METHODS AND KITS

Abstract

A method of detection and identification of one or more microorganism/s in a biological sample comprising the following steps: (a) extracting DNA from the microorganism/s; and (b) amplifying the extracted DNA and indicating the level of extracted DNA in a quantitative PCR; wherein the quantitative PCR is performed using the primer pair of SEQ ID NO.1 and 2 together with the probe of SEQ ID NO. 7, and/or the primer pair of SEQ ID NO. 3 and 4 together with the probe of SEQ ID NO.8.

Claims

1. A method of detection and identification of one or more microorganism/s in a biological sample comprising the following steps: (a) extracting DNA from the microorganism/s; and (b) amplifying the extracted DNA and indicating the level of extracted DNA in a quantitative PCR; wherein the quantitative PCR is performed using the primer pair of SEQ ID NO.1 and 2 together with the probe of SEQ ID NO. 7, the primer pair of SEQ ID NO. 3 and 4 together with the probe of SEQ ID NO.8 or both.

2. A method of detection and identification of one or more microorganism/s in a biological sample comprising the following steps: (a) extracting DNA from the microorganism/s by contacting the microorganism/s with an extraction solution comprising a quaternary ammonium compound including a silicon containing functional group; and (b) amplifying the extracted DNA and indicating the level of extracted DNA in a quantitative PCR.

3. A method according to claim 1 wherein step (a) comprises: extracting DNA from the microorganism/s by contacting the microorganism/s with an extraction solution comprising a quaternary ammonium compound including a silicon containing functional group.

4. A method according to claim 3 wherein step (a) involves contacting the sample with an extraction solution comprising a compound of general formula (I): ##STR00004## or a derivative salt thereof wherein L is a linking group; each of R.sup.2, R.sup.3, R.sup.4, R.sup.5 and R.sup.6 is independently selected from H or an optionally substituted alkyl, alkenyl, aryl or alkoxy group; and n is 0 or 1.

5. A method according to claim 4 wherein step (a) involves contacting the microorganism/s with a compound of formula (II): ##STR00005##

6. A method according to claim 3 wherein the extraction solution further comprises a solubilising agent.

7. A method according to claim 6 wherein the solubilising agent is an alkyl polyglucoside.

8. A method according to claim 3 which includes a step (a2) between step (a) and step (b) of contacting the material obtained in step (a) with a proteinaceous washing agent.

9. A method according to claim 8 wherein the proteinaceous washing agent is bovine serum albumin or acetylated bovine serum albumin.

10. A method according to claim 1 wherein step (b) comprises the addition of at least one reagent for performing the quantitative PCR.

11. A method according to claim 10 wherein the reagent is selected from DNA polymerase, buffer, dNTPs, or a source of Magnesium.

12-15. (canceled)

16. A kit for the detection and identification of one or more microorganism/s in a biological sample, the kit comprising: (a) means for extracting DNA from the microorganism/s; (b) at least one reagent for performing PCR; and (c) one or more of the primer pair of SEQ ID NO.1 and 2 and/or the primer pair of SEQ ID NO.3 and 4; (d) means for visualising PCR products.

17. A pair of single stranded oligonucleotide primers and a probe for quantitative PCR detection of Plasmodium sp. DNA in a biological sample selected from: (a) the forward primer comprising the sequence identified in SEQ ID NO.1, the reverse primer comprising the sequence identified in SEQ ID NO.2 and the probe comprising the sequence identified in SEQ ID NO. 7; (b) the forward primer comprising the sequence identified in SEQ ID NO.3, reverse primer comprising the sequence identified in SEQ ID NO.4 and the probe comprising the sequence identified in SEQ ID NO.8, and (c) the forward primer comprising the sequence identified in SEQ ID NO.5, the reverse primer comprising the sequence identified in SEQ ID NO.6, and the probe comprising the sequence identified in SEQ ID NO.9.

18-21. (canceled)

22. The kit according to claim 16, wherein the means for visualizing PCR products comprises the probe of SEQ ID NO 7, SEQ ID NO: 8, or both.

23. The kit according to claim 16, wherein the means for extracting DNA from the microorganism/s comprises an extraction solution comprising a quaternary ammonium compound including a silicon containing functional group.

Description

[0145] The present invention will now be further described with reference to the following non-limiting examples and figures in which:

[0146] FIG. 1 shows the quantitative PCR results from a clinical blood sample indicating the presence of Protozoan Plasmodium sp. DNA;

[0147] FIG. 2 shows the quantitative PCR results from a clinical blood sample indicating the presence of human RNAse-P DNA (internal control for the process of extraction and subsequent amplification for detection);

[0148] FIG. 3 shows the quantitative PCR results from a clinical blood sample indicating the presence of bacterial E. coli DNA;

[0149] FIG. 4 shows the quantitative PCR results from a clinical blood sample indicating the combined presence of the human RNAse-P control DNA and Protozoan plasmodium sp. DNA

EXAMPLES

[0150] The following examples demonstrate the amplification and identification of microorganism DNA extracted from biological samples together with the use of a human gene control. In each case, the extraction of DNA takes place by using the extraction solution described above. The supernatant isolated from the extraction is fully PCR compliant (either with end point or real time -qPCR-) and was used directly in the PCR methods described below.

Example I Detection and Identification of Protozoan Plasmodium sp. DNA Through qPCR

[0151] FIG. 1 provided for this example was obtained by qPCR using the TaqMan® Universal PCR Master Mix (Applied Biosystems), containing HotStar Taq DNA polymerase, MgCl2 and dNTP's.

[0152] Plasmodium sp. detection and identification required the use of the following oligonucleotide primers for protozoan target amplification during qPCR:

TABLE-US-00004 Primer Protozoan Forward: 5′-GTTAAGGGAGTGAAGACGATCAGA-3′ (SEQ ID NO. 1) Primer Protozoan Reverse: 5′AACCCAAAGACTTTGATTTCTCATAA-3′ (SEQ ID NO. 2)

[0153] Plus the following probe for real time amplification on any qPCR system:

TABLE-US-00005 Probe Plasmodium: (SEQ ID NO. 7) 5′-FAM-ACCGTCGTAATCTTAACCATAAACTATGCCGACTAG-TAMRA- 3′

[0154] The protozoan detection and identification described here in combination with the extraction step complies with the use of any standard PCR mastermix. Using the TaqMan® Universal PCR Master Mix (Applied Biosystems), the following mix was used for the generation of this example:

TABLE-US-00006 PCR Master Mix: 12.5 μl Primer Protozoan F: 0.25 μl Primer Protozoan R: 0.25 μl {close oversize brace} Mixture volume: 20 μl Plasmodium sp. probe: 1.25 μl H.sub.2O: 5.5 μl

[0155] Likewise, the following qPCR conditions were set:

TABLE-US-00007 Mix activation 50° C. 2 min Initial denaturation 95° C. 10 min Denaturation 95° C. 15 sec {close oversize brace} 45 cycles Annealing 60° C. 60 sec

[0156] FIG. 1 shows the increased fluorescence of the Plasmodium probe during qPCR with the extracted DNA from the sample. Thereby indicating the presence of Plasmodium in the blood sample.

Example II Detection and Identification of Human Control DNA Through qPCR

[0157] FIG. 2 provided for this example was obtained using the TagMan® Universal PCR Master Mix (Applied Biosystems), containing HotStar Taq DNA polymerase, MgCl2 and dNTP's. Human RNAse-P detection and identification requires the use of the following oligonucleotide primers for human target amplification:

TABLE-US-00008 Control Primer Forward: 5′-AGATTTGGACCTGCGAGCG-3′ (SEQ ID NO. 5) Control Primer Reverse: 5′-GAGCGGCTGTCTCCACAAGT-3′ (SEQ ID NO. 6)

[0158] Plus the following probe for real time amplification on any qPCR system:

TABLE-US-00009 Probe Human: (SEQ ID NO. 9) 5′-FAM/VIC-TTCTGACCTGAAGGCTCTGCGCG-BHQ1-3′

[0159] The human RNAse-P detection and identification described here in combination with the extraction step complies with the use of any standard PCR mastermix. Using the TagMan® Universal PCR Master Mix (Applied Biosystems), the following mix was used for the generation of this example:

TABLE-US-00010 PCR Master Mix: 12.5 μl Control Primer F: 0.5 μl Control Primer R: 0.5 μl {close oversize brace} Mixture volume: 20 μl Human Probe: 0.5 μl H.sub.2O: 6.0 μl

[0160] Likewise, the following qPCR conditions were set:

TABLE-US-00011 Mix activation 50° C. 2 min Initial denaturation 95° C. 10 min Denaturation 95° C. 15 sec {close oversize brace} 45 cycles Annealing 60° C. 60 sec

[0161] FIG. 2 shows the increased fluorescence of the Human probe during qPCR with the extracted DNA from the sample. Thereby indicating the presence of human RNAse P DNA in the blood sample. This verifies that the sample is human and that the qPCR system is working correctly.

Example III Detection and Identification of Bacterial E. coli DNA Through End Point PCR

[0162] This example uses the same components and set up as required for detection of bacterial targets through qPCR, but products resulting from amplification the extracted DNA by PCR were visualised on agarose gel to show compliance with end point PCR detection. This example used a qPCR approach to generate products of amplification that were fit for end point analysis on agarose gel.

[0163] FIG. 3 provided for this example was obtained using Promega® Universal PCR Master Mix, REF M7502. DNA polymerase, MgCl2 and dNTP's are contained premixed.

[0164] Detection and identification of E. coli DNA requires the use of the following oligonucleotide primers for bacterial target amplification:

TABLE-US-00012 Bacterial Primer F: EcoF 5′-GGAACTGGTGCCGGAACGC-3′ (SEQ ID NO. 3) Bacterial Primer R: 5′-GACTTCGATCAGTTTGACG-3′ (SEQ ID NO. 4)

[0165] Plus the following probe for real time amplification on any qPCR* system:

TABLE-US-00013 Probe E.coli: (SEQ ID NO. 8) 5′-FAM-CGTATCACTGCGCGCCACATTCG-TAMRA-3′

[0166] The E. coli detection and identification described here in combination with extraction step complies with the use of any standard PCR mastermix. Using the components described above, the following mix was used for the generation of this example (*the probe was not added, since it is necessary for qPCR detection and this example aimed to prove end point PCR compliance, making this element redundant) However, this component would be added as per examples 1 and 2 if real time qPCR were being conducted:

TABLE-US-00014 PCR Master Mix: 12.5 μl Primers (each one): 0.5 μl {close oversize brace} Mixture volume: 20 μl H.sub.2O: 6.5 μl

[0167] Likewise, the following qPCR conditions were set:

[0168] Initial denaturation 94° C. 5 min

TABLE-US-00015 Denaturation 94° C. 20 sec Annealing 54° C. 20 sec {close oversize brace} 30 cycles Extension 72° C. 30 sec Final extension 72° C. 5 min Cooling 10° C. ∞

[0169] FIG. 3 shows an agarose gel obtained from a 1% dilution of low electro-endosmosis agarose in TAE buffer as is known in the art. The gel was subjected to the classical electrophoresis field to separate the PCR products into size order, the PCR products are then visualised under a UV light. PCR products corresponding to amplification of the E. coli target are shown at the bottom of all lanes except the last lane on the left next to the molecular weight marker. This lane corresponds to amplification of the same target from the same blood sample subjected to a full Qiagen® purification process as positive control.

Example IV Parallel Detection and Identification of Human Control DNA and Protozoan Plasmodium sp. DNA Through qPCR

[0170] FIG. 4 provided for this example was obtained using the TaqMan® Universal PCR Master Mix (Applied Biosystems), containing HotStar Taq DNA polymerase, MgCl2 and dNTP's. Parallel detection and identification of human RNAse-P gene and plasmodium sp. targets requires the use of the following oligonucleotide primers for combined human control and protozoan target amplification:

TABLE-US-00016 Primer Protozoan Forward: 5′-GTTAAGGGAGTGAAGACGATCAGA-3′ (SEQ ID NO. 1) Primer Protozoan Reverse: 5′AACCCAAAGACTTTGATTTCTCATAA-3′ (SEQ ID NO. 2) Control Primer Forward: 5′-AGA TTTGGACCTGCGAGCG-3′ (SEQ ID NO. 5) Control Primer Reverse: 5′-GAGCGGCTGTCTCCACAAGT-3′ (SEQ ID NO. 6)

[0171] Plus the following probes for real time amplification on any qPCR system:

TABLE-US-00017 Probe Plasmodium: (SEQ ID NO. 7) 5′-FAM-ACCGTCGTAATCTTAACCATAAACTATGCCGACTAG- TAMRA-3′ Probe Human: (SEQ ID NO. 9) 5′-FAM/VIC-TTCTGACCTGAAGGCTCTGCGCG-BHQ1-3′

[0172] The combined human control and protozoan detection and identification described here in combination with the extraction step complies with the use of any standard PCR mastermix. Using the TaqMan® Universal PCR Master Mix (Applied Biosystems), the following mix was used for the generation of this example:

TABLE-US-00018 PCR Master Mix: 12.5 μl ControlPrimer F: 0.5 μl Control Primer R: 0.5 μl Probe human: 0.5 μl Primer FProtozoa.: 0.25 μl {close oversize brace} Mixture volume: 20 μl Primer RProtozoa.: 0.25 μl probe plasmodium: 1.25 μl H.sub.2O: 4.25 μl

[0173] Likewise, the following qPCR conditions were set:

TABLE-US-00019 Mix activation 50° C. 2 min Initial denaturation 95° C. 10 min Denaturation 95° C. 15 sec {close oversize brace} 45 cycles Annealing 60° C. 60 sec

[0174] FIG. 4 shows the increased fluorescence of the Human and plasmodium sp. probes during qPCR with the extracted DNA from the sample. Thereby indicating the presence of human RNAse P DNA and Plasmodium DNA in the blood sample. This verifies that the sample is human and that the qPCR system is working correctly whilst also identifying the microorganism contained in the sample.