Method and Kit of Detecting the Absence of Micro-Organisms

Abstract

Methods of detecting the absence or presence of a micro-organism in a sample comprising: contacting the sample with a nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the micro-organism in the sample, incubating the thus contacted sample under conditions suitable for nucleic acid modifying activity; and specifically determining the absence or presence of a modified nucleic acid molecule resulting from the action of the nucleic acid modifying activity on the substrate nucleic acid molecule to indicate the absence or presence of the micro-organism. Corresponding kits are also provided.

Claims

1. A method of detecting the absence or presence of a micro-organism in a sample comprising: (a) contacting the sample with a nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the micro-organism in the sample, (b) incubating the thus contacted sample under conditions suitable for nucleic acid modifying activity; and (c) specifically determining the absence or presence of a modified nucleic acid molecule resulting from the action of the nucleic acid modifying activity on the substrate nucleic acid molecule to indicate the absence or presence of the micro-organism, characterised in that the nucleic acid molecule is modified so as to protect it from nuclease activity.

2. The method of claim 1 wherein the modification of the nucleic acid molecule is selected from incorporation of synthetic nucleotides, incorporation of methylation and protection of the 3′ and/or 5′ ends.

3. The method of claim 2 wherein the synthetic nucleotides comprise phosphorothioate nucleotides and/or locked nucleic acid nucleotides.

4. The method of claim 1 wherein the action of the nucleic acid modifying activity on the substrate nucleic acid molecule produces an extended nucleic acid molecule.

5. The method of claim 1 wherein the nucleic acid modifying activity comprises polymerase activity.

6. (canceled)

7. (canceled)

8. The method of claim 1 wherein step (a) comprises contacting the sample with a nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the micro-organism in the sample together with an internal positive control (IPC) nucleic acid molecule, wherein the IPC nucleic acid molecule is susceptible to nuclease activity and is used to identify contaminating nuclease activity in the pellet or wherein the IPC nucleic acid molecule is modified so as to protect it from nuclease activity.

9. (canceled)

10. The method of claim 8 wherein the modification of the IPC nucleic acid molecule is selected from incorporation of synthetic nucleotides, incorporation of methylation and protection of the 3′ and/or 5′ ends.

11. The method of claim 10 wherein the synthetic nucleotides comprise phosphorothioate nucleotides and/or locked nucleic acid nucleotides.

12. The method of claim 8 wherein the IPC nucleic acid molecule comprises identical primer binding sites to the nucleic acid molecule such that there is competition for primer binding in step (c).

13. The method of claim 8 wherein a nucleic acid probe is added in step (c) which binds to a target probe sequence within the (sense strand of the) nucleic acid molecule, optionally wherein the nucleic acid probe is labelled.

14. The method of claim 13 wherein a further nucleic acid probe is added in step (c) which binds to a target probe sequence within the IPC nucleic acid molecule, optionally wherein the further nucleic acid probe is labelled.

15. The method of claim 14 wherein the nucleic acid probe does not bind to the IPC nucleic acid molecule and the further nucleic acid probe does not bind to the nucleic acid molecule.

16. The method of claim 14 or claim 15 wherein the nucleic acid probe and further nucleic acid probe are differently labelled.

17. The method of claim 1 wherein the nucleic acid molecule is at least partially double stranded and comprises uracil residues in the complementary strand and step (c) comprises adding Uracil DNA Glycosylase (UDG) to the sample in order to degrade the uracil residues in the complementary strand.

18. The method of claim 17 wherein the complementary strand of the nucleic acid molecule comprises a modification at the 3′ end to prevent extension.

19. The method of claim 18 wherein the modification at the 3′ end comprises incorporation of a non-extendible nucleotide.

20. The method of claim 19 wherein the non-extendible nucleotide is a dideoxy nucleotide triphosphate (ddNTP), optionally wherein the ddNTP is dideoxyCytidine.

21. (canceled)

22. The method of claim 1 wherein step (c) comprises a nucleic acid amplification step.

23.-25. (canceled)

26. The method of claim 1, wherein the sample contains a non-micro-organism source of nucleic acid modifying activity and the method comprises: (a) treating the sample under high pH conditions for no more than 5 minutes in order to inhibit the non-micro-organism source of nucleic acid modifying activity (whilst not affecting the nucleic acid modifying activity of the micro-organism in the sample), (b) contacting the sample with a nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the micro-organism in the sample, (c) incubating the thus contacted sample under conditions suitable for nucleic acid modifying activity; and (d) specifically determining the absence or presence of a modified nucleic acid molecule resulting from the action of the nucleic acid modifying activity on the substrate nucleic acid molecule to indicate the absence or presence of the micro-organism.

27. (canceled)

28. (canceled)

29. The method of claim 26 wherein the treatment under high pH conditions is stopped by adding a reagent to lower the pH.

30.-32. (canceled)

33. The method of claim 1 wherein the sample contains a non-micro-organism source of nucleic acid modifying activity and the method comprises: (a) (i) incubation of the sample with a reagent that lyses non-microorganisms if present in the sample but does not lyse microorganisms in the sample, (ii) optionally separation of the lysed cell material from the intact microorganisms (if any) in the sample, (iii) contacting the (separated) intact microorganisms (if any) in the sample with a high pH reagent and incubating for no more than 5 minutes in order to inhibit the non-micro-organism source of nucleic acid modifying activity (whilst not affecting the nucleic acid modifying activity of the micro-organism in the sample), (iv) adding a pH lowering reagent in order to stop the incubation at high pH, (v) separation of the microorganisms if present in the sample from the pH modifying reagents, (vi) lysis of any separated microorganisms, (vii) contacting the sample with a nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the micro-organism in the sample, (viii) incubating the thus contacted sample under conditions suitable for nucleic acid modifying activity, and (ix) specifically determining the absence or presence of a modified nucleic acid molecule resulting from the action of the nucleic acid modifying activity on the substrate nucleic acid molecule to indicate the absence or presence of the micro-organism; or (b) (i) incubation of the sample with a reagent that lyses non-micro-organisms if present in the sample but does not lyse micro-organisms in the sample, (ii) centrifugation of the sample to form a pellet containing micro-organisms if present in the sample, (iii) removal of the supernatant from the pellet, (iv) re-suspending the pellet in a high pH reagent and incubating for no more than 8 minutes in order to inhibit the non-micro-organism source of nucleic acid modifying activity (whilst not affecting the nucleic acid modifying activity of the micro-organism in the sample), (v) adding a pH lowering reagent in order to stop the incubation at high pH, (vi) a second centrifugation of the sample to form a pellet containing micro-organisms if present in the sample, (vi) removal of the supernatant from the pellet, (vii) lysing any micro-organisms in the pellet, (viii) contacting the sample with a nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the micro-organism in the sample, (ix) incubating the thus contacted sample under conditions suitable for nucleic acid modifying activity, and (x) specifically determining the absence or presence of a modified nucleic acid molecule resulting from the action of the nucleic acid modifying activity on the substrate nucleic acid molecule to indicate the absence or presence of the micro-organism.

34. The method of claim 33 wherein steps (a)(vi) and (a)(vii) or b(vii) and b(viii) are performed together, optionally wherein the nucleic acid molecule is added to the sample together with a lysis reagent.

35.-38. (canceled)

39. The method of claim 1 wherein the sample potentially contains a non-micro-organism source of nuclease activity and the method comprises: (i) incubation of the sample with a reagent that lyses non-micro-organisms if present in the sample but does not lyse micro-organisms in the sample, (ii) separation of the lysed cell material from the intact microorganisms (if any) in the sample and/or inactivation of the lysed cell material, (iii) lysing any microorganisms following the separation and/or inactivation, (iv) contacting the sample with a nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the micro-organism in the sample, (v) incubating the thus contacted sample under conditions suitable for nucleic acid modifying activity, and (vi) specifically determining the absence or presence of a modified nucleic acid molecule resulting from the action of the nucleic acid modifying activity on the substrate nucleic acid molecule to indicate the absence or presence of the micro-organism; or (a) centrifugation of the sample to form a pellet containing micro-organisms if present in the sample, (b) removal of the supernatant from the pellet, (c) lysing any micro-organisms in the pellet, (d) contacting the sample with a nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the micro-organism in the sample, (e) incubating the thus contacted sample under conditions suitable for nucleic acid modifying activity, and (f) specifically determining the absence or presence of a modified nucleic acid molecule resulting from the action of the nucleic acid modifying activity on the substrate nucleic acid molecule to indicate the absence or presence of the micro-organism.

40. The method of claim 39 wherein steps (iii) and (iv) or (c) and (d) respectively are performed together, optionally wherein the nucleic acid molecule is added to the sample together with a lysis reagent.

41. (canceled)

42. A kit comprising: (a) at least one nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the micro-organism in the sample, wherein the at least one nucleic acid molecule is at least partially double stranded and comprises uracil residues in the complementary strand, characterised in that the nucleic acid molecule is modified so as to protect it from nuclease activity, and (b) at least one internal positive control (IPC) nucleic acid molecule which comprises identical primer binding sites to the nucleic acid molecule such that there is competition for primer binding in a nucleic acid amplification reaction containing both the nucleic acid molecule and the IPC.

43.-59. (canceled)

Description

DESCRIPTION OF THE FIGURES

[0403] FIG. 1. Improved ETGA detection of microorganisms by increasing dNTP and substrate concentration. Each chart shows the ct value obtained for the detection of the ETGA target substrate (FAM channel) in ETGA detection experiments for a range of relevant microorganisms. In all cases the negative blood culture control was >39.9 ct units, the negative reagent negative controls were >40 ct units and the positive reagent controls were <20 ct units.

[0404] FIG. 2. Detection of IPC molecule in a blood culture sample prepared with IPC DNA added only in the microbial Lysis mixture (LM) or PCR mastermix (MM) compared to a negative control sample. A quantity of IPC DNA was added to the LM to provide the same ct value as when added to the MM. Data shows that there is a complete loss in detection of the IPC molecule (in a 40 cycle PCR reaction) in the negative blood culture sample compared to the negative control (with no blood) when added to LM and not when added to MM.

[0405] FIG. 3. Increased background caused by adding IPC to LM instead of MM. The data plotted on the chart shows ct value in the FAM channel (detecting ETGA substrate) versus total viable count (TVC) obtained for blood culture samples using a protocol where the IPC had been added to LM (diamonds) and MM (squares). The amount of IPC added to LM was equivalent to 50× higher than in MM for each PCR reaction. The measured background was higher when using 50× the normal concentration of IPC in LM compared to using the standard concentration of IPC in MM. Background levels were measured by an ETGA test procedure with IPC in the MM (blue dashed line) or LM (green dot-dashed line) in blood culture samples that did not contain any added bacteria.

[0406] FIG. 4. ETGA test background reduction and improved test sensitivity. Graphs show fluorescence detected in the FAM channel in qPCR reaction from ETGA tests carried out on a dilution series of C. albicans in blood culture. The qPCR reaction contained a FAM-labelled probe, capable of detecting the modified ETGA substrate, so amplification indicates the presence of a microorganism. FIG. 4a shows the results of a set of ETGA tests carried out using standard substrate and IPC oligos. FIG. 4b shows the results obtained from exactly the same samples using PTO substrate and IPC. Note that background is much lower in FIG. 4b, and that it is possible to detect lower number of yeast cells in the test.

[0407] FIG. 5. Improvement of yeast detection by use of PTO oligos. Graph shows sensitivity of detection of yeast (C. albicans) in an ETGA test using standard oligos compared to an ETGA test using PTO oligos.

[0408] FIG. 6. Detection of less robust microorganisms by ETGA. Chart shows how detection of a delicate strain of H. influenzae is affected by the ETGA test procedure. Pure culture of S. aureus and H. influenzae (10.sup.5 cfu) was added to the general test protocol (10 mL) at different stages. Data shows that detection was significantly reduced when microorganisms are added before the NaOH resuspension step.

[0409] FIG. 7. Controlling exposure to NaOH to improve detection of H. influenzae. Graph shows the effect of controlling the amount of time that a culture sample containing 10.sup.5 cfu H. influenzae is exposed to NaOH in the ETGA test procedure compared to the standard procedure. The general protocol for 10 mL was carried out on a suspension of H. influenzae in BacT/ALERT broth without blood; after resuspension in NaOH and incubation for 0, 0.5, 2.5 and 5 min, 1 mL 200 mM Tris-HCl [pH7.2] was added prior to centrifugation.

[0410] FIG. 8. Improving the 1 ml ETGA protocol with a pH lowering step. Blood culture samples containing A) no spike, B) H. influenzae (10.sup.5 cfu), C) H. influenzae (10.sup.4 cfu), D) S. aureus (10.sup.5 cfu), E) S. aureus (10.sup.4 cfu) were tested with the original 1 ml procedure (based on a resuspension in 1 mL NaOH) and a procedure containing a pH-lowering step (resuspension in 0.75 mL NaOH, 5 min incubation, 0.5 mL Reagent C). The lower ct value indicates that the microorganisms are detected more strongly.

[0411] FIGS. 9A-C. Cognitor Minus results for E. coli spiked blood broth samples and the positive control (Pol(+)). Data is shown for Cognitor Minus samples analysed at time 0, 2 and 20 hours with or without the 95° C. step for (A) experiment 2, (B) experiment 3 and (C) experiment 4. Data for experiment 1 is not shown because samples were only analysed by QPCR at time 0 hours.

[0412] FIG. 10. Ct values for E.coli spiked blood broth samples (n=4) plotted against log transformed total cfu values. Trend lines are plotted for each time point (0 hours, 2 hours and 20 hours) within the 95° C. (+) or 95° C. (−) data sets. Positive control data is not shown here.

[0413] FIGS. 11A-B. Ct values for E. coli spiked blood broth samples and positive controls (Pol+ve) at time 0, 2 and 20 hours for 95° C. (+) and 95° C. (−) samples processed using either (A) unmodified oligonucleotide lysis mix or (B) phosphorothioate oligonucleotide lysis mix. The data shown is from a single experiment (n=1).

[0414] FIGS. 12A-E. Cognitor Minus results for (A) E. coli, (B) S. aureus and (C) C. albicans spiked blood broth samples, (D) positive controls (PC) and (E) no spike controls (NSCs). Ct values from three replicate experiments (n=3) are plotted against sample storage duration. Trend lines are plotted for each sample set: PTO 95° C. (+); PTO 95° C. (−); UMO 95° C. (+); and UMO 95° C. (−). Only UMO LM data are shown for NSC samples because most PTO LM samples produced ‘No Ct’ due to insufficient amplification.

EXPERIMENTAL SECTION

[0415] The invention will be understood with respect to the following non-limiting examples:

EXAMPLE 1

ETGA Test Modifications

[0416] Methods—General protocol

[0417] For each sample, 1 mL of blood culture (with or without microorganisms added, with or without blood) was mixed with 0.333 mL Reagent A (5% w/v Saponin, 5% w/v Tween 20, 8.5 g/L sodium chloride) in a 1.5 mL microcentrifuge tube and incubated at room temperature for 15 min. Each sample was centrifuged for 3 min at 7300 g, then the supernatant was poured away and the rim of the tube was dabbed on clean laboratory tissue paper. Each pellet was then resupended in 0.75 mL of Reagent B (5 mM NaOH) and incubated for 5 min, then pH was lowered by adding 0.5 mL of Reagent C (1.32 g/L ammonium sulphate, 0.49 g/L magnesium sulphate heptahydrate, 0.75 g/L potassium chloride, 20 mM Tris-HCl, pH8.0). After incubation, samples were centrifuged again and the supernatant removed by pouring away. The remaining pellet was resuspended in 0.5 mL of Reagent C and immediately transferred to a new tube containing a mixture of glass beads (0.1mm and 0.5mm glass beads; supplied by CamBio cat 13118-400, and 13116-400 respectively). A further centrifugation was carried out in order to pellet any suspended cells with the glass beads, and again, the supernatant was removed and discarded.

[0418] 50 μL of microbial Lysis Mixture containing the ETGA substrate (LM; containing reagents L1, L2, L3 at a ratio of 7:2:1, see Table 1) was added to the glass beads and placed in a Disruptor Genie (Scientific Industries, Inc.) cell disruptor for 6 min at 2800 rpm to lyse microbial cells. After disruption, samples were placed in a 37° C. heating block and incubated for 20 min, then transferred to another heating block at 95° C. and incubated for 5 min. After incubation, samples were cooled to room temperature whilst the PCR reagents were prepared.

[0419] After cooling, 3 μL of sample supernatant was added to 27 μL of PCR mastermix (MM; containing a general Taq polymerase PCR mastermix (Roche—cat 04902343001), primers for the ETGA substrate, internal positive control—IPC—DNA, FAM-labelled probe for the ETGA substrate, Texas Red labelled probe for the IPC, and (1.2 ul) UDG enzyme (Bioline—cat no BIO-27044)) in a SmartCycler PCR tube (Cepheid). Samples were placed in the SmartCycler PCR and subjected to the following reaction conditions;

[0420] 1 cycle; 40° C. 10 min, 50° C. 10 min, 95° C. 5 min

[0421] 40-50 cycles: 95° C. 5 sec, 61° C. 20 sec, 72° C. 20 sec.

[0422] Amplification was monitored throughout the reaction in real-time in the Texas Red and FAM excitation/detection channels of the SmartCycler.

TABLE-US-00004 TABLE 1 Lysis mixture components L1 Bovine serum albumin   1.5% w/v Triton X100   1.5% v/v Tween 20   1.5% v/v L2 Ammonium sulphate   2.64 g/L Magnesium sulphate   0.98 g/L heptahydrate Potassium chloride   1.5 g/L Tris-HCl, pH 8.0  40 mM dNTP (A,G,C,T) 500 μM L3 ETGA substrate   0.001 μM-0.01 μM Tris-HCl, pH 8.5  20 mM KCl  10 mM EDTA  10 μM

[0423] The sequences of the PCR reaction components are as follows;

TABLE-US-00005 Fam labelled probe (a molecular beacon): (SEQ ID NO: 1) FAM-cgc tgc gac cga ccg ata agc tag aac agg cag cg-BHQ1 Texas red labelled probe (a molecular beacon): (SEQ ID NO: 2) TxR-cgc gat cag cag gcc aca cgt taa aga cat cgc g-BHQ2 IPC (SEQ ID NO: 3) gcc gat atc gga caa cgg ccg aac tgg gaa ggc gag atc agc agg cca cac gtt aaa gac aga gag aca aca acg ctg gcc gtt tgt cac cga cgc cta Forward primer (SEQ ID NO: 4) ccg ata tcg gac aac ggc cga act gg Reverse primer (SEQ ID NO: 5) tag gcg tcg gtg aca aac ggc cag c

[0424] The substrate components are;

TABLE-US-00006 AS (SEQ ID NO: 6) uaggcgucggugacaaacggccagcguuguugucucu-DDC (3′ terminal is a dideoxy-C) S1 (SEQ ID NO: 7) gccgatatcggacaacggccgaactgggaaggcgagactgaccgaccgat aagctagaacagagagacaacaac

[0425] Results and Discussion 1—Increasing Substrate Concentration

[0426] The general protocol was modified by increasing the amount of ETGA substrate in LM by 10-fold (from 0.001 μM to 0.01 μM) and increasing the amount of dNTP 2-fold (from 50 μM to 100 μM).

[0427] The increased quantity of substrate and dNTP enabled improved detection of C. albicans (FIG. 1a), E. coli (FIG. 1b), S. aureus (FIG. 1c), E. faecalis (FIG. 1d), P. aeruginosa (FIG. 1e), or 2 different strains of H. influenzae (including a delicate clinical strain) (FIG. 1f and FIG. 1g). Data is shown in FIG. 1.

[0428] Results and Discussion 2—Improving ETGA Test Sensitivity and Lowering Background by Modifying Oligo Components

[0429] Evidence of Nuclease Activity

[0430] Adding the IPC molecule at the same time as the ETGA substrate molecule was thought to be an improvement on the original protocol. If IPC was added in LM, the IPC would be subject to exactly the same test conditions as the ETGA substrate and therefore provide a more accurate test control. For example, conditions that may negatively impact the substrate molecule such as nuclease activity that could digest the substrate, would also affect the IPC. If the IPC is added later (in MM for example) it would not be subject to the same conditions and may result in false interpretation of the data. If IPC is added at the same time as the ETGA substrate the magnitude of the effect of the test conditions on the nucleic acid templates could be measured.

[0431] To exemplify this, FIG. 2 shows that test conditions could have a negative impact on the detection of the ETGA substrate; when adding comparable amounts of IPC DNA in LM rather than in MM, it was found that there was indeed a loss in the ability to detect the IPC in blood culture specimens compared to negative control samples (without blood). The same loss of detection was not seen when using IPC in MM. Loss of the IPC when added in LM was attributed to nucleases that may have been active during the 37° C. incubation step of the ETGA protocol. Nucleases were most likely to have originated from the blood specimen.

[0432] Clearly, if the IPC molecule was lost during the test, the ETGA substrate molecule could also be lost.

[0433] Loss of the ETGA substrate molecule in a positive blood culture sample would obviously result in reduced detection sensitivity or potentially lead to a false negative result, but, by adding IPC to LM it would be possible to determine (and perhaps quantify) suspected nuclease activity and interpret results accordingly. Samples where a drop in IPC quantity was observed (as seen by a rise in ct value compared to a negative reagent control) could be reported as ‘unresolved’ rather than ‘negative’, thus indicating that the sample was subject to nuclease activity and may, in fact, be positive.

[0434] Adding an IPC molecule to LM could improve the ETGA test. However, depending on how common nuclease activity was found to be in clinical specimens, this could raise the overall number of unresolved results rendering the test less attractive to potential users due to the high perceived failure rate (and thus explaining why IPC was originally added to MM rather than LM).

[0435] Testing showed that the IPC molecule could be added to the test in the LM and still be detected in the presence of presumed nuclease activity by increasing the concentration 20-50 fold, but high levels of background were detected even in negative blood culture samples (FIG. 3). In this case, background may have been caused by the presence of partly digested oligonucleotides that interfere in the detection PCR reaction. Obviously, high levels of background may reduce the sensitivity of the test, especially when attempting to detect very low numbers of bacteria.

[0436] Whilst increasing the amount of IPC in LM may be considered a solution to the problem of loss of target DNA molecules due to nuclease activity, it may only mask the issue and as previously mentioned may contribute to higher background level. Increasing the amount of IPC molecule in samples where contaminating nuclease activity was low could also reduce test sensitivity by increasing competition for reaction components in the detection PCR thereby reducing the ability of the test to detect the target substrate. A better solution to the putative nuclease problem was required.

[0437] Protecting ETGA Substrate and IPC From Nuclease Activity

[0438] Based on the assumption that the IPC molecule should be included in LM, and that nuclease activity may be having a detrimental effect on the ETGA test, attempt was made to protect the DNA targets (IPC and ETGA substrate) from nuclease degradation. DNA can be protected from nuclease activity by various means, by modification (e.g. methylation, end modification) or using non-standard nucleotides during the synthesis of synthetic oligonucleotides (e.g. locked nucleic acids, phopsphorothioate nucleotides).

[0439] ETGA test has been found to be less sensitive to yeasts than bacteria. The reason for this not known but is likely to be due to a combination of reason such as, differences in in vitro activity or absolute quantity of the fungal enzymes in cells compared to the bacterial enzymes, or sensitivity to inhibitors.

[0440] Demonstration of the Use of PTO Oligos in the ETGA Test

[0441] Standard ETGA substrate and IPC oligos were replaced with phosphorothioate oligos (PTO) with the same nucleotide sequence in LM in the general protocol. PTO-modified ETGA substrate was added to LM at 0.01 μM and PTO-modified IPC was added at a sufficient quantity to achieve a ct value of 37-41 in a 50 cycle PCR reaction.

[0442] A dilution series of yeast cells (10.sup.5, 10.sup.4, 10.sup.3 and 0 cfu/ml) in blood culture was tested with the original ETGA test protocol with standard oligos and with PTO modified oligos. Both tests were run on exactly the same spiked blood cultures (FIG. 4).

[0443] Data showed that the ETGA test with standard oligos yielded results that included high levels of background that may have occluded the detection of low levels of yeast cells, whereas results obtained with PTO modified oligos did not suffer from the same effect. In fact, results showed that PTO oligos reduced the overall background to undetectable levels on the same culture specimens, thus allowing the lowering of the threshold level in the qPCR reaction and potentially increasing the sensitivity of the ETGA test. Note that when using standard oligos, the threshold level was set at 50 units due to the amount of background fluorescence detected, but, when using the PTO oligos the threshold level could be lowered to 10 units, or lower if required. PTO oligos were detected later in the PCR reaction than when using standard oligos, but the PTO qPCR reaction could also be run for longer (50 cycles rather than 40) because the level of background was so low. The result of this reduction in background meant that lower levels of microbial load (as low as 10.sup.3 cfu/ml in this example) could be detected with PTO oligos that would have previously been undetectable when using the standard oligos.

[0444] In a further experiment, nuclease resistant PTO versions of the substrate molecules (MWG-Eurofins) were used in an ETGA test. A dilution series of yeast overnight culture was used to artificially spike a blood culture (a sample of the blood culture was then spread on SDA to confirm total viable counts). Each suspension was then tested with an ETGA test using standard oligos and an ETGA test using PTO oligos. Sensitivity of detection of the yeast was much improved by the use of PTO. Note that the PTO results are plotted on a different y-axis to the standard test, due to the differing number of PCR cycles. The dashed lines indicate the Ct value of the negative control (blood culture without any microorganisms). The data from this experiment show that the PTO substrate was shown to improve the detection of yeasts (FIG. 5) by 1000-fold, and furthermore, it did not display the effect of raising background levels. The finding that a PTO substrate could be used to increase the sensitivity of the ETGA test was very significant.

EXAMPLE 2

Reducing False Negative Test Results in the ETGA Test

[0445] General Protocols

[0446] For 10 mL Specimens

[0447] For each sample, 10 mL of blood culture (with or without microorganisms added, with or without blood) was mixed with 3.33 mL Reagent A (5% w/v Saponin, 5% w/v Tween 20, 8.5 g/L sodium chloride) in a 15 mL Falcon tube and incubated at room temperature for 15 min. Each sample was centrifuged for 8 min at 3600 g, then the supernatant was poured away and the rim of the tube was dabbed on clean laboratory tissue paper. Each pellet was then resupended in 5 mL of Reagent B (5 mM NaOH) and incubated for 5 min. After incubation, samples were centrifuged again and the supernatant removed by pouring away. The remaining pellet was resuspended in 1 mL of Reagent C (1.32 g/L ammonium sulphate, 0.49 g/L magnesium sulphate heptahydrate, 0.75 g/L potassium chloride, 20 mM Tris-HCl, pH8.0) and immediately transferred to a 1.5 mL microcentrifuge tube containing a mixture of glass beads. A further centrifugation for 3 min at 7300× g was carried out in order to pellet any suspended cells with the glass beads, and again, the supernatant was removed and discarded.

[0448] 50 μL of microbial Lysis Mixture (LM; containing ETGA substrate see table 1 in Example 1 above) was added to the glass beads and placed in a Disruptor Genie cell disruptor for 6 min at 2800 rpm to lyse microbial cells. After disruption, samples were placed in a 37° C. heating block and incubated for 20 min, then transferred to another heating block at 95° C. and incubated for 5 min. After incubation, samples were cooled to room temperature whilst the PCR reagents were prepared.

[0449] After cooling, 3 μL of sample supernatant was added to 27 μL of PCR mastermix (MM; containing a general Taq polymerase PCR mastermix, primers for the ETGA substrate, internal positive control—IPC—DNA, FAM-labelled probe for the ETGA substrate, Texas Red labelled probe for the IPC, and UDG enzyme) in a SmartCycler PCR tube (Cepheid). Samples were placed in the SmartCycler PCR and subjected to the following reaction conditions;

[0450] 1 cycle; 40° C. 10 min, 50° C. 10 min, 95° C. 5 min

[0451] 40-50 cycles: 95° C. 5 sec, 61° C. 20 sec, 72° C. 20 sec.

[0452] Amplification was monitored throughout the reaction in real-time in the Texas Red and FAM excitation/detection channels of the SmartCycler.

[0453] For 1 mL Specimens

[0454] For each sample, 1 mL of blood culture was mixed with 0.333 mL Reagent A (5% w/v Saponin, 5% w/v Tween 20, 8.5 g/L sodium chloride) in a 1.5 mL microcentrifuge tube and incubated at room temperature for 15 min. Each sample was centrifuged for 3 min at 7300 g, then the supernatant was poured away and the rim of the tube was dabbed on clean laboratory tissue paper. Each pellet was then resupended in 1 mL of Reagent B (5 mM NaOH) and incubated for 5 min. After incubation, samples were centrifuged again and the supernatant removed by pouring away. The remaining pellet was resuspended in 0.5 mL of Reagent C (1.32 g/L ammonium sulphate, 0.49 g/L magnesium sulphate heptahydrate, 0.75 g/L potassium chloride, 20 mM Tris-HCl, pH8.0) and immediately transferred to a new tube containing a mixture of glass beads. A further centrifugation was carried out in order to pellet any suspended cells with the glass beads, and again, the supernatant was removed and discarded.

[0455] Microbial lysis and PCR detection was then carried out as previously described for the 10 mL protocol.

[0456] PCR reaction components and substrates are per Example 1 above.

[0457] Background

[0458] A single clinical microbial isolate, identified as Haemophilus influenzae gave a false negative result in the ETGA test during a clinical performance evaluation. The microorganism was detected by standard automated blood culture in Biomerieux Bact/ALERT blood culture media.

[0459] When a spike of cultured microbial cells was added to different stages of the ETGA test it was found that the H. influenzae strain was only detected when added after the NaOH wash step (Reagent B). Other, more robust bacterial species were found to be detectable when added to the test from the start (see FIG. 6). Results indicated that the strain of H. influenzae was particularly sensitive to the NaOH washing step (or a combination of the steps up to and including the NaOH washing step).

[0460] This result was not typical of all strains of H. influenzae and, to date, has only been associated with this strain. The isolate was used as a model ‘weak’ organism to develop a new ETGA procedure that was better at detecting less robust microorganisms.

[0461] Incubation in NaOH is an essential step in the ETGA protocol that must be carried out in order to inactivate free polymerases, reduce contaminants and reduce background. Attempts were therefore made to reduce the damaging effect of NaOH without detrimentally affecting the test results. The concentration of NaOH could not be lowered because it did not remove sufficient contaminating material, which leads to failure of the test.

[0462] Results—Reducing Sample Exposure to NaOH

[0463] In the general 10 mL protocol, note that the time taken to centrifuge the sample increases the total time that the sample is exposed to NaOH by 8 min. Shortening the length of time could be achieved and controlled by neutralisation of the alkali, or, at least lowering the pH of the sample after an optimal period of incubation time by adding 1 mL of 200 mM Tris-HCl buffer, pH 7.2 to the NaOH.

[0464] The general protocol for 10 mL specimens was carried out on a suspension of H. influenzae in culture media drawn from a BacT/ALERT SA bottle (no blood was added). Results demonstrate that neutralisation (or significant lowering the pH) of the NaOH lead to lower ct values (from identical samples) and therefore improved sensitivity of detection (see FIG. 7).

[0465] Data also suggested that the shorter incubation time would be better, but again, short incubation times did not allow sufficient removal of contaminants to enable reliable PCR amplification, leading to reaction failure or high background levels and false positive results.

[0466] Lowering of the pH of the sample after NaOH treatment could potentially be achieved by adding any suitable buffer or acid. The preferred method of lowering the pH would be to use Reagent C (a Tris-HCl buffer, pH 8) because the reagent is already used in the test.

[0467] Results—Preferred Embodiment, 1 mL

[0468] Detection of both H. influenzae and S. aureus could be improved in the general protocol for 1 mL specimens by replacing the original NaOH step with the more complex steps consisting of resuspension in 0.75 mL NaOH, incubation at room temperature for 5 min then adding 0.5 mL Reagent C to lower the pH. Results, summarised in FIG. 8, showed that the ct values from the ETGA tests on all microorganism-containing samples were lower when using the pH-lowering protocol compared to the original 1 mL protocol, demonstrating that the pH lowering protocol improved detection.

EXAMPLE 3

Analysis of the Importance of the 95° C. and the Use of a PTO Substrate Purpose

[0469] The purpose of the work outlined in this example was to assess the effect of the 95° C. step on the performance of the Cognitor Minus test. The Cognitor Minus test was carried out using bacteria-spiked blood broth samples with or without the 95° C. step to compare the following characteristics: [0470] The Ct values obtained [0471] The stability of ETGA template DNA as measured by QPCR at different time-points following completion of sample preparation [0472] The Ct values obtained and stability of ETGA template DNA when lysis mix (LM) contains unmodified oligonucleotide (UMO) ETGA substrate as opposed to the phosphorothioate modified oligonucleotide (PTO) ETGA substrate.

[0473] Introduction

[0474] The earlier steps in the Cognitor Minus test aim to lyse blood cells and wash away blood-derived proteins such as DNA polymerases, which will produce non-microorganism derived ETGA template DNA, and nuclease enzymes which may digest microorganism-derived ETGA template DNA. Any resulting intact microorganisms are then lysed by the addition of lysis mix (LM) and bead milling. Following microorganism lysis and the ETGA reaction, samples contain a mixture of microorganism proteins, LM components, newly synthesised ETGA template DNA and residual blood cell proteins. The 95° C. step is intended to denature all proteins in order to protect the ETGA template DNA and internal process control (IPC) DNA from nuclease digestion so that it can be successfully detected by QPCR.

[0475] To assess the importance of the 95° C. step, the Cognitor Minus test was carried out using bacteria-spiked blood cultures with or without the 95° C. step. The samples were analysed by QPCR at three time points: immediately after sample preparation; after 2 hours at room temperature; and after a further 18 hours at 4° C. The aim was to compare the Ct values obtained and the consistency of Ct values across the three time points as an indicator of ETGA template DNA stability. An additional experiment was performed using LM containing UMO ETGA substrate to test whether the stability of the resulting ETGA template DNA differs from that of LM containing PTO ETGA substrate.

[0476] Materials and Methods

[0477] Reagents Used

[0478] Reagent A—5% (w/v) Saponin, 5% (v/v) Tween 20 and 146 mM Sodium chloride

[0479] Reagent B—5 mM Sodium hydroxide.

[0480] Reagent C—10 mM Ammonium sulphate, 2 mM Magnesium sulphate heptahydrate, 10 mM Potassium chloride and 20 mM Tris-HCl [pH 8.0].

[0481] Lysis Mix (LM) comprised of L1, L2, L3, dNTPs, PTO-IPC stock: [0482] L1 (252 mL in 360 mL LM)—1.46% (w/v) BSA, 0.15% (v/v) Triton X100 and 0.15% (v/v) Tween 20; [0483] L2 (36 mL in 360 mL LM)—100 mM Ammonium sulphate, 20 mM Magnesium sulphate heptahydrate, 100 mM Potassium chloride and 200 mM Tris-HCl [pH 8.0]; [0484] L3 (36 mL in 360 mL LM)—0.1 μM PTO-AS oligo, 0.1 μM PTO-S1 oligo, 20 mM Tris-HCl [pH 8.5], 10 mM Potassium chloride and 10 μM EDTA; [0485] 10 mM dNTPs (3.6 mL in 360 mL LM) [0486] PTO-IPC stock (˜180 μL in 360 mL LM) *Note: variable concentration [0487] H.sub.2O (˜32.22 mL in 360 mL LM)

[0488] Method 1: The Cognitor Minus Test on E. coli Spiked Blood Broth With and Without the 95° C. Step

[0489] Escherichia coli (ATCC® 25922™) was grown in nutrient broth for 18 hours at 37° C. BacT/ALERT SA blood broth (sheep blood) was inoculated to approximately 1×10.sup.7 cfu/mL, 1×10.sup.6 cfu/mL, 1×10.sup.5 cfu/mL, 1×10.sup.4 cfu/mL, and 1×10.sup.3 cfu/mL with E. coli. Two sets of 1 ml samples were prepared for testing with or without the 95° C. step. Total viable count (TVC) plates were prepared to confirm cfu/mL values. Two sets of blood broth only ‘no spike’ controls (NSCs), positive controls (broth only plus DNA polymerase) and negative controls (broth only) were also prepared giving a total of 16 samples (see Table 2).

[0490] To each 1 mL sample, 330 μL Reagent A was added and mixed by five tube inversions. Samples were incubated for 15 minutes at room temperature (approximately 19° C.) and then centrifuged for 3 minutes at 7300 RCF. Following centrifugation, supernatants were decanted into a clinical waste receptacle and the open tubes blotted on sterile tissue paper. Each pellet was resuspended in 750 μL Reagent B by tip mixing and incubated for 5 minutes at room temperature. Next, 500 μL Reagent C was added to each sample and mixed by three tube inversions. Samples were centrifuged for 3 minutes at 7300 RCF. The resulting supernatants were decanted into a clinical waste receptacle and the open tubes blotted on sterile tissue paper. Each pellet was resuspended in 500 μL Reagent C by tip mixing, transferred to a beadmill tube containing glass beads (0.1 mm and 0.5 mm glass beads), and centrifuged for 3 minutes at 7300 RCF. Following centrifugation, supernatants were transferred to waste by pipette. 50 μL LM was added to each sample and an additional 10 μL of DNA Polymerase solution was added to the positive control samples. Samples were then placed into a Disruptor Genie and run for 6 min at 2800 rpm. After bead milling, samples were transferred to a heat block set at 37° C. and incubated for 20 minutes.

[0491] Following the 37° C. microorganism lysis (ETGA) step, the 95° C. (−) samples (samples 9-16) were progressed immediately to QPCR setup, whilst the 95° C. (+) samples (samples 1-8) were incubated at 95° C. for 5 minutes prior to QPCR setup. Both sets of samples were analysed by QPCR immediately. The same samples were analysed by QPCR again after 2 hours at room temperature (approximately 19° C.), and again after a further 18 hours at 4° C. This experiment was replicated four times to allow statistical analysis of the results.

TABLE-US-00007 TABLE 2 Test Samples 95° C. (+) Samples 1. E. coli 1 × 10.sup.7 cfu/mL 2. E. coli 1 × 10.sup.6 cfu/mL 3. E. coli 1 × 10.sup.5 cfu/mL 4. E. coli 1 × 10.sup.4 cfu/mL 5. E. coli 1 × 10.sup.3 cfu/mL 6. Blood broth only (NSC) 7. Positive control (Pol + ve) 8. Negative control (Pol − ve) 95° C. (−) Samples 9. E. coli 1 × 10.sup.7 cfu/mL 10. E. coli 1 × 10.sup.6 cfu/mL 11. E. coli 1 × 10.sup.5 cfu/mL 12. E. coli 1 × 10.sup.4 cfu/mL 13. E. coli 1 × 10.sup.3 cfu/mL 14. Blood broth only (NSC) 15. Positive control (Pol + ve) 16. Broth only (Pol − ve) NSC: No spike control

[0492] Method 2: PTO ETGA Substrate vs UMO ETGA Substrate

[0493] BacT/ALERT blood broth SA was inoculated to approximately 1×10.sup.7 cfu/mL and 1×10.sup.4 cfu/mL with E. coli. Four sets of 1 ml samples were prepared to compare Cognitor Minus test results with and without the use of PTOs, with and without the 95° C. step. TVC plates were prepared to confirm cfu/mL values. NSCs and positive controls were also prepared (see Table 3). All samples were processed according to the general protocol described above in “Method 1”. Following the 37° C. microorganism lysis (ETGA) step, the 95° C. (−) samples (samples 9-16) were progressed immediately to QPCR setup, whilst the 95° C. (+) samples (samples 1-8) were incubated at 95° C. for 5 minutes before QPCR setup. Both sets of samples were analysed by QPCR immediately. The same samples were analysed by QPCR after 2 hours at room temperature, and again following a further 18 hours at 4° C.

TABLE-US-00008 TABLE 3 Test Samples 95° C. (+) Samples 1. E. coli 1 × 10.sup.7 cfu/mL: UMO LM 2. E. coli 1 × 10.sup.7 cfu/mL: PTO LM 3. E. coli 1 × 10.sup.4 cfu/mL: UMO LM 4. E. coli 1 × 10.sup.4 cfu/mL: PTO LM 5. Blood broth only (NSC): UMO LM 6. Blood broth only (NSC): PTO LM 7. Positive control (Pol + ve): UMO LM 8. Positive control (Pol + ve): PTO LM 95° C. (−) Samples 9. E. coli 1 × 10.sup.7 cfu/mL: UMO LM 10. E. coli 1 × 10.sup.7 cfu/mL: PTO LM 11. E. coli 1 × 10.sup.4 cfu/mL: UMO LM 12. E. coli 1 × 10.sup.4 cfu/mL: PTO LM 13. Blood broth only (NSC): UMO LM 14. Blood broth only (NSC): PTO LM 15. Positive control (Pol + ve): UMO LM 16. Positive control (Pol + ve): PTO LM NSC: no spike control, UMO: unmodified oligonucleotide, PTO: phosphorothioate oligonucleotide, LM: lysis mix

[0494] Results and Discussion

[0495] Results 1: Removal of the 95° C. Step Improves ETGA QPCR Signal Without a Reduction in Signal Over Time

[0496] The results for samples processed with or without the 95° C. step at time 0, 2 and 20 hours following sample preparation (n=3) are shown in FIGS. 9A-C. Within individual experiments, removal of the 95° C. step resulted in reduced Ct values (increased ETGA signal) for all E. coli spiked blood broth dilutions and positive controls (FIGS. 9A-C). The Ct values obtained for the same samples at time 0, 2 and 20 hours are highly consistent across the three replicate experiments with maximum ΔCt values ranging from 0.07 Ct units to 1.07 Ct units (average maximum ΔCt value of 0.30 Ct units) for 95° C. (+) samples and maximum ΔCt values ranging from 0.03 Ct units to 0.58 Ct units (average maximum ΔCt value of 0.38 Ct units) for 95° C. (−) samples. All NSCs and negative controls yielded Ct values greater than 40 or had no QPCR amplification at all (data not shown). FIG. 10 shows all of the data for E. coli spiked blood broth samples plotted together and several trends are apparent. Firstly, there is a clear difference between the Ct values obtained for 95° C. (+) samples compared to 95° C. (−) samples, with approximately a 1.0 Ct unit reduction in Ct values for 95° C. (−) samples. Secondly, there is very little difference between the Ct values obtained at different time points for both 95° C. (+) and 95° C. (−) samples. However, there is a small reduction in Ct value as storage time increases when the 95° C. step is removed, which is apparent from the trend lines in FIG. 10 and is more pronounced in experiment 3 (FIG. 9B).

[0497] Linear modelling was performed (using R) to determine whether there are statistically significant differences between the Ct values obtained for the E. coli spiked blood broth dilution series with or without the 95° C. step and at different time points. Table 4 shows the p-values obtained for different comparisons. Comparison of Ct values for 95° C. (+) samples with 95° C. (−) samples using data from all four experiments produced highly significant p-values (p<0.001) regardless of whether data from individual time points or data from all time points were included in the analysis. Comparison of Ct values for different time points within 95° C. (+) or 95° C. (−) datasets using data from all four experiments produced non-significant p-values (p>0.05). Time point comparisons within the same experiment produced non-significant p-values (p>0.05) for all datasets apart from the 95° C. (−) dataset in experiment 3 (T3), which had a p-value of 0.016. This significant p-value is likely to be due to the more pronounced reduction in Ct value with time that was observed in this particular experiment.

TABLE-US-00009 TABLE 4 Linear Models (using R) to compare Ct value standard curves for the E. coli spiked blood culture dilution series at different time points and with or without the 95° C. step. Data analysed Comparison P-value Significance All data 95° C. (+) vs  .sup. <2 × 10.sup.−16 *** 95° C. (−) Time 0 H: T1-T4 data 95° C. (+) vs 4.82 × 10.sup.−8 *** 95° C. (−) Time 2 H: T2-T4 data 95° C. (+) vs 1.31 × 10.sup.−8 *** 95° C. (−) Time 20 H: T2-T4 data 95° C. (+) vs .sup. 1.47 × 10.sup.−10 *** 95° C. (−) 95° C. (+): T1-T4 data Time 0.730 — 95° C. (−): T1-T4 data Time 0.094 — 95° C. (+): T2 data only Time 0.896 — 95° C. (−): T2 data only Time 0.657 — 95° C. (+): T3 data only Time 0.956 — 95° C. (−): T3 data only Time 0.016 * 95° C. (+): T4 data only Time 0.769 — 95° C. (−): T4 data only Time 0.061 — Significance codes: * p < 0.05, ** p < 0.01, *** p < 0.001. —: not significant. T: experiment.

[0498] Results 2: Cognitor Minus Results are More Consistent Across Different Time Points When Using PTO LM Rather Than UMO LM

[0499] FIGS. 11A-B shows the Ct values obtained for E. coli spiked blood broth samples (1×10.sup.7 cfu/mL and 1×10.sup.4 cfu/mL) and positive controls processed using either UMO LM (FIG. 11A) or PTO LM (FIG. 11B) with and without the 95° C. step. The data shown here is from a single experiment. The Ct values for NSCs using UMO LM were all at least 5 Ct units higher than the Ct values obtained for 1×10.sup.3 cfu/mL E. coli spiked blood broth samples, whilst the PTO LM NSC Ct values were all greater than 42.0 Ct units or had no QPCR amplification at all (data not shown). The results demonstrate that the Ct values for UMO LM samples are on average 10.2 Ct units lower than the Ct values for PTO LM samples. There is also a greater reduction in Ct value for UMO LM samples than there is for PTO LM samples when the 95° C. step is removed. Most importantly, in relation to ETGA template DNA stability, the Ct values obtained for UMO LM samples are notably less consistent across the different time points than the Ct values for PTO LM samples. The Ct values for E. coli spiked blood broth samples in the UMO LM 95° C. (+) dataset increased by approximately 1.0 Ct unit from 2 hours to 20 hours, whilst the corresponding positive control showed a 0.24 Ct unit reduction in Ct value. This indicates that nuclease digestion of ETGA template DNA may be occurring in samples when bacteria and/or host blood cells are present despite the protein denaturing effect of the 95° C. step, but not in the positive control were only DNA polymerase enzyme is added. When the 95° C. step is removed, the Ct values for UMO LM samples decrease with increased sample storage time. This indicates that in the absence of protein denaturation, continued ETGA template generation may out compete any increase in nuclease digestion, hence resulting in increased ETGA QPCR signal. The PTO LM data (FIG. 11B) demonstrate highly consistent Ct values across all time points for both 95° C. (+) and 95° C. (−) samples. These data indicate that ETGA template DNA formed from PTO substrate DNA is more resistant to change by either nuclease degradation and/or additional ETGA template DNA generation following the 37° C. ETGA reaction.

SUMMARY

[0500] The data shown here demonstrate that ETGA template DNA detection is improved when the 95° C. step is removed. Furthermore, ETGA QPCR signal does not deteriorate with increased sample storage time in the absence of the 95° C. step. Comparison of PTO LM with UMO LM indicates that the high stability of ETGA template DNA is dependant of the use of PTO ETGA substrate DNA. All of the data shown here support removal of the 95° C. step from the Cognitor Minus test. It is worth noting that the increased sensitivity of the test may increase the chance of detecting background signal (blood-derived ETGA signal), however, optimisation of other factors such as blood lysis/wash performance and interpretation of QPCR results should eliminate the impact of this.

EXAMPLE 4

Analysis of the Importance of the 95° C. and the Use of a PTO Substrate: Primary Panel Microorganisms

[0501] Purpose

[0502] The purpose of the work presented in this report was to: [0503] 1. Compare Cognitor Minus results with and without the 95° C. step for all primary panel microorganisms (E. coli; S. aureus; and C. albicans) [0504] 2. Confirm the findings of Example 3 with regard to comparison between phosphorothioate modified oligonucleotide (PTO) LM and unmodified oligonucleotide (UMO) LM [0505] 3. Test the effect of extended sample storage duration (up to 72 hours) on QPCR results for each primary panel microorganism using PTO LM and UMO LM

[0506] Introduction

[0507] The earlier steps in the Cognitor Minus test aim to lyse blood cells and wash away blood-derived proteins such as DNA polymerases, which will produce non-microorganism derived ETGA template DNA, and nuclease enzymes which may digest microorganism-derived ETGA template DNA. This process should not harm any microorganisms that are present in the blood sample. Isolated intact microorganisms are then lysed by the addition of lysis mix (LM) and bead milling. After microorganism lysis and the ETGA reaction, samples contain a mixture of microorganism proteins, LM components, newly synthesised ETGA template DNA and residual blood cell proteins. In the current Cognitor Minus test protocol, the 95° C. step is intended to denature all proteins in order to protect ETGA template DNA and internal process control (IPC) DNA from nuclease digestion so that it can be successfully detected by QPCR. The 95° C. step also inactivates DNA polymerases, thereby quenching the ETGA reaction. However, since incorporating the 95° C. step into the protocol, the UMOs used to form the ETGA substrate (and IPC) in the LM have been replaced with PTOs which are nuclease resistant. Whilst the ETGA extension strand that forms the ETGA template is constructed from standard dNTPs, the PTO substrate DNA that it is annealed to may confer protection against nuclease digestion. Due to the benefits of removing the 95° C. step, such as protocol simplification and a reduction in the time required to run the test, it was deemed important to re-evaluate the necessity of the 95° C. step.

[0508] To assess the importance of the 95° C. step, the Cognitor Minus test was carried out using microorganism-spiked blood broth with or without the 95° C. step. Samples were analysed by QPCR at five time points: immediately after sample preparation; after 2 hours stored at room temperature (approximately 19° C.); and after 24 hours, 48 hours and 72 hours stored at 4° C. The Ct values obtained and consistency of Ct values across the five time points were used to assess the effect of the 95° C. step on Cognitor Minus test performance and sample stability for each of the primary panel microorganisms using either PTO LM or UMO LM.

[0509] Materials and Methods

[0510] For details of reagents, see Example 3.

[0511] Escherichia coli (ATCC® 25922™), Staphylococcus aureus (ATCC® 25923™) and Candida albicans (ATCC® 10231™) were grown in liquid media (E. coli and S. aureus in nutrient broth; and C. albicans in Sabouraud media) for approximately 18 hours at 37° C. BacT/ALERT SA blood broth (sheep blood; see Table 6) was inoculated with E. coli, S. aureus and C. albicans to approximately 1×10.sup.4 cfu/mL, 1×10.sup.4 cfu/mL and 1×10.sup.5 cfu/mL respectively. Four sets of 1 ml samples were prepared for testing with or without the 95° C. step for PTO LM and UMO LM. Blood broth only ‘no spike’ controls (NSCs) and positive controls (broth only plus DNA polymerase (PC)) were also prepared giving a total of 20 samples (see Table 5). Total viable count (TVC) plates were prepared to confirm cfu/mL values (see Table 7) and negative blood broth.

[0512] To each 1 mL sample, 330 μL Reagent A was added and mixed by five tube inversions. Samples were incubated for 15 minutes at room temperature (approximately 19° C.) and then centrifuged for 3 minutes at 7300 RCF. Following centrifugation, supernatants were decanted into a clinical waste receptacle and the open tubes blotted on sterile tissue paper. Each pellet was resuspended in 750 μL Reagent B by tip mixing and incubated for 5 minutes at room temperature. Next, 500 μL Reagent C was added to each sample and mixed by three tube inversions. Samples were centrifuged for 3 minutes at 7300 RCF. The resulting supernatants were decanted into a clinical waste receptacle and the open tubes blotted on sterile tissue paper. Each pellet was resuspended in 500 μL Reagent C by tip mixing, transferred to a beadmill tube containing glass beads (0.1 mm and 0.5 mm glass beads), and centrifuged for 3 minutes at 7300 RCF. Following centrifugation, supernatants were transferred to waste by pipette. 50 μL LM was added to each sample and an additional 10 μL of DNA Polymerase solution was added to the positive control samples. Samples were then placed into a Disruptor Genie and run for 6 min at 2800 rpm. After bead milling, samples were transferred to a heat block set at 37° C. and incubated for 20 minutes.

[0513] Following the 37° C. microorganism lysis (ETGA) step, the 95° C. (−) samples (samples 11-20) were progressed immediately to QPCR setup, whilst the 95° C. (+) samples (samples 1-10) were incubated at 95° C. for 5 minutes prior to QPCR setup. Both sets of samples were analysed by QPCR immediately. The same samples were analysed by QPCR again after 2 hours at room temperature, and again after 24 hours, 48 hours and 72 hours at stored at 4° C. This experiment was replicated three times to allow for statistical analysis of the results.

TABLE-US-00010 TABLE 5 Test Samples 95° C. (+) Samples 1. E. coli 1 × 10.sup.4 cfu/mL: PTO LM 2. S. aureus 1 × 10.sup.4 cfu/mL: PTO LM 3. C. albicans 1 × 10.sup.5 cfu/mL: PTO LM 4. PC: PTO LM 5. NSC: PTO LM 6. E. coli 1 × 10.sup.4 cfu/mL: UMO LM 7. S. aureus 1 × 10.sup.4 cfu/mL: UMO LM 8. C. albicans 1 × 10.sup.5 cfu/mL: UMO LM 9. PC: UMO LM 10. NSC: UMO LM 95° C. (−) Samples 11. E. coli 1 × 10.sup.4 cfu/mL: PTO LM 12. S. aureus 1 × 10.sup.4 cfu/mL: PTO LM 13. C. albicans 1 × 10.sup.5 cfu/mL: PTO LM 14. PC: PTO LM 15. NSC: PTO LM 16. E. coli 1 × 10.sup.4 cfu/mL: UMO LM 17. S. aureus 1 × 10.sup.4 cfu/mL: UMO LM 18. C. albicans 1 × 10.sup.5 cfu/mL: UMO LM 19. PC: UMO LM 20. NSC: UMO LM PC: positive control, NSC: no spike control, UMO: unmodified oligonucleotide, PTO: phosphorothioate oligonucleotide, LM: lysis mix

TABLE-US-00011 TABLE 6 Materials Batch/Lot Reagent Supplier number Expiry date Sheep Blood TCS Bioscience 30112000 27 Apr. 2015 (Replicate 1) Sheep Blood TCS Bioscience 30210900 25 May 2015 (Replicate 2) Sheep Blood TCS Bioscience 30255300 8 Jun. 2015 (Replicate 3)

TABLE-US-00012 TABLE 7 Microorganism TVCs Exp. 1 TVC Exp. 2 TVC Exp. 3 TVC Microorganism (cfu total) (cfu total) (cfu total) E. coli 7,200 10,200 28,200 S. aureus 4,200 23,000 23,200 C. albicans 271,000 191,000 246,000

[0514] Results and Discussion

[0515] The results for samples processed using either PTO LM or UMO LM with or without the 95° C. step at time 0, 2, 24, 48 and 72 hours (n=3) are shown in FIGS. 12A-E.

[0516] Removal of the 95° C. Step Improves ETGA QPCR Signal

[0517] All microorganism-spiked blood broth samples and positive control samples processed without the 95° C. step produced lower Ct values (stronger ETGA signal) than corresponding samples processed with the 95° C. step, for both PTO LM and UMO LM. The average ΔCt values (95° C. (+) subtract 95° C. (−)) for PTO LM samples at ‘0 hours’ were 0.98 Ct units, 1.41 Ct units, 0.12 Ct units and 1.21 Ct units for E. coli, S. aureus and C. albicans and positive control respectively. The average ΔCt values (95° C. (+) subtract 95° C. (−)) for UMO LM samples at ‘0 hours’ were 3.91 Ct units, 4.10 Ct units, 5.36 Ct units and 1.20 Ct units for E. coli, S. aureus and C. albicans and positive control respectively. The majority of NSC samples processed with PTO LM did not produce Ct values due to low QPCR amplification, and therefore this data is not shown in FIGS. 12A-E. However, the incidence of sufficient amplification for Ct values was higher for 95° C. (−) samples than 95° C. (+) samples (7/15 Ct values compared to 2/15 Ct values with no obvious trends for storage duration; and all NSC PTO LM Ct values were between 42.0 Ct units and 45.0 Ct units). NSC samples processed with UMO LM produced Ct values at ‘0 hours’ that were on average 0.42 Ct units lower without the 95° C. step. This increase in QPCR signal for NSC samples processed without the 95° C. step is expected given the general increase in signal observed for positive samples.

[0518] Cognitor Minus Results are More Consistent Across Different Time Points When Using PTO LM Rather Than UMO LM

[0519] Within the PTO LM dataset, all microorganism-spiked blood broth samples and positive control samples produced highly consistent Ct values across the 72 hour storage period, regardless of whether samples were processed with the 95° C. step. Within the UMO LM 95° C. (+) dataset, E. coli, S. aureus and positive control samples produced fairly consistent Ct values across the 72-hour storage period, whereas C. albicans and NSC samples showed an increase in Ct values over time (reduction in ETGA signal). This increase in Ct values for C. albicans and NSC samples may be due to a greater impact of nuclease activity on ETGA template DNA concentration when the starting concentration is lower, as indicated by higher Ct values for these samples at ‘0 hours’.

[0520] UMO LM 95° C. (−) samples generally showed a decrease in Ct value (increase in ETGA QPCR signal) over time, except for C. albicans and NSC samples were Ct values were highly consistent across the 72-hour period. This indicates that for E. coli, S. aureus and positive control samples processed without the 95° C. step (protein denaturation), continuation of the ETGA reaction results in increased QPCR signal, out competing any nuclease activity. Whereas, in C. albicans and NSC samples, where the effect of nuclease degradation seems to be more pronounced, the continued ETGA reaction may be counteracting this degradation to provide more stable QPCR signal over time.

[0521] Statistical Analysis of Results

[0522] Linear modelling was performed (using R) to determine whether there are statistically significant differences between the Ct values obtained for PTO LM and UMO LM samples processed with or without the 95° C. step at different time points. For each dataset (e.g. E. coli with PTO LM dataset), ‘Ct’ was modelled against the following explanatory variables and their interactions: ‘Log10 cfu’, ‘Time’, and ‘95° C. step’. Non-significant (p>0.05) interactions and variables were removed from the model in a stepwise manner, resulting in model simplification. However, non-significant variables were not removed from the model if any of their interactions were significant (p<0.05). Therefore, the final model for each dataset contained only significant interactions, significant variables, and non-significant variables that form significant interactions. Significance codes for each variable and interaction are shown in Table 8 (significance codes are based on p-values for each interaction or variable at the point of model simplification, if removed from the model, or from the final model).

TABLE-US-00013 TABLE 8 Significance codes for p-values of each explanatory variable and their interactions using linear modelling in R Log10 Log10 Log10 cfu: cfu: Time: Log10 cfu: Dataset cfu Time 95° C. Time 95° C. 95° C. Time: 95° C. E. coli PTO LM *** NS *** NS ~ NS NS E. coli UMO LM *** ** *** NS NS * NS S. aureus PTO LM *** NS * NS ** NS NS S. aureus UMO LM *** *** *** NS *** ** NS C. albicans PTO LM * NS *** NS ** NS NS C. albicans UMO LM ~ NS ** NS ** NS NS PC PTO LM ~ *** NS PC ~ *** ~ UMO LM NSC UMO LM NS NS *** Significance codes: *** p < 0.001, ** p < 0.01, * p < 0.05, ~ p < 0.1, NS—not significant.

[0523] The 95° C. step has a significant effect on Ct value for all microorganisms and positive controls with PTO LM and UMO LM. Sample storage duration (‘Time’ and ‘Time’ interactions) has no significant effect on Ct value for any microorganism or positive control with PTO LM; and is also non-significant for C. albicans and the positive control with UMO LM. However, sample storage duration is a significant variable for E. coli and S. aureus with UMO LM: most likely due to the observed reduction in Ct value over time for samples processed without the 95° C. step in these sample sets. There is no significant effect of the 95° C. step for the NSC UMO LM dataset; but there is a significant interaction between sample storage duration and the 95° C. step (Time:95° C′). Statistical analysis could not be performed on the NSC PTO LM dataset due to missing Ct values as a result of low QPCR amplification.

[0524] Summary

[0525] The data shown here demonstrate that ETGA template DNA detection is improved when the 95° C. step is removed. Furthermore, ETGA QPCR signal does not deteriorate with increased sample storage duration in the absence of the 95° C. step when samples are processed using PTO ETGA substrate DNA. ETGA signal is not as stable when samples are processed using UMO ETGA substrate DNA: in the absence of the 95° C. step ETGA QPCR signal continues to increase with sample storage duration; and with the 95° C. step ETGA QPCR signal is more likely to deteriorate as a result of nuclease degradation without continued production of ETGA template DNA. These results are consistent with the results presented in Example 3.

[0526] All of the data shown here support removal of the 95° C. step from the Cognitor Minus test, and confirm the importance of using PTO ETGA substrate DNA in the LM. These results also verify that samples processed using PTO LM can be stored for up to 72 hours at 4° C., without being detrimental to test results. It is worth noting that the increased sensitivity of the test upon removal of the 95° C. step may increase the chance of detecting background signal (blood-derived ETGA QPCR signal). However, whilst NSC PTO LM samples did not provide a complete set of Ct values for comparison (due to low amplification), the NSC UMO LM dataset demonstrates that the increase in ETGA QPCR signal associated with removal of the 95° C. is lower for NSCs than it is for positive samples. Furthermore, optimisation of other factors such as blood lysis/wash performance and interpretation of QPCR results should eliminate the impact of this.

[0527] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. Moreover, all embodiments described herein are considered to be broadly applicable and combinable with any and all other consistent embodiments, as appropriate.

[0528] Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties.