incubated at 37 ° c . for 20 minutes and 3 μl of each reaction were subsequently placed into qpcr . s . aureus and e . coli cultures were grown , washed and diluted as described above . to demonstrate ddctp - dependent termination of microbial dna polymerase , 5 μl of bacterial stock were added to bead lysis tubes containing 50 μl of reaction buffer with either 50 μm dctp or 50 μm ddctp . lysis , incubation and qpcr were performed as described above . five micro - liters of each bacterial stock were plated to determine more accurate cfu input levels . gene specific pcr of genomic dna was also performed on the same lysates used for dna polymerase detection . s . aureus and e . coli cultures were grown , washed and diluted as described above . five micro - liters of bacterial stock were added to bead lysis tubes containing 50 μl of reaction buffer with 50 μm ddctp . prior to lysis , 1 μl of dctp at 2 . 5 mm , 0 . 25 mm 0 . 025 mm 0 . 0025 mm was added to ddctp - containing reactions . reactions containing 50 μm dctp alone and ddctp alone were run in parallel as “ non - terminated ” and “ terminated ” comparators . lysis , incubation and qpcr were performed as described above . five micro - liters of each bacterial stock were plated to determine more accurate cfu input levels . gene - specific pcr was also performed on the same lysates used for dna polymerase detection . s . aureus and e . coli cultures were grown , washed and diluted as described above . two hundred micro - liters of bacterial stocks at approximately 2000 cfu / μl ( in t . e .) were incubated at 25 ° c ., 45 ° c ., 65 ° c ., 85 ° c . and 105 ° c . for 20 minutes . after heating , samples were cooled to room temperature and 5 μl of each bacterial stock were added to bead lysis tubes containing 50 ia of reaction buffer . lysis , incubation and qpcr were performed as described above . five micro - liters of each bacterial stock ( treated at various temperatures ) were also added to 1 ml of t . e . and 50 μl were plated for colony count determination . gene specific pcr was also performed on the same lysates used for dna polymerase detection . in the development of the present invention , it was set out to develop a rapid , simple , highly sensitive and quantitative assay capable of measuring dna polymerase extension activity derived from purified commercial sources or freshly lysed cells , which would improve upon and overcome the disadvantages of the foregoing described methodologies of the know art . fig1 shows a schematic overview of the mechanisms involved in coupling dna polymerase extension activity to qpcr . notably , oligo 2 ( see fig1 , step c ) is removed by uracil dna glycosylase ( udg ) prior to taq activation , thus preventing non - specific extension of the substrate just prior to pcr cycling . a microbial detection method linking t4 dna ligase activity to pcr amplification has been previously reported ( 18 ), which contains similarities to our dpe - pcr assay and is another example of an etga methodology . however , during the development of the present invention a modified version of this method , aimed at detecting nad - dependent dna ligase activity , suffered from various limitations ( unpublished data ), leading to the development of the novel dna polymerase - based approach of the invention as described herein . an experiment was performed to determine the approximate analytical sensitivity of the dpe - pcr assay using commercially available dna polymerase i . as shown in fig2 a , detection of dna polymerase i was achieved over a wide range of input enzyme . in fact , measurement of dna polymerase i extension activity is achieved down to as little as 2 × 10 − 11 units ( u ) of enzyme ( equivalent to approximately 50 molecules of polymerase ). to our knowledge , detection of dna polymerase extension activity at this level is unrivaled in existing dna polymerase assays . in theory , this level of sensitivity could enable single microbe detection as e . coli has been reported to contain approximately 400 dna polymerase i molecules per cell ( 11 ). regression analysis also showed a strong positive linear correlation ( r 2 = 0 . 992 ) between the dpe - pcr cycle threshold ( ct ) values and units of input commercial dna polymerase i after graphing data from two independent limit of detection experiments ( fig2 b ). after sensitivity and linearity experiments were performed , it was important to determine if the dpe - pcr assay signal was independent of intrinsic exonuclease activity . to this end , we subsequently compared signals generated by 2 × 10 − 7 u of dna polymerase i to those generated from dna polymerase i lacking 5 ′→ 3 ′ exonuclease activity ( klenow ) and another version of the enzyme lacking all exonuclease activity ( klenow exo −). for additional specificity and background signal determination , e . coli dna ligase at 2 × 10 − 7 u and a no input control ( nic ) were tested in parallel . as shown in fig2 c , both klenow and klenow exo − were detected at similar levels when compared to wild type dna polymerase i , providing evidence that the dpe - pcr assay signal is derived from dna polymerase - dependent extension and not intrinsic exonuclease activity . in addition to using exonuclease free polymerases , we set out to further demonstrate that dpe - pcr assay signal is derived from dna polymerase - dependent extension of the dna substrate prior to qpcr . since incorporation of dideoxy nucleotides is a well - established method used for termination of dna polymerase chain extension activities ( 19 , 20 ), we chose to substitute dctp with dideoxyctp ( ddctp ) within our reaction mix . the schematic shown in fig2 d reveals the first possible position within the substrate that ddctp can be incorporated by dna polymerase . if ddctp is incorporated into this position , the extension product of oligo 1 would be insufficient in length for subsequent detection by qpcr primer 1 ( fig1 ). as shown in fig2 d , substitution of dctp with ddctp eliminates signal generated by dna polymerase i , thus demonstrating that the dpe - pcr assay signal is dependent upon dna polymerase extension of the substrate prior to qpcr . the presence of a low copy internal amplification control confirms that qpcr was not inhibited by the presence of low amounts of ddctp that are carried over from the dna polymerase assay reagents ( supplemental fig1 c ). in addition , we feel it is important to note that we have sporadically observed a weak , but detectable signal in the absence of input - dna polymerase ( no input control ). due to the exquisite sensitivity of the dpe - pcr assay , we have demonstrated that weak background noise signals can be derived from several potential sources such as , but not limited to , dna polymerase contamination present in the reagents prior to reaction assembly , dna polymerase introduced by the operator during experimental setup and / or incomplete degradation of oligo 2 ( fig1 ) prior to activation of taq ( unpublished data ). notably , these irregular sources of background noise are controllable by instituting stricter reagent preparation procedures and good aseptic technique . sensitive universal detection of microbes via measurement of endogenous dna polymerase extension activity directly from cell lysates in addition to detecting purified polymerase activity , a simple universal method that measures microbial - derived dna polymerase activity would be highly desirable . if achieved , such a method could enable the screening of candidate antimicrobial agents in actively growing cultures , thus allowing comparison of dna polymerase extension activity to organism proliferation . additionally , measurement of dna polymerase extension activity could be used to screen environmental or biological samples for the presence of any microorganism harboring active dna polymerase . to this end , we developed a simple method that couples microbial lysis to a dpe - pcr assay provided by the invention . as shown in fig3 , a liquid sample known to contain , or suspected of containing , microbes is added to a bead mill lysis tube , disrupted and immediately transitioned into the dpe - pcr assay . we chose one gram negative bacteria ( e . coli ) and one gram positive bacteria ( s . aureus ) to demonstrate the ability of our assay to measure microbial - derived dna polymerase extension activity in crude cellular lysates . as shown in fig4 a , when linked with bead mill lysis , the dpe - pcr assay is capable of detecting a wide dynamic range of input e . coli , down to and below 10 colony forming units ( cfu ) per lysis tube . linear regression analysis of e . coli detection was also performed down to 10 cfu of input bacteria and showed a strong positive linear correlation between input cfu and dna polymerase extension activity signal as indicated by an r 2 value of 0 . 999 ( fig4 b ). colony count plating and e . coli - gene specific qpcr ( gspcr ) were run in parallel , confirming both the input level of cfu per reaction and the ability to monitor intact genomic dna from the exact same lysates . dna polymerase extension activity from s . aureus lysates was detected to a similar input level ( fig4 c ). s . aureus detection was plotted down to 10 cfu of input bacteria and also showed a strong linear correlation between input cfu and dna polymerase extension activity signal ( r 2 = 0 . 999 , fig4 d ). colony count plating and gspcr were performed in parallel to confirm the amount of s . aureus present in each bead lysis tube , as well as the presence of directly analyzable genomic dna . complete tables of plating , gspcr and dna polymerase activity results for both e . coli and s . aureus can be found in fig3 and 4 . we subsequently tested the ability of the dpe - pcr assay to measure dna polymerase activity from seventeen additional clinically relevant microorganisms . as shown in table 1 , we were able to detect dna polymerase activity from all seventeen additional organisms including six gram - negative bacteria , six gram - positive bacteria and five candida species . detection of the seventeen additional microbes exhibited a strong positive linear correlation to input cfu with impressive low limits of detection . to date , and without failure we have similarly tested and detected a total of 31 different microbial species ( data not shown ). the upper linear dynamic ranges have yet to be fully characterized . more results containing parallel plating data and dpe - pcr results for each of 17 additional microbes are presented in fig8 . together , these data support the notion that the performance of dpe - pcr in accordance with the teachings of the present invention has the potential to be useful as a universal “ pan ” test for the sensitive detection of any microbe in a normally sterile environment . as shown in fig2 d , substitution of dctp with ddctp in the reaction mix represents a powerful tool for blocking the detection of dna polymerase - dependent extension activity within our assay . to demonstrate that the signal derived from bacterial spikes was dependent upon their dna polymerase extension activity , and not the other endogenous bacterial enzyme activities present in the lysates , we set up an experiment to compare dpe - pcr signals obtained from e . coli and s . aureus using a standard dna polymerase reaction mix containing ( datp , dttp , dgtp , dctp ) versus a reaction mix containing ( datp , dttp , dgtp , ddctp ). as shown in fig5 a , when compared to the standard reaction mix , substitution of ddctp blocked the generation of signal derived from e . coli cfu spikes ( fig5 a ). a dctp rescue experiment was subsequently performed by comparing dna polymerase extension activity from bacteria lysed in a reaction mix containing 100 % ddctp ( 50 μm ), to those containing 50 μm ddctp spiked with increasing amounts of dctp ( see materials and methods for a detailed description of rescue experiments ). fig5 b demonstrates the rescue effect that increasing amounts of dctp has on quantifiable dna polymerase extension activity derived from e . coli lysates . in addition to measuring microbial dna polymerase extension activity , gspcr was run in parallel to verify that equivalent amounts of e . coli were present in each of the assayed lysates . a graphical comparison of dna polymerase activity versus presence of genomic dna is presented in fig5 c . signal termination ( via ddctp ) and dctp rescue experiments were subsequently repeated with s . aureus and similar results were obtained ( fig5 d - f ). tables containing dpe - pcr and gspcr data for both e . coli and s . aureus can be found in fig6 and 7 . qpcr internal control values are provided to demonstrate that low levels of ddctp carried over into qpcr are not inhibitory , and thus are not responsible for the disappearance of dna polymerase activity signal ( fig6 a and 7a ). together , the data presented in fig5 strongly support the claim that the dpe - pcr assay is specifically detecting microbial dna polymerase extension activity and signal is not derived from substrate modification via enzymatic activities other than dna polymerase . measurement of dna polymerase extension activity as an indicator of bacterial viability traditional methods for determining bacterial viability are dependent upon growth and visualization of a particular microbe on solid medium ( 21 ). although bacterial growth and visualization is the current industry gold standard , the traditional cfu viability determination methods are undesirable due to the length of time required for cfu formation . furthermore , the ability to grow on solid media or in liquid culture can vary dramatically from one microbe to another , thus potentially limiting the detection of certain fastidious organisms ( 22 ). due to the aforementioned limitations of traditional methods , there is a growing need in a wide variety of pharmaceutical ( 23 ), environmental , food processing and clinical testing arenas for the rapid assessment of microbial viability . consequently , numerous molecular methods have been developed in an effort to quickly assess microbial viability status within a given matrix ( 24 ). despite being rapid and sensitive , molecular methods that detect the presence of nucleic acid often fall short of representing an accurate measurement of cell viability . for example , amplification of endogenous dna or rna is a poor indicator of bacterial viability , due to the persistence of nucleic acid after cell death ( 25 , 26 ). we set out to determine the feasibility of using dna polymerase extension activity as an indicator of bacterial viability . to this end , an experiment was designed to compare detection of dna polymerase extension activity and pcr - mediated detection of genomic dna as indicators of bacterial viability following various amounts of heat treatment . to begin , e . coli suspensions were treated at increasing temperatures for a fixed period of time . after heat treatment , bacteria were subsequently assayed for the presence of both dna polymerase extension activity and genomic dna . heat treated and non - heat treated bacterial stocks were also plated in parallel to monitor bacterial viability via the presence of visible cfu . fig6 a represents the levels of e . coli dna polymerase extension activity measured after the indicated amounts of heat treatment . notably , a significant drop in e . coli dna polymerase extension activity was observed after incubation of bacterial suspensions between 45 ° c . and 65 ° c . ( fig6 a ). in contrast , gspcr signal obtained from the same lysates remained relatively constant at all temperatures and is graphically compared to dna polymerase activity in fig6 b . plating results presented below the graph further demonstrate that increasing levels of heat treatment are sufficient to prevent cfu formation and are paralleled by a dramatic loss of dna polymerase activity ; however , dead cells still contribute genomic dna levels very close to their original input levels confirming that gspcr is a poor indicator of the presence of viable cells ( fig6 b ). in fig6 c , the bar graphs further highlight the relative abilities of dpe - pcr and gspcr to monitor the disappearance of cfu in response to lethal amounts of heat treatment . subsequently , we wanted to test whether measurement of dna polymerase extension activity could be used to indicate the viability status of a gram positive organism as well . the previous e . coli experiments were repeated with s . aureus under the same conditions . fig7 a - c show similar results obtained from heat treatment experiments repeated with s . aureus . collectively , the strong concordance between the presence of cfu and dna polymerase extension activity shown in fig4 , 6 , 7 , and table 1 of fig8 , demonstrates that dpe - pcr performed according to this invention has potential to be used as a general indicator of cell viability , and may additionally present the possibility of measuring dna polymerase extension activity from microbes exposed to other clinically or pharmaceutically relevant agents ( bacteriostatic and bactericidal ) aimed at reducing cell proliferation or viability . in summary , in accordance with the present invention there has been developed a novel , highly sensitive , quantitative and rapid dpe - pcr assay . in addition to quantitative detection of extremely low levels of purified enzyme , we have demonstrated the ability of dpe - pcr to reproducibly measure dna polymerase extension activity from less than 10 cfu of bacteria via coupling to bead lysis . we have also demonstrated the potential for dpe - pcr to universally detect microbes by testing a panel of microorganisms comprised of seven gram - negative bacteria , seven gram - positive bacteria and five candida species . furthermore , preliminary evidence that the dpe - pcr assay can be used to assess bacterial viability was provided via the reproducibly strong correlation between dna polymerase extension activity and proliferation as indicated by the presence of cfu . considering the data disclosed herein , it is presently believed that the novel methods and techniques of the invention such as the preferred dpe - pcr assay as disclosed herein , has the potential to become a useful tool for a wide range of testing applications within pharmaceutical , environmental , food and clinical settings . the contents of all references , patents and published patent applications cited throughout this application , are incorporated herein by reference to the same extent as if each individual publication , patent or patent application was specifically and individually indicated to be incorporated by reference . the foregoing detailed description has been given for clearness of understanding only and no unnecessary limitations should be inferred therefrom as modifications will be obvious to those skilled in the art . it is not an admission that any of the information provided herein is prior art or relevant to the presently claimed inventions , or that any publication specifically or implicitly referenced is prior art . unless defined otherwise , all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs . while the invention has been described in connection with specific embodiments thereof , it will be understood that it is capable of further modifications and this application is intended to cover any variations , uses , or adaptations of the invention following , in general , the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth . 1 . rothwell , p . j and waksman , g . 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