High-sensitivity real-time polymerase chain reaction for detection of nucleic acids

• US 20030219788A1
• Filed: 02/11/2003
• Published: 11/27/2003
• Est. Priority Date: 02/11/2002
• Status: Active Grant

• Current Assignee: Auburn University
• Original Assignee: Auburn University
• Inventors: Kaltenboeck, Bernhard
• Granted Patent: US 7,252,937 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

The invention features methods based on PCR that are capable of detecting single target molecules in a sample input volume of, e.g., 5 μl, and of quantifying organismal, e.g., chlamydial, DNA with high accuracy. Desirably, these methods employ a single tube format coupled with real-time fluorescent detection of amplicons. This approach facilitates the application of quantitative PCR (qPCR) to microbiological diagnosis in clinical settings. The invention also features primers and probes for the detection of Chlamidia. The use of specific hybridization probes with qPCR amplification provides the ability for identification of individual species or strains of microorganisms.

Claims

1. A method of determining an amount of a target nucleic acid in a sample, said method comprising the steps of:

a) providing said sample;

b) contacting an aliquot of said sample with a solid support, wherein nucleic acid in said aliquot binds to said solid support;

c) eluting said nucleic acid bound to said solid support with at least two sequential volumes of 5-25 μ

l to produce a concentrated nucleic acid sample;

d) contacting said concentrated nucleic acid sample of step (c) with oligonucleotide primers capable of amplifying said target nucleic acid under appropriate conditions, a first probe labeled with a donor fluorophore, and a second probe labeled with an acceptor fluorophore to produce an amplification sample, wherein said first and second probes hybridize proximally to one another on said target nucleic acid, and wherein energy transferred from said donor fluorophore induces fluorescence from said acceptor fluorophore; and

e) performing an amplification on said amplification sample to produce an amplification product, wherein said amplification comprises a series of step-down cycles and a series of fluorescence acquisition cycles, wherein each of said fluorescence acquisition cycles comprises detection of fluorescence from said acceptor fluorophore,

wherein the kinetics of an increase in intensity of fluorescence from said acceptor fluorophore are indicative of the amount of said target nucleic acid in said sample.

7. The method of any of claims 1-6, wherein said solid support comprises glass fiber fleece.

8. The method of any of claims 1-6, wherein said series of step-down cycles comprises six cycles having a first annealing temperature of the melting temperature of said primers, nine cycles having a second annealing temperature of 2°

C. lower than said first annealing temperature, and three cycles having a third annealing temperature of 4°

C. lower than said first annealing temperature.

9. The method of any of claims 1-6, wherein said series of fluorescence acquisition cycles comprises six or more cycles having an annealing temperature lower than the melting temperature of said probes, wherein in each cycle, fluorescence detection of said acceptor fluorophore occurs under conditions in which said first and second probes hybridize to said target nucleic acid.

10. The method of any of claims 1-6, wherein said donor fluorophore comprises fluorescein or carboxyfluorescein and said acceptor fluorophore comprises BODIPY®

630/650, Cy5.5, LIGHTCYCLER®

Red-640, or LIGHTCYCLER®

Red-705.

11. The method of any of claims 1-6, further comprising the step of contacting said concentrated nucleic acid sample with reverse transcriptase under conditions appropriate to reverse transcribe any RNA in said concentrated nucleic acid sample before said amplification.

12. The method of claim 11, wherein said reverse transcriptase is stable at ≦

65°

C.

13. The method of claim 11, wherein prior to reverse transcription, said nucleic acid sample is incubated at between 65°

C. and 80°

C.

14. The method of any claims 1-6, wherein said target nucleic acid is present in a single genome in two or more copies.

15. The method of any of claims 1-6, wherein said target nucleic acid is DNA from Chlamydia.

16. The method of claim 15, wherein said DNA from Chlamydia comprises a 23S rRNA gene.

17. The method of any of claims 1-6, wherein said primers are genus-specific.

18. The method of any of claims 1-6, wherein one of said first or second probes is genus-specific and the other of said probes is species- or strain-specific.

19. The method of any of claims 1-6, wherein said amplification occurs in a glass capillary.

2. A method of determining an amount of a target nucleic acid in a sample, said method comprising the steps of:

a) providing said sample;

b) contacting an aliquot of said sample with a solid support, wherein nucleic acid in said aliquot binds to said solid support;

c) eluting said nucleic acid bound to said solid support in step (b) to produce a volume of eluate and reducing said volume by centrifugal ultrafiltration to produce a concentrated nucleic acid sample;

d) contacting said concentrated nucleic acid sample of step (c) with oligonucleotide primers capable of amplifying said target nucleic acid under appropriate conditions, a first probe labeled with a donor fluorophore, and a second probe labeled with an acceptor fluorophore to produce an amplification sample, wherein said first and second probes hybridize proximally to one another on said target nucleic acid, and wherein energy transferred from said donor fluorophore induces fluorescence from said acceptor fluorophore; and

e) performing an amplification on said amplification sample to produce an amplification product, wherein said amplification comprises a series of step-down cycles and a series of fluorescence acquisition cycles, wherein each of said fluorescence acquisition cycles comprises detection of fluorescence from said acceptor fluorophore,

wherein the kinetics of an increase intensity of fluorescence from said acceptor fluorophore are indicative of the amount of said target nucleic acid in said sample.

3. A method of determining an amount of a target nucleic acid in a sample, said method comprising the steps of:

a) providing a sample;

b) contacting a plurality of aliquots of said sample with a plurality of solid supports, wherein nucleic acid in said plurality of aliquots binds to said plurality of solid supports;

c) eluting said nucleic acid bound to each of said plurality of solid supports in step (b) to produce a plurality of volumes of eluate;

d) combining said volumes of (c) to produce a combined volume;

e) reducing said combined volume by centrifugal ultrafiltration to produce a concentrated nucleic acid sample;

f) contacting said concentrated nucleic acid sample of step (e) with oligonucleotide primers capable of amplifying said target nucleic acid under appropriate conditions, a first probe labeled with a donor fluorophore, and a second probe labeled with an acceptor fluorophore to produce an amplification sample, wherein said first and second probes hybridize proximally to one another on said target nucleic acid, and wherein energy transferred from said donor fluorophore induces fluorescence from said acceptor fluorophore; and

g) performing an amplification on said amplification sample to produce an amplification product, wherein said amplification comprises a series of step-down cycles and a series of fluorescence acquisition cycles, wherein each of said fluorescence acquisition cycles comprises detection of fluorescence from said acceptor fluorophore,

wherein the kinetics of an increase in intensity of fluorescence from said acceptor fluorophore are indicative of the amount of said target nucleic acid in said sample.

4. A method of diagnosing a microbial infection in a mammal, said method comprising the steps of:

a) providing a sample of a biological material from said mammal;

b) preparing said sample for nucleic acid extraction to produce a nucleic acid sample;

c) contacting an aliquot of said nucleic acid sample with a solid support, wherein nucleic acid in said aliquot binds to said solid support;

d) eluting said nucleic acid from said solid support with at least two sequential volumes of 5-25 μ

l to produce a concentrated nucleic acid sample;

e) contacting said concentrated nucleic acid sample of step (d) with oligonucleotide primers capable of amplifying a target nucleic acid under appropriate conditions, a first probe labeled with a donor fluorophore, and a second probe labeled with an acceptor fluorophore to produce an amplification sample, wherein said first and second probes hybridize proximally to one another on said target nucleic acid, and wherein energy transferred from said donor fluorophore induces fluorescence from said acceptor fluorophore; and

f) performing an amplification on said amplification sample to produce an amplification product, wherein said amplification comprises a series of step-down cycles and a series of fluorescence acquisition cycles, wherein each of said fluorescence acquisition cycles comprises detection of fluorescence from said acceptor fluorophore, wherein fluorescence from said acceptor fluorophore is indicative of a presence of said microbial infection, and an absence of fluorescence from said acceptor fluorophore is indicative of an absence of said microbial infection.

20. The method of claim 4, 5, or 6, wherein said mammal is a human.

5. A method of diagnosing a microbial infection in a mammal, said method comprising the steps of:

a) providing a sample of a biological material from said mammal;

b) preparing said sample for nucleic acid extraction to produce a nucleic acid sample;

c) contacting an aliquot of said nucleic acid sample with a solid support, wherein nucleic acid in said aliquot binds to said solid support;

d) eluting said nucleic acid bound to said solid support in step (b) to produce a volume of eluate and reducing said volume by centrifugal ultrafiltration to produce said concentrated nucleic acid sample;

e) contacting said concentrated nucleic acid sample of step (d) with oligonucleotide primers capable of amplifying a target nucleic acid under appropriate conditions, a first probe labeled with a donor fluorophore, and a second probe labeled with an acceptor fluorophore to produce an amplification sample, wherein said first and second probes hybridize proximally to one another on said target nucleic acid, and wherein energy transferred from said donor fluorophore induces fluorescence from said acceptor fluorophore; and

f) performing an amplification on said amplification sample to produce an amplification product, wherein said amplification comprises a series of step-down cycles and a series of fluorescence acquisition cycles, wherein each of said fluorescence acquisition cycles comprises detection of fluorescence from said acceptor fluorophore, wherein fluorescence from said acceptor fluorophore is indicative of a presence of said microbial infection, and an absence of fluorescence from said acceptor fluorophore is indicative of an absence of said microbial infection.

6. A method of diagnosing a microbial infection in a mammal, said method comprising the steps of:

a) providing a sample of a biological material from said mammal;

b) preparing said sample for nucleic acid extraction to produce a nucleic acid sample;

c) contacting a plurality of aliquots of said sample with a plurality of solid supports, wherein nucleic acid in said plurality of aliquots binds to said plurality of solid supports;

d) eluting said nucleic acid bound to each of said plurality of solid supports in step (c) to produce a plurality of volumes of eluate;

e) combining said volumes of (d) to produce a combined volume;

fi) reducing said combined volume by centrifugal ultrafiltration to produce a concentrated nucleic acid sample;

g) contacting said concentrated nucleic acid sample of step (f) with oligonucleotide primers capable of amplifying a target nucleic acid under appropriate conditions, a first probe labeled with a donor fluorophore, and a second probe labeled with an acceptor fluorophore to produce an amplification sample, wherein said first and second probes hybridize proximally to one another on said target nucleic acid, and wherein energy transferred from said donor fluorophore induces fluorescence from said acceptor fluorophore; and

h) performing an amplification on said amplification sample to produce an amplification product, wherein said amplification comprises a series of step-down cycles and a series of fluorescence acquisition cycles, wherein each of said fluorescence acquisition cycles comprises detection of fluorescence from said acceptor fluorophore, wherein fluorescence from said acceptor fluorophore is indicative of a presence of said microbial infection, and an absence of fluorescence from said acceptor fluorophore is indicative of an absence of said microbial infection.

21. A primer having a nucleic acid sequence consisting of 5′

-GGGGTTGTAGGGTYGAGRAIAWRRGATC-3′

(SEQ ID NO;

     6);

5′

-GAGAGTGGTCTCCCCAGATTCARACTA-3′

(SEQ ID NO;

     7);

5′

-TGACTAGGTTGRGCAAGYRTYT-3′

(SEQ ID NO;

     13);

or 5′

-AAAGACAIATAYTCTTAAACGTCTATTATTAT-3′

(SEQ ID NO;

     14), wherein I is inosine;

K is G or T;

M is C or A;

R is G or A;

W is A or T; and

Y is C or T.

22. A probe having a nucleic acid sequence consisting of 5′

-AACGTTKARTTCTTGAACGCGAGGTTT-3′

(SEQ ID NO;

     5);

5′

-GRAYGAHACAGGGTGATAGTCCCGTA-3′

(SEQ ID NO;

     9);

5′

-ACGAAARAACAARAGACKCTAWTCGAT-3′

(SEQ ID NO;

     10);

5′

-ACGAAAAAACAAAAGACGCTAATCGAT-3′

(SEQ ID NO;

     11);

5′

-ACGAAAGGAGAKMAAGACYGACCTCAAC-3′

(SEQ ID NO;

     12);

or 5′

-CGTAAACTAAATGTTTTGGAAACAA-3′

(SEQ ID NO;

     16), wherein H is A or C or T;

I is inosine;

K is G or T;

M is C or A;

R is G or A;

W is A or T; and

Y is C or T.

23. The probe of claim 22 further comprising a donor fluorophore conjugated to the 3′

end of said probe.

24. The probe of claim 22, wherein said probe is 5′

-ACGAAARAACAARAGACKCTAWTCGAT-3′

(SEQ ID NO;

     10);

5′

-ACGAAAAAACAAAAGACGCTAATCGAT-3′

(SEQ ID NO;

     11);

or 5′

-ACGAAAGGAGAKMAAGACYGACCTCAAC-3′

(SEQ ID NO;

     12), and wherein said probe further comprises an acceptor fluorophore conjugated to the 5′

end of said probe and a 3′

phosphate group.

25. A probe having a nucleic acid sequence consisting of 5′

-CCTGAGTAGRRCTAGACACGTGAAAC-3′

(SEQ ID NO;

     8) or 5′

-CTTAACAAIRCAAAWGAAATAGAAT-3′

(SEQ ID NO;

     15), wherein I is inosine;

R is G or A; and

W is A or T.

26. The probe of claim 25 further comprising an acceptor fluorophore conjugated to the 5′

end of said probe and a 3′

phosphate group.