Source: https://patents.justia.com/patent/9175353
Timestamp: 2020-08-09 17:51:21
Document Index: 711359859

Matched Legal Cases: ['§371', 'Application No. 61', '§1', '§9', 'Application No. 2009313808', 'Application No. 09826888']

US Patent for Compositions, kits and methods for detection of campylobacter nucleic acid Patent (Patent # 9,175,353 issued November 3, 2015) - Justia Patents Search
Justia Patents Probes For Detection Of Microbial Nucleotide SequencesUS Patent for Compositions, kits and methods for detection of campylobacter nucleic acid Patent (Patent # 9,175,353)
Nov 16, 2009 - Gen-Probe Incorporated
This application is the national stage pursuant to 35 U.S.C. §371 of PCT International Application No. PCT/US09/064516, which has an international filing date of Nov. 16, 2009, which designated the United States of America, and which claims the benefit of priority to U.S. Provisional Application No. 61/114,547, filed on Nov. 14, 2008, the contents of each of these applications are incorporated herein by reference in their entirety.
In one embodiment, the compositions, kits, and/or methods may further include or use a detection oligonucleotide, preferably a linear or hairpin detection oligonucleotide, more preferably a torch oligonucleotide or molecular beacon oligonucleotide. In one aspect, the detection oligonucleotide stably hybridizes to an amplification product generated from a Campylobacter target nucleic acid using at least one amplification oligomer described herein, and more preferably using two amplification oligomers described herein. In one aspect, the detection oligonucleotide stably hybridizes to amplification products generated from a C. jejuni target nucleic acid and a C. coli, a C. lari or a C. coli and a C. Ian target nucleic acid using at least one amplification oligomer described herein, and more preferably using two amplification oligomers described herein. In one aspect, the detection oligonucleotide is from 10 to 70 nucleotides in length and stably hybridizes to the “+” or “−” strand of a region of SEQ ID NO:91, or RNA equivalent thereof. In one aspect, the detection oligonucleotide is a torch oligonucleotide selected from the sequences of SEQ ID NOS:59-70 and their complements. In one aspect, the detection oligonucleotide is a torch oligonucleotide selected from the sequences of SEQ ID NOS:62, 63, 65, 66, 67, and 68, and their complements. In one aspect, the detection oligonucleotide is a torch oligonucleotide selected from the sequences of SEQ ID NO:68, SEQ ID NO:67, and their complements. In one aspect, the detection oligonucleotide is a torch oligonucleotide having the sequence of SEQ ID NO:67 or its complement.
A detecting step may be performed after the amplification reaction is completed, or may be performed simultaneous with amplifying the target region, e.g., in real time. In one embodiment, the detection step allows homogeneous detection, e.g., detection of the hybridized probe without removal of unhybridized probe from the mixture (e.g., U.S. Pat. Nos. 5,639,604 and 5,283,174). In embodiments that detect the amplified product near or at the end of the amplification step, a linear probe may be used to provide a signal to indicate hybridization of the probe to the amplified product. In other embodiments that use real-time detection, the probe may be a linear probe, such as a dual labeled TaqMan probe detected upon 5′→3′ exonuclease degradation of the probe, or may be a hairpin probe, such as a molecular beacon, molecular torch, or hybridization switch probe labeled with a reporter moiety that is detected when the hairpin probe binds to amplified product. Various forms of such probes have been described previously (e.g., U.S. Pat. Nos. 5,118,801; 5,210,015; 5,312,728; 5,538,848; 5,925,517; 6,150,097; 6,849,412; 6,835,542; 6,534,274; and 6,361,945; and US Pub. Nos. 20060068417A1; and US Pub. No. 20060194240A1).
The term “fragment” as used herein in reference to the Campylobacter targeted nucleic acid sequence refers to a piece of contiguous nucleic acid. In certain embodiments, the fragment includes contiguous nucleotides from a Campylobacter species'target nucleic acid, wherein the number of contiguous nucleotides in the fragment are less than that for the entire 16S rRNA or its encoding gene.
“Probe,” “detection probe” or “detection oligonucleotide” are terms referring to a nucleic acid oligomer that hybridizes specifically to a target sequence in a nucleic acid, or in an amplified nucleic acid, under conditions that promote hybridization to allow detection of the target sequence or amplified nucleic acid. Detection may either be direct (e.g., a probe hybridized directly to its target sequence) or indirect (e.g., a probe linked to its target via an intermediate molecular structure). Probes may be DNA, RNA, analogs thereof or combinations thereof and they may be labeled or unlabeled. A probe's “target sequence” generally refers to a smaller nucleic acid sequence within a larger nucleic acid sequence that hybridizes specifically to at least a portion of a probe oligomer by standard base pairing. A probe may comprise target-specific sequences and other sequences that contribute to the three-dimensional conformation of the probe (e.g., U.S. Pat. Nos. 5,118,801; 5,312,728; 6,849,412; 6,835,542; 6,534,274; and 6,361,945; and U.S. Pub. No. 20060068417).
As used herein, a “label” refers to a moiety or compound joined directly or indirectly to a probe that is detected or leads to a detectable signal. Direct labeling can occur through bonds or interactions that link the label to the probe, including covalent bonds or non-covalent interactions, e.g. hydrogen bonds, hydrophobic and ionic interactions, or formation of chelates or coordination complexes. Indirect labeling can occur through use of a bridging moiety or “linker” such as a binding pair member, an antibody or additional oligomer, which is either directly or indirectly labeled, and which may amplify the detectable signal. Labels include any detectable moiety, such as a radionuclide, ligand (e.g., biotin, avidin), enzyme or enzyme substrate, reactive group, or chromophore (e.g., dye, particle, or bead that imparts detectable color), luminescent compound (e.g., bioluminescent, phosphorescent, or chemiluminescent labels), or fluorophore. Labels may be detectable in a homogeneous assay in which bound labeled probe in a mixture exhibits a detectable change different from that of an unbound labeled probe, e.g., instability or differential degradation properties. A “homogeneous detectable label” can be detected without physically removing bound from unbound forms of the label or labeled probe (e.g., U.S. Pat. Nos. 5,283,174, 5,656,207, and 5,658,737). Labels include chemiluminescent compounds, e.g., acridinium ester (“AE”) compounds that include standard AE and derivatives (e.g., U.S. Pat. Nos. 5,656,207, 5,658,737, and 5,639,604). Synthesis and methods of attaching labels to nucleic acids and detecting labels are well known (e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), Chapter 10; U.S. Pat. Nos. 5,658,737, 5,656,207, 5,547,842, 5,283,174, and 4,581,333). More than one label, and more than one type of label, may be present on a particular probe, or detection may use a mixture of probes in which each probe is labeled with a compound that produces a detectable signal (e.g., U.S. Pat. Nos. 6,180,340 and 6,350,579).
As used herein, a “capture oligonucleotide” or “capture probe” refers to a nucleic acid oligomer that specifically hybridizes to a target sequence in a target nucleic acid by standard base pairing and joins to a binding partner on an immobilized probe to capture the target nucleic acid to a support. One example of a capture oligomer includes an oligonucleotide comprising two binding regions: a target hybridizing sequence and an immobilized probe-binding region. A variation of this example, the two regions may be present on two different oligomers joined together by one or more linkers. Another embodiment of a capture oligomer the target hybridizing sequence is a sequence that includes random or non-random poly-GU, poly-GT, or poly U sequences to bind non-specifically to a target nucleic acid and link it to an immobilized probe on a support. (PCT Pub No. WO 2008/016988). The immobilized probe binding region can be a nucleic acid sequence; referred to as a tail. Tails include a substantially homopolymeric tail of about 10 to 40 nucleotides (e.g., A10 to A40), or of about 14 to 33 nt (e.g., T3A14 to T3A30), that bind to a complementary immobilized sequence attached to the support particle or support matrix. Another example of a of a capture oligomer comprises two regions, a target hybridizing sequence and a binding pair member that is not a nucleic acid sequence.
By “preferentially hybridize” is meant that under stringent hybridization assay conditions, an oligonucleotide hybridizes to its target sequences, or replicates thereof, to form stable oligonucleotide: target sequence hybrid, while at the same time formation of stable oligonucleotide: non-target sequence hybrid is minimized. For example, a probe oligonucleotide preferentially hybridizes to a target sequence or replicate thereof to a sufficiently greater extent than to a non-target sequence, to enable one having ordinary skill in the art to accurately detect the RNA replicates or complementary DNA (cDNA) of the target sequence formed during the amplification. Appropriate hybridization conditions are well known in the art for probe, amplification, target capture, blocker and other oligonucleotides, may be predicted based on sequence composition, or can be determined by using routine testing methods (e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) at §§1.90-1.91, 7.37-7.57, 9.47-9.51 and 11.47-11.57, particularly §§9.50-9.51, 11.12-11.13, 11.45-11.47 and 11.55-11.57).
Oligonucleotides were designed by first comparing 16S rRNA sequences (or gene sequences encoding 16S rRNA) of a number of Campylobacter species (e.g., C. jejuni, C. lari, C. coli, C. fetus, C. upsaliensis, C. hyointestinalis, C. curvus, C. hominis, C. insulaenigrae, C. lanienae, C. mucosalis, C. rectus, C. sputorum, and C. concisus) and a number of other bacterial species within the order of Campylobacterales (e.g., Arcobacter, Helicobacter, Flexispira, Geospirillum, Sulfurospirillum, Sulfuricurvum, and Hydrogenimonas). Oligonucleotides were synthesized in vitro, and in some embodiment oligomers can be characterized by determining the Tm and hybridization characteristics of the C. jejuni, C. lari, or C. coli oligomers with complementary target sequences (synthetic or purified rRNA from bacteria) using standard laboratory methods. Then, selected oligomer sequences were further tested against 16S rRNA sequences. This test used different combinations of amplification oligomers (selected from those shown in Table 1). The amplification reactions included 16S rRNA targets from lysates or purified from various Campylobacter species grown in culture. The amplification reaction determined the efficiency of amplification of the 16S rRNA target sequences using the various amplification oligo combinations. Amplification oligomers include those that may function as primer, promoter primer, and promoter provider oligomers. The relative efficiencies of different combinations of amplification oligomers were monitored by detecting the amplified products of the amplification reactions, generally by binding a labeled probe (Table 2) to the amplified products and detecting the relative amount of signal that indicated the amount of amplified product made.
SEQ Use ID NO: Sequence (5′ → 3′)
Primer 1 CACCGAAAAACTTTCCCTACTCAAC Primer 2 CTACACCGAAAAACTTTCCCTACTCAAC Primer 3 CCTACACCGAAAAACTTTCCCTACTCAAC Primer 4 CACCGAAAAACTTTCCCTACTC Primer 5 CTACACCGAAAAACTTTCCCTACTC Primer 6 CCTACACCGAAAAACTTTCCCTACTC Primer 7 GTCTCATCCTACACCGAAAAAC Primer 8 CTATATAGTCTCATCCTACACCG Primer 9 GCTGATACTATATAGTCTCATCCTACACCG Primer 10 CTATATAGTCTCATCCTACACC Primer 11 GCTGATACTATATAGTCTCATCCTACACC Primer 12 CTATATAGTCTCATCCTACAC Primer 13 CCAACTAGCTGATACTATATAGTCTCATCCTACAC Primer 14 GCTGATACTATATAGTCTCATCC Primer 15 GTGCGCCACTAATCCACTTC Primer 16 CGTGCGCCACTAATCCACTT Primer 17 CGTGCGCCACTAATCCACT T7 Provider*/Primer 18 aatttaatacgactcactatagggagaCCTACACAAGAGGACAACA GTTG Target Hybridizing 19 CCTACACAAGAGGACAACAGTTG Seq. T7 Provider/Primer 20 aatttaatacgactcactatagggagaCCTACACAAGAGGACAACA GTTGG Target Hybridizing 21 CCTACACAAGAGGACAACAGTTGG Seq. T7 Provider/Primer 22 aatttaatacgactcactatagggagaCACAAGAGGACAACAGTTG GAAACG Target Hybridizing 23 CACAAGAGGACAACAGTTGGAAACG Seq. T7 Provider/Primer 24 aatttaatacgactcactatagggagaCACAAGAGGACAACAGTTG GAAACGAC Target Hybridizing 25 CACAAGAGGACAACAGTTGGAAACGAC Seq. T7 Provider/Primer 26 aatttaatacgactcactatagggagaAAGAGGACAACAGTTGGAA AC Target Hybridizing 27 AAGAGGACAACAGTTGGAAAC Seq. T7 Provider/Primer 28 aatttaatacgactcactatagggagaGGACAACAGTTGGAAACGA CTGCTAATACTCT Target Hybridizing 29 GGACAACAGTTGGAAACGACTGCTAATACTCT Seq. T7 Provider/Primer 30 aatttaatacgactcactatagggagaCAACAGTTGGAAACGACTG CTAATACTCT Target Hybridizing 31 CAACAGTTGGAAACGACTGCTAATACTCT Seq. T7 Provider/Primer 32 aatttaatacgactcactatagggagaCAGTTGGAAACGACTGCTA ATACTCT Target Hybridizing 33 CAGTTGGAAACGACTGCTAATACTCT Seq. T7 Provider/Primer 34 aatttaatacgactcactatagggagaGGCGTGCCTAATACATGCA AGTCG Target Hybridizing 35 GGCGTGCCTAATACATGCAAGTCG Seq. T7 Provider/Primer 36 aatttaatacgactcactatagggagaCGTGCCTAATACATGCAAG TCG Target Hybridizing 37 CGTGCCTAATACATGCAAGTCG Seq. T7 Provider/Primer 38 aatttaatacgactcactatagggagaGCCTAATACATGCAAGTCG AAC Target Hybridizing 39 GCCTAATACATGCAAGTCGAAC Seq. T7 Provider/Primer 40 aatttaatacgactcactatagggagaCCTAATACATGCAAGTCGA ACG Target Hybridizing 41 CCTAATACATGCAAGTCGAACG Seq. T7 Provider/Primer 42 aatttaatacgactcactatagggagaGCTAGAAGTGGATTAGTGG CGCAC Target Hybridizing 43 GCTAGAAGTGGATTAGTGGCGCAC Seq. T7 Provider/Primer 44 aatttaatacgactcactatagggagaGGATTAGTGGCGCACGGGT GAG Target Hybridizing 45 GGATTAGTGGCGCACGGGTGAG Seq. T7 Provider/Primer 46 aatttaatacgactcactatagggagaGGGTGAGTAAGGTATAG TTAATCTGC Target Hybridizing 47 GGGTGAGTAAGGTATAGTTAATCTGC Seq. T7 Provider/Primer 48 aatttaatacgactcactatagggagaGGTGAGTAAGGTATAGTTA ATCTGCC Target Hybridizing 49 GGTGAGTAAGGTATAGTTAATCTGCC Seq. Blocker 50 CAACTGTTGTCCTCTTGTG Blocker 51 CTAGCAAGCTAGAAGCTTC Blocker 52 CTAATCCACTTCTAGCAAGC Blocker 53 TCACCCGTGCGCCACTAATC Blocker 54 TTAGGCACGCCGCCAG Blocker 55 ATTAGGCACGCCGCCAG Blocker 56 GTAGGGCAGATTAACTATAC Blocker 57 CTTGTGTAGGGCAGATTAAC
For the sequences listed in Table 2, the lowercase letters indicate the nucleotides in the sequence that form part of the closing domain, but are not part of the target binding sequence. Embodiments of the hairpin probe oligomers were synthesized with a fluorescent label attached at one end of the sequence and a quencher compound attached at the other end of the sequence. Some embodiments of hairpin oligomers also include a non-nucleotide linker moiety at selected positions within the sequence. Examples of such embodiments include those that include an abasic 9-carbon (“C9”) linker between residues 6 and 7 of SEQ ID NO.: 70, between residues 15 and 16 of SEQ ID NO.: 68, between residues 16 and 17 of SEQ ID NO.: 61, between residues 17 and 18 of SEQ ID NO.: 67, between residues 18 and 19 of SEQ ID NO.: 69, between residues 20 and 21 of SEQ ID NO.: 66, between residues 21 and 22 of SEQ ID NO.: 63, between residues 23 and 25 of SEQ ID NO.: 62, between residues 24 and 25 of SEQ ID NO.: 65, between residues 25 and 26 of SEQ ID NOS.: 59 and 64, and between residues 26 and 27 of SEQ ID NO.: 60. Detection probes may be used with helper probes that are unlabeled and facilitate binding of the labeled probe to its target as previously described (U.S. Pat. No. 5,030,557).
SEQ Use ID NO: Sequence (5′ → 3′) Torch 59 cGGAGTATAGAGTATTAGCAGTCGTcTCCg Torch 60 GCAGGAGTATAGAGTATTAGCAGTCGcctgc Torch 61 GCAGGAGTATAGAGTAcctGC Torch 62 ccGTGTTAAGCAGGAGTATAGAGcacgg Torch 63 ccGTGTTAAGCAGGAGTATAGcacgg Torch 64 cTCCCTACTCAACTTGTGTTAAGCAGG gag Torch 65 cTCCCTACTCAACTTGTGTTAAGCgGGAG Torch 66 cTCCCTACTCAACTTGTGTTggGag Torch 67 cTCCCTACTCAACTTGTGggag Torch 68 cTCCCTACTCAACTTGggag Torch 69 ccGCTAGAAGCTTCATCGagcgg Torch 70 cggaaGCAAGCTAGAAGCTTCcg
SEQ Use ID NO: Sequence (5′ → 3′) Target Capture 71 GCGTCAGGGTTTCCCCCATTGCGTTTAAAAAAAAAAAAAAA AAAAAAAAAAAAAAA Target Capture 72 GCGTCAGGGTTTCCCCCATTGCG[binding partner] Target Capture 73 GCTTATTCCTTAGGTACCGTCAGTTTAAAAAAAAAAAAAAA AAAAAAAAAAAAAAA Target Capture 74 GCTTATTCCTTAGGTACCGTCAG[binding partner]
Reagents used in target capture and amplification steps in the examples described herein generally included one or more of the following. Probe Matrix Lysis Reagent contained 0.1% (w/v) Lithium Lauryl Sulfate (LLS), 20 mM Lithium Succinate, and 1 mM EDTA. Lysis Reagent contained 1% (w/v) LLS, 100 mM Tris, 2.5 mM succinic acid, 10 mM EDTA, and 500 mM lithium chloride (LiCl) at pH 6.5. Target Capture Reagent contained 300 mM HEPES, 1.88 M lithium chloride, 100 mM EDTA, at pH 6.4, and 250 .micro.g/ml of paramagnetic particles (0.7-1.05 micron particles, SERA-MAG.sup.™ MG-CM, Seradyn, Inc., Indianapolis, Ind.) with (dT).sub.14 oligomers covalently bound thereto. Wash Solution used in target capture contained 10 mM HEPES, 150 mM NaCl, 1 mM EDTA, and 0.1% (w/v) sodium dodecyl sulfate, at pH 7.5. Amplification Reagent was mixed with other reaction components produce a solution containing 50 mM HEPES, 33 mM KCl, 30 mM MgCl.sub.2, 0.5 mM of each dNTP (dATP, dCTP, dGTP, dTTP), 10 mM ATP, 2 mM CTP, 12.7 mM UTP, 2 mM GTP, at pH 7.7. Amplification oligonucleotides (primers 0.125 pmol/.micro.L, promoter primers 0.125 pmol/.micro.L, blocker oligonucleotides 0.0125 pmol/.micro.L, promoter provider oligonucleotides) 0.125 pmol/.micro.L, and optionally probes 0.3 pmol/.micro.L, may be added to the reaction mixture in the amplification reagent or separate from the amplification reagent. Enzyme Reagent contained 360 RTU/.micro.l of Moloney murine leukemia virus (MMLV) reverse transcriptase (RT) and about 80 PU/.micro.l of T7 RNA polymerase, 75 mM HEPES, 120 mM KCl, 10% TRITON® X-100, 160 mM N-acetyl-L-cysteine, and 1 mM EDTA at pH 7.0, where 1 RTU of RT incorporates 1 nmol of dTTP in 20 min at 37.deg.C., and 1 PU of T7 RNA polymerase produces 5 nmol of RNA transcript in 20 min at 37.deg.C. Equipment and Material generally included: KingFisher® 96 (Thermo Electron, Waltham, Mass.); KingFisher® mL (Thermo Electron, Waltham, Mass.); PTI-FP-2® FluoDia Plate Reader (Photon Technology International, Birmingham, N.J.); Eppendorf® Thermomixer R 022670565 (Eppendorf Corporation, Westbury, N.Y.); Hard-Shell Thin-Wall 96-Well Skirted PCR Plates, colored shell/white well, Catalog numbers: HSP-9615, HSP-9625, HSP-9635) (BioRad Hercules, Calif.); KingFisher® 96 tip comb for DW magnets (Catalog number: 97002534) Thermo Electron, Waltham, Mass.); DW 96 plate, V bottom, Polypropylene, sterile 25 pcs/case (Axygen Catalog number: P-2ML-SQ-C-S; VWR catalog number 47749-874); KingFisher® 96 KF plate (200 microliters) (Catalog number: 97002540); KingFisher® mL tip comb (Catalog number 97002111); and KingFisher® mL tubes (Catalog number 97002121).
Fluorescence was measured at regular time intervals (e.g., every 72 sec) during the amplification reaction for a total of 63 reads. Two colors were read at each interval. Monitoring was done using a real time fluorescent detection system (e.g., Bio-Rad Laboratory's Opticon™ or Chromo4™, or a FluoDia® T70 instrument). Real time algorithms are well known in the art, examples are described in Biochem Biophys Res Commun. 2002 Jun. 7; 294(2):347-53 and Nucleic Acids Research 2004 32(22):e178. The negative controls were tested to determine background fluorescent signal levels. Raw data were gathered and analyzed to calculate signal emergence time (TTime) and range relative fluorescent units (RFUs). Oligo combinations were then grouped based upon TTime with TTime under 23 minutes being considered as the best combinations, TTimes between 23 minutes and 29 minutes being considered as adequate oligo combinations and TTimes of 30 or more minutes being unacceptable. Positive criterion depended on the instrument used. For instance, positive criterion was set at 0.05 RFU and was assessed over a 67-minute time course for the MJ Chromo4, whereas positive criterion was set at 1000 RFU and was assessed over a 75-minute time course using a different plate reader instrument. The oligomer combinations listed in Table 4 produced a fluorescent curve with a TTime less than or equal to 23 minutes. The oligomer combinations listed in Table 5 produced a fluorescent curve with a TTime between 23 and 30 minutes. From Tables 4 and 5, a preferred set of oligonucleotide combinations was selected for secondary screening. In addition to TTime, oligo combinations were also looked at to detect design flaws such as the number of mismatched nucleobases compared to Campylobacter species and the degree of burden for specificity placed upon each oligonucleotide in a combination. Other factors impacting the selection of oligo combinations for secondary screening included poor oligo compatibility and poor performance in amplifying and/or detecting target.
Oligo C. fetus, Combination C. jejuni; C. fetus Number C. coli; ssp (from and C. lari venerealis C. upsaliensis Neg Table 6) detection detection detection Control 2-1 (+) (+) (+) Low Negative 2-2 (+) (+) (+) Low Negative 2-3 (+) (+) Negative Negative 2-4 (+) (+) (+) Low Negative 2-5 (+) (+) Negative Negative 2-6 (+) Low Slope Negative Negative Negative 2-7 (+) (+) (+) Low (+) Low 2-8 (+) Negative Negative Negative 2-9 (+) (+) (+) Very Low (+) Low 2-10 (+) (+) (+) Very Low Negative 2-11 (+) (+) (+) Very Low Negative 2-12 (+) (+) (+) Very Low Negative 2-13 (+) Negative (+) Very Low Negative 2-14 (+) Negative (+) Low Negative 2-15 (+) Negative (+) Low Negative 2-16 (+) Negative (+) Low Negative 2-17 (+) Negative (+) Low Negative 2-18 (+) Negative (+) Low Negative 2-19 (+) (+) (+) Low Negative 2-20 (+) (+) (+) Low Negative 2-21 (+) (+) Low Negative Negative 2-22 (+) Negative Negative Negative
An additional screen was performed using oligo combination from Table 6 to identify torches with sensitivity towards C jejuni. C. lari, and C. coli and discrimination against C. upsaliensis and C. fetus. Positive criterion was set at 0.05 RFU on a MJ Chrom4 instrument. These oligonucleotide sets were tested against 1E5 copies of target organism per reaction. Results of this additional screen indicated that oligo combinations comprising Torch oligonucleotides SEQ ID NOS:63 & 65 cross-react with C. fetus, while those comprising Torch SEQ ID NO:66 had only a very low signal to C. fetus. Oligo combinations comprising SEQ ID NOS:67-68 showed very good sensitivity towards C. jejuni. C. lari, and C. coli and discrimination against C. upsaliensis and C. fetus.
1 1 Capture Heat 5 min-85.deg. C. Very slow No action 2 1 Capture Heat 15 min-65.deg. C. Very slow No action 3 2 Cool Heat 30 min-25.deg. C. No action No action (table rotated to empty position) 4 1 Mix prior to Mix No action 1 min-Very slow Collect beads- collect/collect count 20 Sample 1 5 3 Release to Wash Wash Release 30 s Slow 30 s Slow No action 6 1 Capture Wash Release 30 s Very 30 s Very Slow Collect beads- Sample 2 Slow (mix only) count 20 7 3 Release to Wash Release 30 s Slow 30 s Slow Collect beads- Wash 2 count 20 8 4 Capture and Wash Release 30 s Slow 30 s Slow No action release into Amp Soln
Copies of Target Avg TTime (Times (avg of TCO Target 10.sup.5) 4 samples)
SEQ ID NO: 71 C. fetus 5 n/a C. jejuni 0 n/a C. jejuni 1 19.5 C. jejuni 2 17.9 C. jejuni 3 15.6 C. jejuni 4 13.3 C. jejuni 5 11.3 C. ups 5 n/a SEQ ID NO: 73 C. fetus 5 n/a C. jejuni 0 n/a C. jejuni 1 19.8 C. jejuni 2 17.4 C. jejuni 3 15.3 C. jejuni 4 13.2 C. jejuni 5 10.9 C. ups 5 n/a SEQ ID NO: 58 C. fetus 5 n/a C. jejuni 0 n/a C. jejuni 1 19.1 C. jejuni 2 16.8 C. jejuni 3 14.8 C. jejuni 4 12.8 C. jejuni 5 11.2 C. ups 5 n/a SEQ ID NO: 71 C. jejuni 5 10.4 C. lari 0 n/a C. lari 1 17.4 C. lari 2 16.0 C. lari 3 12.7 C. lari 4 11.0 C. lari 5 8.4 C. ups 5 21.2* SEQ ID NO: 73 C. jejuni 5 10.0 C. lari 0 n/a C. lari 1 18.2 C. lari 2 15.5 C. lari 3 12.9 C. lari 4 10.3 C. lari 5 8.1 C. ups 5 21.6* SEQ ID NO: 58 C. jejuni 5 10.5 C. lari 0 n/a C. lari 1 18.1 C. lari 2 15.6 C. lari 3 12.8 C. lari 4 10.2 C. lari 5 8.3 C. ups 5 n/a C. coli 0 n/a C. coli 1 16.6 C. coli 2 14.4 C. coli 3 12.0 C. coli 4 10.1 C. coli 5 7.5 C. ups 5 n/a SEQ ID NO: 73 C. jejuni 5 7.9 C. coli 0 n/a C. coli 1 16.7 C. coli 2 14.5 C. coli 3 12.4 C. coli 4 10.0 C. coli 5 7.8 C. ups 5 n/a SEQ ID NO: 58 C. jejuni 5 8.1 C. coli 0 n/a C. coli 1 15.9 C. coli 2 13.5 C. coli 3 11.6 C. coli 4 9.2 C. coli 5 7.1 C. ups 5 n/a *1 of 4 samples showed a positive TTime for both test conditions.
Specificity Testing. Organisms that were closely related to the target organism but were genotypically distinct by rRNA analysis were selected as negatives. Seven challenge organisms were tested at 1E5 copies per reaction using the KingFisher mL instrument for target capture, an Eppendorf thermomixer for annealing the primers and for enzyme addition, and the PTI-FP-2® FluoDia plate reader was used for detection. Challenge organisms were C. fetus ssp venerealis; C. fetus ssp fetus; C. fecalis; E. coli; C. upsaliensis; S. enteritidis; and H. pylori . The positive control was C. jejuni (ATCC 33560) at 1E4 copies per positive control reaction. Lysis solution was used as negative control. All organisms were tested in replicate reactions. The positive criterion was set at 1000 RFU. Less than or equal to 7 of 140 reaction should be positive. Twenty-eight negative control reactions were included. The input for target capture was 1 mL; the output for target capture was 100 .micro.L, of which 30 .micro.L was used in the amplification. Testing for specificity at this stage yielded about a 98% rate of success in discriminating against the challenge organisms (3/140 were positive; all E. coli reaction wells) and false positive reactions were detected in 2 of 84 negative reaction wells. Thus, false positive rates in the E. coli wells are similar those in the negative control wells. The positive control was 100% positive. Table 11.
Copies per Percent Organism Reaction Positive C. fetus ssp venerealis 1E5 0% C. fetus ssp. fetus 1E5 0% C. fecalis 1E5 0% E. coli 1E5 0.5% C. upsaliensis 1E5 0% S. enteritidis 1E5 0% H. pylori 1E5 0% C. jejuni (positive) 1E4 100% Negative 0 2.4%
Various T7 Providers
SEQ ID NO: Sequence (5′ → 3′) 32 aatttaatacgactcactatagggagaCAGTTGGAAACGACTGCTAATACTCT 84 aatttaatacgactcactatagggagaCAGTTGGAAACGACTGCTAAT 85 aatttaatacgactcactatagggagaCAGTTGGAAACGACTGCTA 86 aatttaatacgactcactatagggagaCAATTGGAAACGACTGCTAATACTCT 87 aatttaatacgactcactatagggagaCAGTTGGAAACGACTGCCAACACTCT 88 aatttaatacgactcactatagggagaCAGTTGGAAACGACTGCTAACAC 26 aatttaatacgactcactatagggagaAAGAGGACAACAGTTGGAAAC
The amplification oligo combinations were screened for sensitivity to C. jejuni, C. coli and C. lari target nucleic acids. A subset of these combinations of amplification oligomers was then screened for interference and cross-reactivity by nearest neighbors. Sensitivity assays were set up as has been generally described herein. C. jejuni (ATCC 33291), C. coli (ATCC 33559) and C. lari (ATCC 35221) were tested at a level of 0, 1E2, 1E3 or 1E4 copies per reaction. Negative control was oligoless amplification reagent. Positive selection criterion was at least 2 of 3 samples having 1000 RFU for the 1E2 reaction and 3 of 3 samples having a TTime for RFU 1000 no greater than 20 minutes for the 1E4 reaction. Three amplification oligo combinations performed well for sensitivity testing.
Analytical Testing. The number of organisms tested was expanded to include twenty-five organisms for detection and fifteen challenge organisms. The organisms were tested at 1E5 copies/assay (approximately 100 CFU). Testing was performed on the Kingfisher 96 instrument for target capture and the PTI reader for detection. All were tested in replicate reactions of 3, from which one 30.micro.L replicate was amplified. For the inclusives, a positive reading in at least 2 of 3 replicates and for the exclusives, no more than 1 of 3 replicates should be positive. If these criteria are not met for any organism, testing for that species/strain will be repeated in replicates of 12. The inclusive organisms tested are C. jejuni, C. jejuni ssp doylei, C. coli, and C. lari. The exclusive organisms tested are E. coli, E. coli O157:H7, E. vulnaris, E. hermannii, Enterobacter cloacae, S. enteriditis, Edwardsiella hoshinae, P. mirabilis, Citrobacter brakii, Pseudomonas fluorescens, Shigella flexneri, Aeromonas hydrophila, Arcobacter butzleri, Campylobacter upsaliensis and Campylobacter fetus ssp fetus. Positive control was C. jejuni (ATCC 33560). All of the inclusive organisms yielded 100% positive detection except for two of 16 tested strains of C. jejuni (ATCC 35920 and 35925). These two strains of C. jejuni yielded 0 of 3 positives. None of the exclusive organisms yielded any positive results. Positive controls were 100% and there were no false positives.
Sample ID Avg TTime
In summary, real-time TMA technology was suitable for rapid, highly sensitive detection of food-borne pathogens. The assay had a sensitivity of 5E3 target nucleic acid copies/assay (approximately 10 CFU) for the desired species, C. jejuni, C. coli and C. lari, while excluding various nearest neighbors and potentially co-contaminating flora at 1E7 target nucleic acids copies/assay (approximately 10,000 CFU). The data demonstrated a rapid test format that allowed screening of food samples for Campylobacter within a single 8-hour work shift.
(a) contacting a sample suspected of containing at least a Campylobacter target nucleic acid with at least two amplification oligomers that stably hybridize to a C. jejuni target nucleic acid and a C. coli, a C. lari, or a C. coli and a C. lari target nucleic acid, wherein a first of said amplification oligomers comprises SEQ ID NO:26 or SEQ ID NO:32 and wherein a second of said amplification oligomers comprises a target hybridizing sequence 15 to 45 nucleotides in length and configured to target a sequence in a region of a Campylobacter 16S rRNA gene corresponding to nucleotides 170 to 226 of GenBank Accession No.: AF393202.1, gi:20378208;
2. The method of claim 1, wherein said second of said amplification oligomers comprises a target hybridizing sequence configured to target a sequence in a region of a Campylobacter 16S rRNA gene selected from the group consisting of: a region corresponding to nucleotides 170 to 212 of GenBank Accession No.: AF393202.1, gi:20378208; a region corresponding to nucleotides 184 to 212 of GenBank Accession No.: AF393202.1, gi:20378208; and a region corresponding to nucleotides 170 to 205 of GenBank Accession No.: AF393202.1, gi:20378208; or wherein said second amplification oligomer is selected from the group consisting of: SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6; SEQ ID NO:7; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; SEQ ID NO:11; SEQ ID NO:12; SEQ ID NO:13; and SEQ ID NO:14.
3. The method of claim 1, wherein said first of said amplification oligomers comprises SEQ ID NO:26.
4. The method of claim 1, wherein said first of said amplification oligomers comprises SEQ ID NO:32.
5. The method of claim 1, wherein said first amplification oligomer is SEQ ID NO:26 and said second amplification oligomer is selected from the group consisting of SEQ ID NOS:1 through 14.
6. The method of claim 1, wherein said second amplification oligomer consists essentially of SEQ ID NO:7.
7. The method of claim 1, wherein step b. further comprises contacting said sample with a blocker oligomer.
8. The method of claim 7, wherein said blocker oligomer consists essentially of SEQ ID NO:50.
9. The method of claim 1, wherein said detection reaction comprises contacting said amplification product with a detection probe oligomer configured to hybridize to a portion of said amplification product.
10. The method of claim 9, wherein said detection is real-time detection.
11. The method of claim 9, wherein said detection oligomer is selected from the group consisting of SEQ ID NOS:63 through 68.
12. The method of claim 9, wherein said detection oligomer consists essentially of SEQ ID NO:67.
13. The method of claim 9, wherein said detection oligomer consists essentially of SEQ ID NO:68.
14. The method of claim 9, wherein said second amplification oligomer is SEQ ID NO:7.
15. The method of claim 14, wherein said detection reaction comprises contacting said amplification product with a detection probe oligomer selected from the group consisting of SEQ ID NO:67 and SEQ ID NO:68.
16. The method of claim 15, wherein said first amplification oligomer is SEQ ID NO:26, and wherein step b. further comprises contacting said sample with a blocker consisting essentially of SEQ ID NO:50.
17. The method of claim 15, wherein said first amplification oligomer is SEQ ID NO:32, and wherein step b. further comprises contacting said sample with a blocker consisting essentially of SEQ ID NO:50.
18. The method of claim 1, further comprising the step of contacting said sample suspected of containing a Campylobacter target nucleic acid with a target capture oligomer.
19. The method of claim 18, wherein said target capture oligomer is selected from the group consisting of SEQ ID NOS:58, 71 and 73.
20. The method of claim 1, wherein said sample further contains nucleic acid from one or more bacteria closely related to C. jejuni; C. coli, or C. lari and said target nucleic acid is specifically detected in said sample.
21. The method of claim 20, wherein said one or more bacteria is at least one of: C. Fetus, ssp. fetus, C. fetus ssp venerealis, C upsaliensis, E. coli, C. fecalis, S. enteridis, H. pylori, E. vulnaris, E. hermannii, Enterobacter cloacae, Edwardsiella hoshinae, P. miribalis, Citrobacter brakii, Pseudomonas fluorescens, Shigella flexneri, Aeromonas hydrophila, A. butzleri, C. Fetus, ssp. fetus, C. fetus ssp venerealis, and C. upsaliensis.
22. The method of claim 1, wherein at step c said target nucleic acid is specifically amplified in the presence of a nucleic acid from one or more bacteria closely related to C. jejuni; C. coli, or C. lari.
23. The method of claim 22, wherein said one or more bacteria is at least one of: C. Fetus, ssp. fetus, C. fetus ssp venerealis, C upsaliensis, E. coli, C. fecalis, S. enteridis, H. pylori, E. vulnaris, E. hermannii, Enterobacter cloacae, Edwardsiella hoshinae, P. miribalis, Citrobacter brakii, Pseudomonas fluorescens, Shigella flexneri, Aeromonas hydrophila, A. butzleri, C. Fetus, ssp. fetus, C. fetus ssp venerealis, and C. upsaliensis.
24. A method for specifically detecting a Campylobacter target nucleic acid in a sample comprising the steps of:
(a) contacting a sample suspected of containing at least a Campylobacter target nucleic acid with at least two amplification oligomers that stably hybridize to a C. jejuni target nucleic acid and a C. coli, a C. lari, or a C. coli and a C. lari target nucleic acid, wherein a first of said amplification oligomers is a promoter based amplification oligomer comprising a target hybridizing sequence that is SEQ ID NO:27 or SEQ ID NO:33 and further comprising a 5′ promoter sequence that is SEQ ID NO:75 and wherein a second of said amplification oligomers comprises a target hybridizing sequence 15 to 45 nucleotides in length and configured to target a sequence in a region of a Campylobacter 16S rRNA gene corresponding to nucleotides 170 to 226 of GenBank Accession No.: AF393202.1, gi:20378208;
5004682 April 2, 1991 Roberts et al.
5447848 September 5, 1995 Barns et al.
5529910 June 25, 1996 Ohashi et al.
5571674 November 5, 1996 Hoshina et al.
5610060 March 11, 1997 Ward et al.
5639602 June 17, 1997 Rashtchian et al.
5663049 September 2, 1997 Barns et al.
5674684 October 7, 1997 Hogan et al.
5691138 November 25, 1997 Guesdon et al.
5695931 December 9, 1997 Labigne
5695960 December 9, 1997 Chan et al.
5738847 April 14, 1998 Braun
5756701 May 26, 1998 Wu et al.
5811237 September 22, 1998 Labigne
5874300 February 23, 1999 Blaser et al.
5945282 August 31, 1999 Rossau et al.
5981189 November 9, 1999 Chan et al.
5998138 December 7, 1999 Stonnet et al.
6001565 December 14, 1999 Fox et al.
6013501 January 11, 2000 Chan et al.
6066461 May 23, 2000 McMillian et al.
6080547 June 27, 2000 Fox et al.
6087105 July 11, 2000 Chan et al.
6156546 December 5, 2000 Konkel et al.
6166196 December 26, 2000 McMillian et al.
6221582 April 24, 2001 Giesendorf et al.
6245516 June 12, 2001 Al Rashid et al.
6277577 August 21, 2001 Rossau et al.
6355435 March 12, 2002 Wilson et al.
6372424 April 16, 2002 Brow et al.
6468743 October 22, 2002 Romick et al.
6593114 July 15, 2003 Kunsch et al.
6608190 August 19, 2003 Bramucci et al.
6656689 December 2, 2003 Rossau et al.
6673616 January 6, 2004 Dahlberg et al.
6737248 May 18, 2004 Kunsch et al.
7094893 August 22, 2006 Bramucci et al.
7214492 May 8, 2007 Rublee et al.
7303870 December 4, 2007 Hunter et al.
20020048762 April 25, 2002 Rossau et al.
20020055116 May 9, 2002 Cunningham et al.
20020061569 May 23, 2002 Haselbeck et al.
20030054338 March 20, 2003 Brow et al.
20030113757 June 19, 2003 Czajka
20030152916 August 14, 2003 Kacian et al.
20040053320 March 18, 2004 Rossau et al.
20040185446 September 23, 2004 Jones et al.
20040214302 October 28, 2004 Anthony et al.
20050053962 March 10, 2005 Blackburn et al.
20050123946 June 9, 2005 Snaidr et al.
20050123954 June 9, 2005 Feldsine
20050153282 July 14, 2005 Linnen et al.
20050158716 July 21, 2005 Dahlberg et al.
20050260603 November 24, 2005 Denise et al.
20050260619 November 24, 2005 Brousseau et al.
20060051752 March 9, 2006 Wang et al.
20070269813 November 22, 2007 Dewhirst et al.
20080038244 February 14, 2008 Glover et al.
20080124733 May 29, 2008 Fukui et al.
20080268452 October 30, 2008 Kaplan et al.
0 350 392 July 1989 EP
0 425 217 October 1990 EP
1921158 May 2008 EP
5-276999 October 1993 JP
06090795 April 1994 JP
06090796 April 1994 JP
2003164282 June 2003 JP
2003284559 October 2003 JP
2007068413 March 2007 JP
WO9010716 September 1990 WO
98/42842 October 1998 WO
99/24578 May 1999 WO
00/22430 April 2000 WO
00/58505 October 2000 WO
01/40497 June 2001 WO
01/57182 August 2001 WO
01/57274 August 2001 WO
01/59063 August 2001 WO
01/64835 September 2001 WO
02/077186 October 2002 WO
02/079476 October 2002 WO
02/086097 October 2002 WO
02/092818 November 2002 WO
02/097031 December 2002 WO
02/099109 December 2002 WO
03/000865 January 2003 WO
03/004623 January 2003 WO
03/106676 December 2003 WO
2004/024944 March 2004 WO
2005/027731 March 2005 WO
2005/083122 September 2005 WO
2006/029522 March 2006 WO
2006/115199 November 2006 WO
2007/047912 April 2007 WO
2008/016988 February 2008 WO
2008/041354 April 2008 WO
Savill et al. (J Appl. Micro, 2001, vol. 91, p. 38-46).
Lowe et al. (Nucleic Acids Research, 1990, 18(7):1757-1761).
Genbank Accession No. BX119966, “Zebrafish DNA sequence from clone CH211-266N15, complete sequence,” Dec. 6, 2003 [Retrieved from the Internet Feb. 10, 2010: <http://ncbi.nlm.nih.gov/nuccore/BX119966?log$=ACCN>].
Genbank Accession No. AC185373, “NISC Comparative Sequencing Initiative,” May 11, 2006 [Retrieved from the Internet Feb. 11, 2010: <http://ncbi.nlm.nih.gov/nuccore/95102109>].
Genbank Accession No. AC187781, “The sequence of Zea mays bac clone CH201-162N3,” Sep. 13, 2007 [Retrieved from the Internet Feb. 11, 2010: <http://ncbi.nlm.nih.gov/nuccore/157151864>].
Intl. Search Rpt. and Written Opinion in corresponding PCT application for PCT/US09/64516 dated Mar. 3, 2010.
Klena et al., “Differentiation of Campylobacter coli, Campylobacter jejuni, Campylobacter lari, and Campylobacter upsaliensis by a Multiplex PCR Developed from the Nucleotide Sequence of the Lipid A Gene IpxA,” J. Clin. Microbiol., Dec. 2004, pp. 5549-5557, vol. 42(12), American Society for Microbiology, Washington, D.C.
Chan et al., “The Absence of Intervening Sequences in 23S rRNA Genes of Campylobacter coli isolates from Turkeys is a unique attribute of a cluster of related strains which also lack re3sistance to erythromycin,” J. Clin. Microbiol., Feb. 2007, pp. 1208-1214, vol. 73(4), American Society for Microbiology, Washington, D.C.
Hannis et al., “High-Resolution Genotyping of Campylobacter Species by use of PCR and High-Throughput Mass Spectrometry,” J. Clin. Microbiol., Aug. 2008, pp. 1220-1225, vol. 46(4), American Society for Microbiology, Washington, D.C.
Hogan et al., “P-000 Rapid Method of Detecting Listeria genus, Salmonella genus, and Camplobacter using Real Time Transcription-mediated amplification assays targeted to ribosomal RNA,” Abstract and Poster, American Society for Microbiology 108th General Meeting, Jun. 1-5, 2008, Boston MA.
Patent Examination Report No. 1, Australian Patent Application No. 2009313808, mailed Sep. 6, 2012.
Extended European Search Report, European Patent Application No. 09826888.1, mailed Sep. 27, 2012.
P. S. Lubeck et al: “Toward an International Standard for PCR-Based Detection of Food-Borne Thermotolerant Campylobacters: Assay Development and Analytical Validation”, Applied and Environmental Microbiology, vol. 69, No. 9, Sep. 1, 2003, pp. 5664-5669, XP55037859.
Olsen J et al: “Probes and polymerase chain reaction for detection of food-borne bacterial pathogens”, International Journal of Food Microbiology, vol. 28, No. 1, Nov. 1, 1995, pp. 1-78, XP027287750.
Perelle S et al: “A LightCycler real-time PCR hybridization probe assay for detecting food-borne thermophilic Campylobacter”, Molecular and Cellular Probes, vol. 18, No. 5, Oct. 1, 2004, pp. 321-327, XP004523907.
Lehtola M J et al: “Advantages of peptide nucleic acid oligonucleotides for sensitive site directed 16S rRNA fluorescence in situ hybridization (FISH) detection of Campylobacter jejuni, Campylobacter coli and Campylobacter lari”, Journal of Microbiological Methods, vol. 62, No. 2, Aug. 1, 2005, pp. 211-219, XP027746438.
Database Geneseq [Online] Oct. 19, 2006, “Intestine tract pathogenic bacteria detection DNA probe—Seq Id 40.”, XP002683341, retrieved from EBI Database accession AEJ89751; & CN 1 683565 A (Radiation Medical Inst Academy [CN]) Oct. 19, 2005.
Database Geneseq [Online] May 24, 1994, “Campylobacter bacteria probe.”, XP002683342, retrieved from EBI Database accession No. AAQ51 650; & J P 5 276999 A Oct. 26, 1993.
Burnett, TA. et al. Speciating Campylobacter jejuni and Campylobacter Coli isolates from poultry and humans using six PCR-based assays. FEMS Microbiology Letters, vol. 216, p. 201-209, 2002.
Churruca, E. et al. Detection of campylobacter jejuni and campylobacter coli in chicken meat samples by real-time nucleic acid sequence-based amplification with molecular beacons. Int. J. Food Microbiol., vol. 117, p. 85-90, 2007.
PCT International Preliminary Report of Patentability, International Application No. PCT/US2009/064516, May 26, 2011.
Notice of Allowance, U.S. Appl. No. 12/618,910, mailed, Sep. 20, 2013.
Final Rejection, U.S. Appl. No. 12/618,910, mailed Apr. 26, 2013.
Non-final Office Action, U.S. Appl. No. 12/618,910, mailed Sep. 26, 2012.
Non-final Office Action (Restriction Requirement), U.S. Appl. No. 12/618,910, mailed Apr. 19, 2012.
Patent Publication Number: 20120052496
Application Number: 13/128,384
Current U.S. Class: Probes For Detection Of Microbial Nucleotide Sequences (536/24.32)