Nucleic acids and methods for the detection of <i>Enterobacter sakazakii </i>(<i>Cronobacter </i>spp.)

Abstract

Provided are detection means and method specific for the genus Cronobacter (E. sakazakii) which are sensitive by using a target molecule which is multiple existent in Cronobacter cells.

Claims

1. A method of amplifying DNA, comprising, providing a sample comprising bacteria, amplifying DNA from the bacteria with primers in an amplification step, wherein the primers used in the amplification step comprise a first primer comprising SEQ ID NO:2 or a variant thereof, together with a second primer comprising the complement of SEQ ID NO:3 or a variant thereof, wherein the variant of the first primer is a sequence which is identical to SEQ ID NO:2 other than the substitution of one nucleotide base within SEQ ID NO:2 with a different base and the variant of the second primer is a sequence which is identical to the complement of SEQ ID NO:3 other than the substitution of one nucleotide base within the complement of SEQ ID NO:3 with a different nucleotide base, wherein the sample comprises Cronobacter sakazakii strain DSM 4485 and Enterobacter turicensis strain DSM 18397, and wherein the primers amplify DNA from Cronobacter sakazakii strain DSM 4485, but do not amplify DNA from Enterobacter turicensis strain DSM 18397.

2. The method of claim 1, wherein the sample further comprises Enterobacter helveticus strain DSM 18396, and wherein the primers also do not amplify DNA from Enterobacter helveticus strain DSM 183966.

3. The method of claim 2, wherein the sample further comprises Enterobacter pulveris strain DSM 19144, and wherein the primers also do not amplify DNA from Enterobacter pulveris strain DSM 19144.

Description

FIGURES OF THE INVENTION

(1) FIG. 1 shows the DNA region as well as the hybridization positions of the oligonucleotides of this invention.

(2) FIG. 2 shows the Comparison of the 23S-ITS2-5S region of the type strain of C. sakazakii (DSM 4485) with sequences of the species E. helveticus, E. pulveris and E. turicensis.

(3) FIG. 3 shows a melting curve analysis for differentiation of C. turicensis from other Cronobacter spp

(4) FIGS. 4A and 4B respectively show shows the simultaneous detection of Enterobacteriaceae and Cronobacter spp. by probe-based multiplex real-time PCR.

(5) FIG. 5 shows the selective amplification of DNA from viable Cronobacter cells after treatment with EMA. Enrichment cultures with 10.sup.6/ml viable or dead C. sakazakii cells were prepared with or without treatment with ethidium monoazide bromide (EMA). The resulting DNA extracts were tested with the multiplex real-time PCR for Cronobacter and Enterobacteriaceae detection.

EXAMPLES

Example 1: Comparison of Three Primer Pairs Targeting the Ribosomal Operon Regarding Detection Sensitivity of DNA of Cronobacter spp.

(6) Genomic DNA was isolated from pure cultures of the bacteria listed in Table 1 using known standard methods. Dilutions (DNA concentration 100 pg/μl and 1 pg/μl) of these preparations were then added to three different PCR mixes with the following compositions:

(7) Mix A: with primer pair es-16s-for (CAA GTC GAA CGG TAA CAG GG) (SEQ ID NO: 35)/es-16s-rev (GTC CCC CAC TTT GGT CCG) (SEQ ID NO: 36) targeting the 16S rDNA gene published by Malorny & Wagner, 2005 (J. Food Prot. 68: 1623-1627).

(8) Mix B: with primer pair ESFor (ATC TCA AAA MTG ACT GTA AAG TCA CGT T) (SEQ ID NO: 37)/ESRevB (CCG AAR AAG TMT TCG KGC TGC GA) (SEQ ID NO: 38) targeting the region between tRNA-glu- and 23S rDNA-gene published by Derzelle & Dilasser, 2006 (BMC Microbiol. 6: 100).
Mix C: with primer pair SEQ ID NO: 2/SEQ ID NO: 3 targeting the 23S-ITS2-5S region of this invention.

(9) TABLE-US-00001 Final Final Final Conc. Conc. Conc. Component Mix A Mix B Mix C H.sub.2O — — — PCR buffer 1 x conc. 1 x conc. 1 x conc. MgCl.sub.2 4.5 mM 3 mM 3.75 mM dNTP-mix 200 μM each 200 μM each 200 μM each Primer 400 nM — — es-16s-for Primer 400 nM — — es-16s-rev Primer ESFor — 500 nM — Primer — 500 nM — ESRevB SEQ ID — — 600 nM NO: 2 SEQ ID — — 600 nM NO: 3.sup.a) Taq DNA 0.06 U/μl 0.06 U/μl 0.06 U/μl Polymerase Sybr Green ® 0.25 x conc 0.25 x conc 0.25 x conc Sample DNA Var. Var. Var. .sup.a)Complement sequence was used

(10) As described by Derzelle & Dilasser the “FastStart Taq Polymerase” (Roche Diagnostics) was used to prevent unspecific amplification. The DNA intercalating fluorescent dye Sybr Green® was added to estimate the PCR efficiency by measuring the CT-values on a real-time PCR instrument.

(11) The PCR was carried out on a LightCycler® 480 instrument (Roche Diagnostics) according to the following protocols:

(12) TABLE-US-00002 Protocol 1 for mix A and C Initial denaturation 95° C. 10 min. Amplification (45 cycles) 95° C. 15 s. 66° C. 30 s. 72° C. 5 s. 84° C. 10 s. (fluorescence acquisition) Protocol 2 for mix B and C Initial denaturation 95° C. 10 min. Amplification (45 cycles) 95° C. 15 s. 60° C. 10 s. 72° C. 25 s. (fluorescence acquisition)

(13) TABLE-US-00003 TABLE 1 Detection sensitivity of primer pair SEQ ID NO 2 and SEQ ID NO 3 (mix C) in comparison to two other primer pairs targeting the ribosomal operon CP-Value.sup.1 Mix A Mix B Mix C Mix C DNA Proto- Proto- Proto- Proto- Strain amount col 1 col 2 col 1 col 2 C. sakazakii 250 pg 15.5 22.1 15.9 16.3 DSM 4485 C. sakazakii 2.5 pg 23.5 33.7 22.7 24.6 DSM 4485 C. dublinensis 250 pg 29.4 21.8 16.7 16.9 subsp. dublinensis DSM 18705 C. dublinensis 2.5 pg 31.8 31.1 23.5 24.3 subsp. dublinensis DSM 18705 C. dublinensis 250 pg 18.8 23.1 15.7 16.1 subsp. lactaridi DSM 18707 C. dublinensis 2.5 pg 25.4 33.4 22.6 23.8 subsp. lactaridi DSM 18707 C. malonaticus 250 pg 16.3 24.0 16.5 17.8 DSM 18702 C. malonaticus 2.5 pg 23.5 34.4 23.5 24.6 DSM 18702 C. muytjensii 250 pg 34.5 23.7 15.7 16.0 ATCC 51329 C. muytjensii 2.5 pg negative 33.8 22.5 22.9 ATCC 51329 C. turicensis 250 pg 20.8 32.0 15.6 16.2 DSM 18703 C. turicensis 2.5 pg 25.3 35.2 22.5 22.7 DSM 18703 .sup.1CP-Value: The crossing point is calculated by the LightCycler Software as the cycle at which PCR amplification begins its detectable exponential phase

Example 2: Discrimination of C. turicensis from Other Cronobacter Spp. by Melting Curve Analysis

(14) Genomic DNA was isolated from pure cultures of the bacteria listed in FIG. 2 using known standard methods. The DNA of these preparations was then added to the PCR mix with the following composition (final volume 20 NI):

(15) TABLE-US-00004 Component final Concentration H.sub.2O — PCR buffer 1 x conc. MgCl.sub.2 3.75 mM dNTP-mix 200 μM each SEQ ID NO: 2 1 μM SEQ ID NO: 3.sup.a) 300 nM SEQ ID NO: 4-7.sup.b) 45 nM each SEQ ID NO: 8-9.sup.c) 100 nM each Taq DNA Polymerase 0.06 U/μl Sample DNA 125 fg/μl .sup.a)Complement sequence .sup.b)3′-labeled with fluorescein, complement sequence .sup.c)5′-labeled with Cy5, complement sequence

(16) FIG. 2 shows the Comparison of the 23S-ITS2-5S region of the type strain of C. sakazakii (DSM 4485) with sequences of the species E. helveticus, E. pulveris and E. turicensis.

(17) The PCR was carried out on a LightCycler® 2.0 instrument (Roche Diagnostics) according to the following protocol:

(18) TABLE-US-00005 Initial denaturation 95° C. 10 min. Amplification (45 cycles) 95° C. 0 s. 59° C. 30 s. 72° C. 5 s. Melting curve analysis 95° C. 0 s. (1 cycle) 40° C. 45 s. {close oversize brace} ramp rate 0.1°/s 75° C. 0 s. Cooling 40° C. 30 s.

(19) The melting curve analysis for differentiation of C. turicensis from other Cronobacter spp is shown in FIG. 3. Coupled to fluorescent molecules oligonucleotides of SEQ ID NO: 4-7 and SEQ ID NO: 8-9 which hybridise adjacently enable the differentiation of C. turicensis from other Cronobacter species. In addition it is possible to discriminate between different strains of C. turicensis.

Example 3: Specific Detection of a Subgroup of C. turicensis

(20) Genomic DNA was isolated from pure cultures of the bacteria listed in Table 2 using known standard methods. The DNA of these preparations was then added to the PCR mix with the following composition (final volume 20 μl):

(21) TABLE-US-00006 Component Final Concentration H.sub.2O — PCR buffer 1 x conc. MgCl.sub.2 3.75 mM dNTP-mix 200 μM each SEQ ID NO: 2 600 nM SEQ ID NO: 3.sup.a) 600 nM SEQ ID NO: 20.sup.b) 180 nM SEQ ID NO: 21.sup.c) 200 nM Taq DNA Polymerase 0.06 U/μl Sample DNA 125 fg/μl .sup.a)Complement sequence .sup.b)3′-labeled with fluorescein, complement sequence .sup.c)5′-labeled with Cy5, complement sequence

(22) The PCR was carried out on a LightCycler® 2.0 instrument (Roche Diagnostics) according to the following protocol:

(23) TABLE-US-00007 Initial denaturation 95° C. 10 min. Amplification (45 cycles) 95° C. 0 s. 59° C. 30 s. (fluorescence acquisition) 72° C. 5 s. Cooling 40° C. 30 s.

(24) The results of the real-time PCR using the oligonucleotides SEQ ID NO: 20 and SEQ ID NO: 21 as probes are shown in table 2. Only the two C. turicensis strains LMG 2790 and E609 were detected.

(25) TABLE-US-00008 TABLE 2 Specific PCR detection of a C. turicensis subgroup using SEQ ID NO 2, 3, 20 and 21 Species Strain PCR result C. turicensis LMG 2790 + C. turicensis E609 + C. turicensis DSM 18703 − C. turicensis BCD 15786 − C. dublinensis subsp. dublinensis DSM 18705 − C. dublinensis subsp. lactaridi DSM 18707 − C. malonaticus DSM 18702 − C. muytjensii ATCC 51329 − C. sakazakii DSM 4485 − C. genomospecies 1 NCTC 9529 −

Example 4: Detection of Cronobacter Spp. by Probe-Based Real-Time PCR

(26) Genomic DNA was isolated from pure cultures of the bacteria listed in Table 3 using known standard methods. The DNA of these preparations (concentration in the area of approx. 10 ng-100 pg) as well as the amplification control DNA were then added to the PCR mix with the following composition (final volume 20 μl):

(27) TABLE-US-00009 Component final Concentration H.sub.2O — PCR buffer 1 x conc. MgCl.sub.2 3.75 mM dNTP-mix 200 μM each SEQ ID NO: 2 600 nM SEQ ID NO: 3.sup.a) 600 nM SEQ ID NO: 4-7.sup.b) 45 nM each SEQ ID NO: 20.sup.b) 180 nM SEQ ID NO: 8-9.sup.c) 100 nM each SEQ ID NO: 21.sup.c) 200 nM SEQ ID NO: 22.sup.d) 200 nM Taq DNA Polymerase 0.06 U/μl Control DNA 0.25 fg/μl Sample DNA var. .sup.a)Complement sequence .sup.b)3′-labeled with fluorescein, complement sequence .sup.c)5′-labeled with Cy5, complement sequence .sup.d)5′-labeled with LC-Red ® 610

(28) The PCR was carried out on a LightCycler® 2.0 instrument (Roche Diagnostics) according to the following protocol:

(29) TABLE-US-00010 Initial denaturation 95° C. 10 min. Amplification (45 cycles) 95° C. 0 s. 59° C. 30 s. (fluorescence acquisition) 72° C. 5 s. Cooling 40° C. 30 s.

(30) Amplification of Cronobacter spp. DNA was detected in channel 670/back 530. In cases of a negative result amplification of the control DNA should be visible in channel 610/back 530. The results of the probe-based real-time PCR are shown in table 3. All tested Cronobacter strains comprising all species and subspecies as well as C. genomospecies 1 were detected. In contrast, none of the tested bacterial strains not belonging to this genus was registered with this system.

(31) The amplification control was detected in channel 610/back 530 for all samples with a negative result for Cronobacter DNA. The quality of these non-Cronobacter DNA samples was checked with consensus PCR system specific for all eubacteria targeting the 16S rDNA (Barry et al., 1990, Biotechnology 8: 233-236).

(32) TABLE-US-00011 TABLE 3 Specific PCR detection of the genus Cronobacter using SEQ ID NO 2, 3, 4-7, 8-9, 20 and 21 Species Strain.sup.1 PCR result Cronobacter dublinensis subsp. dublinensis DSM 18705 + Cronobacter dublinensis subsp. lactaridi DSM 18707 + Cronobacter dublinensis subsp. NCTC 9844 + Cronobacter malonaticus DSM 18702 + Cronobacter muytjensii ATCC 51329 + Cronobacter sakazakii DSM 4485 + Cronobacter turicensis DSM 18703 + Cronobacter turicensis LMG 2790 + Cronobacter turicensis E609 + Cronobacter turicensis BCD 15786 + Cronobacter genomospecies 1 NCTC 9529 + Enterobacter sakazakii BCD 15300 + Enterobacter sakazakii BCD 15301 + Enterobacter sakazakii BCD 15361 + Enterobacter sakazakii BCD 15381 + Enterobacter sakazakii BCD 15410 + Enterobacter sakazakii BCD 15428 + Enterobacter sakazakii BCD 15429 + Enterobacter sakazakii BCD 15430 + Enterobacter sakazakii BCD 15431 + Enterobacter sakazakii BCD 15432 + Enterobacter sakazakii BCD 15433 + Enterobacter sakazakii BCD 15434 + Enterobacter sakazakii BCD 15435 + Enterobacter sakazakii BCD 15436 + Enterobacter sakazakii BCD 15437 + Enterobacter sakazakii BCD 15438 + Enterobacter sakazakii BCD 15439 + Enterobacter sakazakii BCD 15440 + Enterobacter sakazakii BCD 15441 + Enterobacter sakazakii BCD 15442 + Enterobacter sakazakii BCD 15443 + Enterobacter sakazakii BCD 15444 + Enterobacter sakazakii BCD 15445 + Enterobacter sakazakii BCD 15446 + Enterobacter sakazakii BCD 15447 + Enterobacter sakazakii BCD 15448 + Enterobacter sakazakii BCD 15449 + Enterobacter sakazakii BCD 15450 + Enterobacter sakazakii BCD 15451 + Enterobacter sakazakii BCD 15452 + Enterobacter sakazakii BCD 15459 + Enterobacter sakazakii BCD 15460 + Enterobacter sakazakii BCD 15461 + Enterobacter sakazakii BCD 15462 + Enterobacter sakazakii BCD 15463 + Enterobacter sakazakii BCD 15464 + Enterobacter sakazakii BCD 15469 + Enterobacter sakazakii BCD 15470 + Enterobacter sakazakii BCD 15471 + Enterobacter sakazakii BCD 15472 + Enterobacter sakazakii BCD 15473 + Enterobacter sakazakii BCD 15474 + Enterobacter sakazakii BCD 15475 + Enterobacter sakazakii BCD 15476 + Enterobacter sakazakii BCD 15477 + Enterobacter sakazakii BCD 15478 + Enterobacter sakazakii BCD 15488 + Enterobacter sakazakii BCD 15489 + Enterobacter sakazakii BCD 15490 + Enterobacter sakazakii BCD 15492 + Enterobacter sakazakii BCD 15495 + Enterobacter sakazakii BCD 15497 + Enterobacter sakazakii BCD 15498 + Enterobacter sakazakii BCD 15499 + Enterobacter sakazakii BCD 15500 + Enterobacter sakazakii BCD 15501 + Budvicia aquatica DSM 5075 − Buttiauxella agrestis DSM 4586 − Cedecea davisae DSM 4568 − Citrobacter amalonaticus DSM 4593 − Citrobacter freundii BCD 4696 − Citrobacter koseri DSM 4595 − Edwardsiella tarda DSM 30052 − Enterobacter aerogenes DSM 30053 − Enterobacter amnigenus DSM 4486 − Enterobacter cancerogenus CCM 2421 − Enterobacter cloacae DSM 30054 − Enterobacter cowanii CCM 7015 − Enterobacter gergoviae BCD 674 − Enterobacter helveticus DSM 18396 − Enterbacter hormaechei DSM 12409 − Enterobacter pulveris DSM 19144 − Enterobacter pyrinus DSM 12410 − Enterobacter turicensis DSM 18397 − Erwinia carotovora DSM 30168 − Erwinia crysanthemi DSM 4610 − Escherichia coli DSM 30083 − Escherichia hermannii DSM 4560 − Escherichia vulneris DSM 4564 − Hafnia alvei DSM 30163 − Klebsiella oxytoca DSM 5175 − Klebsiella pneumoniae DSM 30102 − Kluyvera ascorbata DSM 4611 − Kluyvera cryocrescens DSM 4588 − Leclercia adecarboxylata DSM 5077 − Morganella (Proteus) morganii DSM 30164 − Pantoea agglomerans DSM 3493 − Pantoea ananatis DSM 30070 − Pantoea dispersa DSM 30073 − Proteus hauseri DSM 30118 − Proteus mirabilis DSM 788 − Providencia alcalifaciens DSM 30120 − Providencia stuartii DSM 4539 − Rahnella aquatilis DSM 4594 − Raoultella (Klebsiella) planticola DSM 4617 − Raoultella (Klebsiella) terrigena DSM 2687 − Salmonella Abony DSM 4224 − Salmonella bongori BCD 14407 − Serratia ficaria DSM 4569 − Serratia marcescens DSM 1636 − Serratia proteamaculans DSM 4487 − Serratia rubidaea DSM 4480 − Shigella boydii DSM 7532 − Yersinia aldovae ATCC 35236 − Yersinia enterocolitica DSM 4780 − Yersinia frederiksenii ATCC 33641 − For all negative tested strains was the amplification control positive. .sup.1Abbreviations stands for the following culture collections: ATCC: American Type Culture Collection, Manassas, USA BCD: BIOTECON Diagnostics culture collection, Potsdam, Germany CCM: Czech Collection of Microorganisms, Brno, Czech Republic DSM: German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany LMG: BCCM/LMG Bacteria Collection, Gent, Belgium NCTC: National Collection of Type Cultures, Salisbury, UK

Example 5: Simultaneous Detection of Cronobacter Spp. and Enterobacteriaceae by Probe-Based Multiplex Real-Time PCR

(33) Genomic DNA was isolated from pure cultures of the bacteria listed in FIG. 3 using known standard methods. The DNA of these preparations (concentration approx. 5-50 ng) as well as the amplification control DNA was then added to the multiplex PCR mix with the following composition (final volume 25 μl):

(34) TABLE-US-00012 Component Final Concentration H.sub.2O — PCR buffer 1 x conc. MgCl.sub.2 3.75 mM dNTP-mix 200 μM each SEQ ID NO: 2 600 nM SEQ ID NO: 3.sup.a) 600 nM SEQ ID NO: 8-9.sup.b) 125 nM each SEQ ID NO: 21.sup.b) 250 nM Enterobacteriaceae forward-primer 600 nM Enterobacteriaceae reverse-primer 600 nM Enterobacteriaceae probes 1-4.sup.c) 150 nM each Taq DNA Polymerase 0.06 U/μl Control DNA 0.25 fg/μl Sample DNA var. .sup.a)Complement sequence .sup.b)5′-labeled with FAM, 3′-labeled with a quencher, complement sequence .sup.c)5′-labeled with HEX, 3′-labeled with a quencher

(35) The Enterobacteriaceae forward-primer is equivalent to SEQ ID NO 2 (TTC GGG TTG TCATGC CAA TG), the Enterobacteriaceae reverse-primer to the complement sequence of SEQ ID NO 78 (ACC CGT GAG GCT TAA CCT TAC AAC ACC GAA) of the invention WO 01/023606, and the Enterobacteriaceae probes 1-4 are equivalent to SEQ ID NO 3 (CTG AAA GCA TCT AAG CGC GAA ACT TG), SEQ ID NO 4 (CTG AAA GCA TCT AAG CGG GAA ACT TG), SEQ ID NO 5 (CTG AAA GCA TCT AAG CAC GAA CTT G) and SEQ ID NO 6 (CTG AAA GCA TCT AAG CAC GAA ACT TG) of the invention WO 01/023606.

(36) The PCR was carried out on a LightCycler® 480 instrument (Roche Diagnostics) according to the following protocols:

(37) TABLE-US-00013 Initial denaturation 95° C. 5 min. Amplification (40 cycles) 95° C. 10 s. 65° C.-61° C. 70 s. (fluorescence acquisition) step down each cycle by 0.1° C.

(38) The results of the probe-based multiplex real-time PCR are shown in FIG. 4, which shows the simultaneous detection of Cronobacter spp. and Enterobacteriaceae by probe-based multiplex real-time PCR. The specific detection of Cronobacter DNA occurred in the FAM-whereas the DNA amplification of the complete Enterobacteriaceae family could be detected in the HEX-channel.

Example 6: Selective Amplification of DNA from Viable Cronobacter Cells

(39) An according to ISO/TS 22964 pre-enriched sample of infant formula was subcultivated in buffered peptone water (BPW). This subculture were inoculated to 10.sup.6 dead C. sakazakii cells per ml. Inactivation was done by incubation of a C. sakazakii culture with 5×10.sup.6/ml for 10 min at 70° C. (inactivation checked by plating). As controls additional samples were inoculated with viable C. sakazakii cells.

(40) For selective inactivation of DNA from dead cells 100 μl of each sample were mixed with 300 μl of a solution containing 20 μg/ml ethidium bromide monoazide (EMA). After incubation for 5 minutes in dark, the samples were placed on ice, exposed for 5 minutes to a 500 W halogen light bulb in approx. 15-20 cm distance. Afterwards the mixtures were centrifuged (5 min. at 10 000×g) and the supernatant discarded. DNA isolation was performed by resuspension of the cell pellet in 200 μl lysis solution containing Chelex x-100 and incubation at 95-100° C. for 10 minutes. As controls the DNA from 100 μl of all spiked samples was isolated without a treatment with EMA.

(41) PCR mix and protocol were the same as in example 5. The results of this experiment are shown in FIG. 5.

(42) As shown in FIG. 5 the addition of DNA from dead Cronobacter cells without a treatment with EMA to the Cronobacter specific PCR system results in clearly visible fluorescence curves, whereas in the treated samples no amplification is detectable. On viable cells the PCR detection sensitivity is only slightly reduced by this EMA-procedure. Therefore the PCR-based method of this invention could be combined with the EMA-technique and enables a sensitive detection of only viable Cronobacter cells.