Patent Application
20080044816
The ability of the disclosed combination of primers and probes to amplify SARS-CoV nucleic acid was demonstrated using a plasmid DNA clone of the target sequence corresponding to nucleotides 17936-18024 of SARS-CoV strain BJ03 (GenBank Accession No. AY278490). Linearized plasmid DNA was quantified using PicoGreen® dsDNA Quantitation Reagent (Molecular Probes, Inc., Eugene, Oreg.) and diluted to a working concentration with water containing 7 ng/mL salmon sperm DNA. Four replicate SDA reactions were run at each of six target levels, including negative controls that contained water in place of target DNA.
In brief, DNA target was added to SDA Buffer and denatured by heating in a boiling water bath for 5 min. One hundred and ten microliters of the denatured sample was then added to Priming Microwells containing 40 μL of a solution of SDA Primers, Reporter Probe and nucleotides. Following an incubation at ambient temperature for 20 min., the Priming Microwells were transferred to a heat block at 72° C., while corresponding Amplification Microwells containing dried Bst polymerase and BsoBI restriction enzyme were pre-warmed at 54° C. After 10 min. incubation, 100 μL of the priming mixture were transferred from the Priming to the Amplification Microwells, which were then sealed and loaded into a BD ProbeTec ET reader set at 52.5° C. Fluorescent signals were monitored over the course of 1 hour and analyzed using the Passes After Threshold (PAT) algorithm developed for this instrument. (Wolfe D M, Wang S S, Thornton K, Kuhn A M, Nadeau J G, Hellyer T J. Homogeneous strand displacement amplification. In: DNA amplification—current technologies and applications, Demidov V V and Broude N E (Eds.), Horizon Bioscience, Wymondham, UK.) The PAT scores represent the number of instrument passes remaining after the fluorescent readings achieve a pre-defined threshold value. Final SDA reaction conditions were as follows: 50 nM pUC19-based Bumper Primer AB (SEQ ID NO.: 20); 100 nM SDA Primer SarARP (SEQ ID NO.: 3); 500 nM SDA Primer SarAFP (SEQ ID NO.: 2); 250 nM Signal Primer SarAAd-TBD16 (SEQ ID NO.: 4); 500 nM Reporter Probe TBD16 D/R (SEQ ID NO.: 8); 500 μM deoxycytidine 5′-O-(1-Thiotriphosphate), S-isomer (dCsTP); 100 μM each of dATP, dGTP and dTTP; 12.5% DMSO; 25 mM KiPO4; 82 mM KOH; 143 mM bicine; 12 U Bst polymerase; 30 U BsoBI restriction enzyme; 5 mM magnesium acetate.
Positive results were obtained with as little 25 copies of the target plasmid per reaction while no false-positive results were observed in any of the negative controls (Table 6). These data demonstrate that the disclosed combination of primers and Reporter Probe is capable of detecting a SARS-CoV-specific nucleic acid target sequence with a high degree of analytical sensitivity.
A second experiment was conducted to demonstrate the analytical sensitivity of the disclosed primers for the detection of SARS-CoV-specific nucleic acid. In contrast to the previous experiment, Reporter Probe MPC D/R (SEQ ID NO.: 10) was used together with Signal Primer SarAAd-MPC (SEQ ID NO.: 5).
Briefly, DNA target was added to SDA Buffer and denatured by heating in a boiling water bath for 5 min. One hundred and ten microliters of the denatured sample was then added to Priming Microwells containing 40 μL of a solution of SDA Primers, Reporter Probe and nucleotides. The Priming Microwells were allowed to sit for 20 min. at ambient temperature, before being transferred to a heat block at 72° C. At the same time, corresponding Amplification Microwells containing dried Bst polymerase and BsoBI restriction enzyme were pre-warmed at 54° C. Following a 10 min. incubation, 100 μL of the priming mixture were transferred from the Priming to the Amplification Microwells, which were then sealed and placed at 52.5° C. in a BD ProbeTec ET reader. Fluorescent signals were monitored over the course of 1 hour and analyzed using the PAT algorithm developed for this instrument. The PAT scores represent the number of instrument passes remaining after the fluorescent readings achieve a pre-defined threshold value. Final SDA reaction conditions were as follows: 50 nM pUC19-based Bumper Primer AB (SEQ ID NO.: 20); 100 nM SDA Primer SarARP (SEQ ID NO.: 3); 500 nM SDA Primer SarAFP (SEQ ID NO.: 2); 250 nM Signal Primer SarAAd-MPC (SEQ ID NO.: 5); 500 nM Reporter Probe MPC D/R (SEQ ID NO.: 10); 500 μM dCsTP; 100 μM each of dATP, dGTP and dTTP; 12.5% DMSO; 25 mM KiPO4; 82 mM KOH; 143 mM bicine; 12 U Bst polymerase; 30 U BsoBI restriction enzyme; 5 mM magnesium acetate.
Results are summarized in Table 7. All reactions containing 100 copies of plasmid DNA were positive. In contrast, none of the reactions containing water in place of plasmid DNA yielded positive results, thereby demonstrating the analytical sensitivity and specificity of the disclosed of primers and Reporter Probe combination for the detection of the SARS-CoV-specific nucleic acid target sequence.
The analytical specificity of the disclosed primers and probes was verified by testing a panel of 43 bacteria and fungi that are likely to be found in respiratory and/or gastrointestinal specimens. Because all these organisms have genomes comprised of DNA rather than RNA, no reverse transcription step was included in these reactions. A suspension of each organism was prepared in Phosphate-Buffered Saline containing Bovine Serum Albumin (PBS/BSA) at a concentration of approximately 107-108 cells/mL. Fifteen microliters of each suspension were mixed with 150 μL SDA Buffer and heated in a boiling water bath for 5 min. to lyse the organisms and denature the DNA. After cooling to room temperature, 110 μL of denatured sample were added to a Priming Microwell containing containing 40 μL of a solution of SDA Primers, Reporter Probe and nucleotides. The Priming Microwells were incubated at ambient temperature for 20 min. and then transferred to a heat block at 72° C., while corresponding Amplification Microwells were pre-warmed at 54° C. After 10 min., 100 μL of the priming mixture were transferred from the Priming to the Amplification Microwells, which were then sealed and loaded into a BD ProbeTec ET reader set at 52.5° C. Fluorescence was monitored over the course of 1 hour and analyzed using the PAT algorithm developed for this instrument. Final SDA conditions were as follows: 50 nM pUC19-based Bumper Primer AB (SEQ ID NO.: 20); 100 nM SDA Primer SarARP (SEQ ID NO.: 3); 500 nM SDA Primer SarAFP (SEQ ID NO.: 2); 250 nM Signal Primer SarAAd-TBD16 (SEQ ID NO.: 4); 500 nM Reporter Probe TBD16 D/R (SEQ ID NO.: 8); 500 μM dCsTP; 100 μM each of dATP, dGTP and dTTP; 12.5% DMSO; 25 mM KiPO4; 82 mM KOH; 143 mM bicine; 12 U Bst polymerase; 30 U BsoBI restriction enzyme; 5 mM magnesium acetate.
Results are summarized in Table 8. No positive results were obtained except from a plasmid clone of the SARS-CoV target sequence that was run as a positive control, thereby demonstrating the specificity of the disclosed primers and Reporter Probe for the detection of SARS-CoV.
Acinetobacter calcoaceticus
Actinomyces israelii
Aeromonas hydrophila
Alcaligenes faecalis
Bacteroides fragilis
Bordetella pertussis
Candida albicans
Chlamydophila pneumoniae
Citrobacter freundii
Corynebacterium diphtheriae
Corynebacterium jeikeium
Cryptococcus neoformans
Edwardsiella tarda
Eikenella corrodens
Enterobacter aerogenes
Enterococcus faecalis
Escherichia coli
Fusobacterium nucleatum
Haemophilus influenzae
Haemophilus parainfluenzae
Kingella kingae
Klebsiella pneumoniae subsp.
pneumoniae
Lactobacillus acidophilus
Legionella pneumophila
Morganella morganii
Neisseria mucosa
Peptostreptococcus anaerobius
Plesiomonas shigelloides
Porphyromonas asaccharolytica
Proteus mirabilis
Pseudomonas aeruginosa
Serratia marcescens
Staphylococcus aureus
Staphylococcus epidermidis
Stenotrophomonas maltophila
Streptococcus mutans
Streptococcus pneumoniae
Streptococcus pyogenes
Veillonella parvula
Yersinia enterolitica
Yersinia ruckeri
The ability of the disclosed combination of primers and probes to amplify SARS-CoV nucleic acid was demonstrated using a plasmid DNA clone of the target sequence corresponding to nucleotides 15068-15138 of SARS-CoV strain BJ03 (GenBank Accession No. AY278490). Linearized plasmid DNA was quantified using PicoGreen dsDNA Quantitation Reagent (Molecular Probes, Inc., Eugene, Oreg.) and diluted to a working concentration with water containing 7 ng/μL salmon sperm DNA. Eight replicate SDA reactions were run at each of three target levels, including negative controls that contained water in place of target DNA.
In brief, DNA target was added to SDA Buffer and denatured for by heating in a boiling water bath for 5 min. One hundred and fifty microliters of the denatured sample was then added to Priming Microwells containing dried SDA Primers, Reporter Probe and nucleotides. Following an incubation at ambient temperature for 20 min., the Priming Microwells were transferred to a heat block at 72° C., while corresponding Amplification Microwells containing dried Bst polymerase and BsoBI restriction enzyme were pre-warmed at 54° C. After 10 min., 100 μL of the priming mixture were transferred from the Priming to the Amplification Microwells, which were then sealed and loaded into a BD ProbeTec ET reader set at 52.5° C. Fluorescent signals were monitored over the course of 1 hour and analyzed using the PAT algorithm developed for this instrument. The PAT scores represent the number of instrument passes remaining after the fluorescent readings achieve a pre-defined threshold value. Final SDA reaction conditions were as follows: 50 nM pUC19-based Bumper Primer AB (SEQ ID NO.: 20); 500 nM SDA Primer SarBRP (SEQ ID NO.: 15); 100 nM SDA Primer SarBFP (SEQ ID NO.: 14); 250 nM Signal Primer SarBAd-MPC (SEQ ID NO.: 4); 300 nM Reporter Probe MPC D/R (SEQ ID NO.: 8); 500 mM deoxycytidine 5′-O-(1-Thiotriphosphate), S-isomer (dC8TP); 100 μM each of dATP, dGTP and dTTP; 12.5% DMSO; 25 mM KiPO4; 82 mM KOH; 143 mM bicine; 12 U Bst polymerase; 30 U BsoBI restriction enzyme; 5 mM magnesium acetate.
Positive results were obtained with as little 15 copies of the target plasmid per reaction while no false-positive results were observed in any of the negative controls (Table 9). These data demonstrate that the disclosed combination of primers and Reporter Probe is capable of detecting the targeted SARS-CoV-specific nucleic acid sequence with a high degree of analytical sensitivity.
The analytical specificity of the disclosed primers and probes was verified by testing a panel of 43 bacteria and fungi that are likely to be found in respiratory and/or gastrointestinal specimens. Because all these organisms have genomes comprised of DNA rather than RNA, no reverse transcription step was included in these reactions. A suspension of each organism was prepared in PBS/BSA at a concentration of approximately 107-108 cells/mL. Fifteen microliters of each suspension were mixed with 150 μL SDA Buffer and heated in a boiling water bath for 5 min. to lyse the organisms and denature the DNA. After cooling to room temperature, 110 μL of denatured sample were added to a Priming Microwell containing containing 40 μL of a solution of SDA Primers, Reporter Probe and nucleotides. The Priming Microwells were allowed to sit at ambient temperature for 20 min. and then transferred to a heat block at 72° C., while corresponding Amplification Microwells were pre-warmed at 54° C. After a 10 min. incubation, 100 μL of the priming mixture were transferred from the Priming to the Amplification Microwells, which were then sealed and loaded into a BD ProbeTec ET reader set at 52.5° C. Fluorescence was monitored over the course of 1 hour and analyzed using the PAT algorithm developed for this instrument. Final SDA conditions were as follows: 50 nM pUC19-based Bumper Primer AB (SEQ ID NO.: 20); 500 nM SDA Primer SarBRP (SEQ ID NO.: 15); 100 nM SDA Primer SarBFP (SEQ ID NO.: 14); 250 nM Signal Primer SarBAd-MPC (SEQ ID NO.: 17); 500 nM Reporter Probe MPC D/R (SEQ ID NO.: 10); 500 mM dCsTP; 100 μM each of dATP, dGTP and dTTP; 12.5% DMSO; 25 mM KiPO4; 82 mM KOH; 143 mM bicine; 12 U Bst polymerase; 30 U BsoBI restriction enzyme; 5 mM magnesium acetate.
As illustrated in Table 10, no positive results were obtained except from a plasmid clone of the SARS-CoV target sequence that was run as a positive control. This demonstrates the specificity of the disclosed primers and Reporter Probe for the detection of SARS-CoV.
Acinetobacter calcoaceticus
Actinomyces israelii
Aeromonas hydrophila
Alcaligenes faecalis
Bacteroides fragilis
Blastomyces dermatitidis
Bordetella pertussis
Branhamella catarrhalis
Candida albicans
Chlamydophila pneumoniae
Citrobacter freundii
Clostridium perfringens
Corynebacterium diphtheriae
Corynebacterium jeikeium
Cryptococcus neoformans
Edwardsiella tarda
Eikenella corrodens
Enterobacter aerogenes
Enterococcus faecalis
Escherichia coli
Fusobacterium nucleatum
Haemophilus influenzae
Haemophilus parainfluenzae
Histoplasma capsulatum
Kingella kingae
Klebsiella pneumoniae subsp.
pneumoniae
Lactobacillus acidophilus
Legionella pneumophila
Moraxella osloensis
Morganella morganii
Mycobacterium tuberculosis
Mycoplasma pneumoniae
Neisseria meningitides
Neisseria mucosa
Peptostreptococcus anaerobius
Plesiomonas shigelloides
Porphyromonas asaccharolytica
Proteus mirabilis
Providencia stuartii
Pseudomonas aeruginosa
Serratia marcescens
Salmonella cholerasuis
Staphylococcus aureus
Staphylococcus epidermidis
Stenotrophomonas maltophila
Streptococcus mitis
Streptococcus mutans
Streptococcus pneumoniae
Streptococcus pyogenes
Veillonella parvula
Vibrio parahaemolyticus
Yersinia enterolitica
The present application claims priority to U.S. Provisional Application Ser. No. 60/502,279, filed Sep. 12, 2003, which is herein incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US04/29691 | 9/13/2004 | WO | 00 | 3/21/2007 |
| Number | Date | Country | |
|---|---|---|---|
| 60502279 | Sep 2003 | US |
