CA2286512A1 - Oligonucleotide primers for the specific pcr detection of enterobacteriaceae species dna using rfe-rff gene templates - Google Patents
Oligonucleotide primers for the specific pcr detection of enterobacteriaceae species dna using rfe-rff gene templates Download PDFInfo
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Abstract
Oligonucleotide primers were designed for the PCR-based detection of rfe-rff genes involved in the biosynthesis pathway leading to the production of enterobacterial common antigen (ECA). E. coli DNA was detected, using specific rfe (WecA) and rffT
(WecF) gene primers. Moreover, rffT primers allowed the detection of most members of the Enterobacteriaceae family in biological fluids such as blood and urine with sensitivity as low as 120 bacteria per ml of water. Thus, these primers represent an important step in the molecular diagnosis of major Enterobacteriaceae infections and routine testing of contamination in drinking water and food. The invention relates to particular oligonucleotides, the corresponding primers, the use of these primers for PCR detection of Enterobacteriaceae species, the related method for PCR
detection and the related diagnostic assay.
(WecF) gene primers. Moreover, rffT primers allowed the detection of most members of the Enterobacteriaceae family in biological fluids such as blood and urine with sensitivity as low as 120 bacteria per ml of water. Thus, these primers represent an important step in the molecular diagnosis of major Enterobacteriaceae infections and routine testing of contamination in drinking water and food. The invention relates to particular oligonucleotides, the corresponding primers, the use of these primers for PCR detection of Enterobacteriaceae species, the related method for PCR
detection and the related diagnostic assay.
Description
t Oligonucleotide primers for the specific PCR detection of enterobacteriaceae species DNA using rfe-rff gene templates FIELD OF THE INVENTION
The present invention relates to novel PCR oligonucleotide primers for the detection of Enterobacteriaceae species. More particularly, these oligonucleotide primers are directed to rfff gene of Enterobacteriaceae species. The present invention also relates to uses of these oligonucleotide primers for PCR detection of Enterobacteriaceae species and methods for detecting Enterobacteriaceae species by PCR using these oligonucleotide primers.
BACKGROUND OF THE INVENTION
Enterobacteriaceae are gram-negative bacilli usually found in the gastrointestinal tract and are frequently associated with septicemia, meningitis, pneumonia, peritonitis and urinary tract infections. This family of bacteria includes Escherichia coli, Klebsiella pneumoniae, Enterobacter sp., Serratia sp., Salmonella sp., Shigella sp., Citrobacter sp., Yersinia sp., Proteus sp., and Providencia sp. (Farmer, J.J.
Enterobacteriaceae Introduction and Identification. In Murray P.R. et aL,Manual of Clinical Microbiology 1995, pp. 438-464). A specific common feature of this family (Kuhn, H.M. et al., ECA, the Enterobacterial Common Antigen. FEMS Microbiology Reviews. 1988, 54 , pp.
195-222) is the synthesis of enterobacterial common antigen (ECA).
ECA, a glycophospholipid surface antigen of the enterobacteriaceae family, is composed of carbohydrates linked to L-glycerophosphatidyl residues. The carbohydrates comprise N-acetyl-D-glucosamine (GIcNAc), N-acetyl-D-mannosaminuronic (ManNAcA), 4-acetamido-4 and 6-dideoxy-D-galactose (Fuc4NAc). These amino-sugars constitute linear polysaccharide chains of repetitive trisaccharide units. The polysaccharide chains and the lipid component are respectively responsible for serological specificity and anchorage of ECA to the external membrane of enterobacteriaceae (Meier-Dieter U. et al., Nucleotide Sequence of the Escherichia coli rfe Gene Involved in the Synthesis of Enterobacterial Common Antigen. J. Biol. Chem. 1992 , 267 , pp. 746-753; and Ohta M.O. et al., Cloning and expression of the rfe-rff Gene Cluster of Escherichia coli. Mol.
Microbiol.
1991, 5, pp. 1853-1862). ECA has immunologic properties (Peter, H. et al.
Monoclonal Antibodies to Enterobacterial Common Antigen and to Escherichia coli Lipopolysaccharide Outer Core: Demonstration of an Antigen Determinant Shared by Enterobacterial Common Antigen and E. coli K5 Capsular Polysaccharide. Infect.
Immun. 1995, 50, pp. 459-466). A protective action has not been demonstrated in humans but its immunologic properties have been used for diagnostic purposes.
The most frequent diagnostic methods of ECA detection rely on enzyme immunoassay and the passive hemagglutination test (Hubner I. et al. Rapid Determination of Members of the Family Enterobacteriaceae in Drinking Water by an Immunological Assay Using a Monoclonal Antibody Against Enterobacterial Common Antigen. Appl. Environ. Microb. 1992, 58, pp. 3187-3191; and Bayardelle P. and Ranger S. Rapid Detection of Enterobacteriaceae in Urine with Passive Hemagglutination Test. American Society for Microbiology, 95th Meeting.
Washington.
May 1995). The Sensitivity of the enzyme immunoassay is low for Enterobacteriaceae and varies according to the ECA concentration of the species. The detection limit is about 3,9X105 colony-forming units per ml (CFU/ml) of water and is higher for other species (1,3X10' CFU/ml). Although specific, the test presents false positive in 0.3%
of samples. Passive hemagglutination can be used for urine samples with 100,000 CFU/ml but gave false positive results at 4.6%.
Although ECA genes have been well characterized (Meier-Dieter U. et al. J.
Biol.
Chem. 1992, 267, pp. 746-753; Ohta M.O. et al. Mol. Microbiol. 1991, 5, pp.
1853-1862; Marolda C.L. and Valvano M.A. Genetic Analysis of the dTDP-Rhamnose Biosynthesis Region of the Escherichia coli VW187 (07:KI) rfb Gene Cluster:
Identification of Fonctional Homologs of rfb8 and rfbA in the rff Cluster and Correct Location of the rffE Gene. J. Bacteriol. 1995, 177, pp. 5539-5546; Alexander D.C. and Valvano M.A. Role of the rfe Gene in the Biosynthesis of the Escherichia coli 07-Specific Lipopolysaccharide and Other O-Specific Polysaccharides Containing N-Acetylglucosamine. J. Bacteriol. 1994, 176, pp. 7079-7084; Whitfield C. et al.
Modulation of the Surface Architecture of Gram-negative Bacteria by the Action of Surface Polymer: Lipid A-core Ligase and by Determinants of Polymer Chain Length.
Mol. Microbiol. 1997, 23, pp. 629-638; Amor P.A. and Whitfield. Molecular and Functional Analysis of Genes Required for Expression of Group IB K Antigens in Escherichia coli: Characterization of the his-region Containing Gene Clusters for Multiple Cell-surface Polysaccharides. Mol. Microbiol. 1997, 26 pp.145-161) and new nomenclature implemented (Reeves P.R. et al. Bacterial Polysaccharide Synthesis and Gene Nomenclature. Trends in Microbiology. 1996, 4, pp. 495-503), no PCR-based diagnostic test has yet been developed. Some previous PCR primers designed to determine the function of certain genes are species-specific (Marolda C.L.
and Valvano M.A. J. Bacteriol. 1995, 177, pp. 5539-5546). In the new nomenclature, rfe and rff gene cluster is now identified wec gene cluster.
The detection of pathogen-specific genomic DNA was attempted several years ago from diverse infectious agents in clinical specimens (Song J.H et al.
Detection of Salmonella typhi in the Blood of Patients with Typhoid Fever by Polymerase Chain Reaction. J.Clin. Microbiol. 1993, 31, pp. 1439-1443; Zhang Y. et al.
Detection of Streptococcus pneumoniae in Whole Blood by PCR. J. Clin. Microbiol. 1995, 33, pp.
596-601; Greisen,K. et al. PCR Primers and Probes for the 16S rRNA Gene of Most Species of Pathogenic Bacteria, Including Bacteria found in Cerebrospinal fluid. J.
Clin. Microbiol. 1994, 32, pp. 335-351; Whelen A.C. and Persing D.H. The Role of Nucleic Acid Amplification and Detection in the Clinical Microbiology Laboratory.
Annual Review of Microbiology. 1996, 50, pp. 349-373; Hashimoto Y. et al.
Development of Nested PCR Based on the Viab Sequence To Detect Salmonella typhi. J. Clin. Microbiol. 1995, 33, pp. 775-777). Broad-range PCR primers for rRNA have been used to amplify either gram-positive or gram-negative bacteria but this technique calls for a combination of PCR and Southern blot hybridization which is time-consuming and does not allow specific identification of Enterobacteriaceae species (Greisen,K., M. et al. PCR Primers and Probes for the 16S rRNA Gene of Most Species of Pathogenic Bacteria, Including Bacteria Found in Cerebrospinal fluid.
a J. Clin. Microbiol. 1994, 32, pp. 335-351; Song J.H et al. Detection of Salmonella typhi in the Blood of Patients with Typhoid Fever by Polymerase Chain Reaction.
J.Clin. Microbiol. 1993, 31, pp. 1439-1443).
It has been hypothesized that n'e and rff genes, implicated in the synthesis of ECA, represent good targets for the broad spectrum detection of Enterobacteriaceae.
The rfe gene is essential for ECA synthesis although its precise function has not been determined. It appears to encode for a transferase which catalyzes the synthesis of GIcNAc-pyrophosphorylundecaprol (lipid I), the essential first intermediate lipid involved in ECA synthesis, while the rftT gene encodes for a transferase which reassembles Fuc4NAc and ManNAcA, thus completing the repetitive trisaccharide unit of ECA.
It is an object of the present invention to develop a fast PCR-based diagnostic test and to evaluate its sensitivity and specificity as well as its applicability to the routine detection of enterobacteriaceae in biological fluids such as patient blood and urine.
SUMMARY OF THE INVENTION
A first aspect of the invention provides an oligonucleotide selected from the group consisting of:
- RFFT11 having the nucleotide sequence of SEQ ID NO. 1;
- RFFT20 having the nucleotide sequence of SEQ ID NO. 2;
- RFFT17 having the nucleotide sequence of SEQ ID NO. 3;
- RFFT21 having the nucleotide sequence of SEQ ID NO. 4;
- RFFT22 having the nucleotide sequence of SEQ ID NO. 5;
- RFFT7 having the nucleotide sequence of SEQ ID NO. 6;
- RFFT8 having the nucleotide sequence of SEQ ID NO. 7; and RFFT18 having the nucleotide sequence of SEQ ID NO. 8.
A second aspect of the invention provides an oligonucleotide primer selected from the group consisting of RFFT11 (SEQ ID NO. 1); RFFT20 (SEQ ID NO. 2); RFFT17 (SEQ
r ID NO. 3); RFFT21 (SEQ ID N0.4); RFFT22 (SEQ ID NO. 5); RFFT7 (SEQ ID
NO. 6); RFFT8 (SEQ ID NO. 7); and RFFT18 (SEQ ID NO. 8).
A third aspect of the invention provides an oligonucleotide primer comprising the nucleotide sequence of the oligonucleotide selected from the above-defined group or a substantial part thereof.
A fourth aspect of the invention provides an oligonucleotide primer having a nucleotide sequence of about 21 nucleotides being complementary or identical to a sequence of rfff gene of enterobacteriaceae species where the sequence of rfff gene is located between position +31995 and position +32266 of the rfff gene.
A fifth aspect of the invention provides a use of a pair of oligonucleotide primers including a forward primer and a reverse primer for PCR detection of enterobacteriaceae species, wherein the forward primer is selected from the group consisting of RFFT11 (SEQ ID N0. 1); RFFT20 (SEQ ID N0. 2); RFFT17 (SEQ ID
NO. 3); RFFT21 (SEQ ID N0.4); RFFT22 (SEQ ID N0.5); and RFFT7 (SEQ ID
NO. 6); and the reverse primer is selected from the group consisting of RFFT8 (SEQ
ID NO. 7); and RFFT18 (SEQ ID NO. 8).
A sixth aspect of the invention provides a use of an oligonucleotide primer as provided by the above-mentioned third or fourth aspect of the invention, for PCR
detection of enterobacteriaceae species.
A seventh aspect of the invention provides a method of detection of Enterobacteriaceae species comprising the steps of:
a) extracting DNA from a sample;
b) amplifying a fragment of the extracted DNA by PCR using a forward oligonucleotide primer selected from the group consisting of RFFT11 (SEQ ID
NO. 1);
RFFT20 (SEQ ID NO. 2); RFFT17 (SEQ ID NO. 3); RFFT21 (SEQ ID NO. 4); RFFT22 (SEQ ID NO. 5); and RFFT7 (SEQ ID NO. 6); and a reverse oligonucleotide primer selected from the group consisting of RFFT8 (SEQ ID NO. 7); and RFFT18 (SEQ ID
NO. 8) and c) detecting the amplified fragment of the DNA.
An eighth aspect of the invention provides a method of detection of enterobacteriaceae species by nested PCR comprising the steps of:
a) extracting DNA from a sample;
b) amplifying a fragment of the extracted DNA by PCR using RFFT17 (SEQ ID
NO. 3) as forward oligonucleotide primer and RFFT18 (SEQ ID NO. 8) as reverse oligonucleotide primer;
b-1 ) amplifying a portion of the fragment amplified in step (b), by PCR using RFFT7 (SEQ ID NO. 6) as forward primer and RFFT8 (SEQ ID NO. 7) as reverse primer; and c) detecting the portion of the DNA amplified in step (b-1 ).
A ninth aspect of the invention provides a method of detection of Enterobacteriaceae species comprising the steps of:
a) extracting DNA from a sample;
b) amplifying a fragment of the extracted DNA by PCR using a forward oligonucleotide primer comprising the nucleotide sequence of the oligonucleotide selected from the group consisting of RFFT11 (SEQ ID N0. 1); RFFT20 (SEQ ID
NO. 2); RFFT17 (SEQ ID NO. 3); RFFT21 (SEQ ID NO. 4); RFFT22 (SEQ ID NO. 5);
and RFFT7 (SEQ ID N0.6) or a substantial part thereof; and a reverse oligonucleotide primer comprising the nucleotide sequence of the oligonucleotide selected from the group consisting of RFFT8 (SEQ ID NO. 7); and RFFT18 (SEQ ID
NO. 8) or a substantial part thereof; and c) detecting the amplified fragment of the DNA.
A tenth aspect of the invention provides a diagnostic assay for diagnosing Enterobacteriaceae species in a sample. The assay comprising:
- means for extracting DNA from the sample;
- a solution for detecting Enterobacteriaceae species DNA from the extracted DNA
by PCR, the solution comprising a PCR master mixture and a pair of primers including a forward primer and a reverse primer selected from the primers above-mentioned in the second, third or fourth aspect of the invention where the reverse primer is in a downstream position from the forward primer.
The present invention provides a simple and rapid technique of detection of Enterobacteriaceae species.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 a shows the nucleotide sequence of the n'e gene and the respective position of the oligonucleotide primers ECA1, ECA2, K1, K6 and KA7.
Figure 1 b shows the nucleotide sequence of the rffTgene and the respective position of the oligonucleotide primers RFFT11, RFFT20, RFFT17, RFFT21, RFFT22, RFFT7, RFFT8 and RFFT18.
Figure 2 represents a gel of electrophoresis showing the specificities of the primer sets tested with E. coli DNA strain ATCC 25922. Agarose 1.5% gel electrophoresis run with 10 pl of PCR product and stained with ethydium bromide. Lanes: 1, 250 by ladder of DNA molecular weight markers (Pharmacia Biotech) ; 2, primers ECA1/ECA2 (763 bp) ; 3, primers rffTl7/rffTl8 (178 bp) ; 4, 50 by ladder; 5, primers KA7/K6 (122 bp); 6, primers KI/K6 (197 bp); 7, 50 by ladder; 8, primers rffT7/rffT8 (103 bp); 9, primers n'fTll/rfff8 (243 bp); 10, primers rffT20/rffT8 (192 bp);
11, primers rffT21/rffT8 (126bp); 12, primers rffT22/rffT8 (110 bp); 13 negative control with master mix without DNA.
Figure 3 represents a gel of electrophoresis showing sensitivity of nested PCR
evaluated by serial dilution of E. coli. Template primer pairs were studied with the primers RFFT17 /RFFT18 and ECA1/ECA2. Panel A: agarose (1.5%) gel electrophoresis run with 10 NI of PCR product and stained with ethydium bromide. A
g 110-by PCR product was detected with dilutions of E. coli. and the rffT7/rffT8 primer pairs. Lane: 1, 50 by ladder of DNA molecular weight markers; 2, 40,000 bacteria; 3, 7.000 bacteria; 4, 120 bacteria; 5, 8 bacteria; 6, negative control (blank Qiagen without DNA); 7, negative control (master mix without DNA).
Figure 4 represents a gel of electrophoresis showing the specificities of primers RFFT7 and RFFT8 to detect several Entobacteriaceae species. Agarose (1.5%) gel electrophoresis run with 10 pl of PCR product and stained with ethydium bromide. The primers RFFT7 and RFFT8 yield a 103-by product. Lane: 1, E. coli; 2, Klebsiella pneumoniae; 3, Salmonella typhimurium; 4, Citrobacter freundii; 5, Enterobacter cloacae; 6, Proteus mirabilis; 7, Providencia rettgeri; 8, Serratia marcescens; 9, Shigella sonnei; 10, Yersinia enterocolitica; 11, Pseudomonas aeruginosa; 12, positive control of E. coli using ECA1iECA2 primers; 13, 14, negative controls (master mix without DNA); 15, 50 by ladder of DNA molecular weight markers (Pharmacia Biotech).
Figure 5 represents a gel of electrophoresis showing the detection of Enterobacteriaceae species in urine samples with the primers RFFT7/RFFTB.
Agarose (1.5%) gel electrophoresis run with 10 ul of PCR product and stained with ethydium bromide. A 103-by product was present only with positive specimens with enterobacteriaceae. Lane: 1., 50-by ladder of DNA molecular weight markers (Pharmacia Biotech): 2, Klebsiella pneumoniae; 3-6, E, coli; 7, Klebsiella pneumoniae;
8, 9, E. coli; 10, 11, 12, Klebsiella pneumoniae; 13, 14 Enterococcus; 15, negative control (blank Qiagen without DNA); 16, negative control (master mix without DNA).
Figure 6 represents a gel of electrophoresis showing the detection of Enterobacteriaceae species in blood samples with the primers RFFT7 and RFFTB.
Agarose (1.5%) gel electrophoresis run with 10 pl of PCR product and stained with ethydium bromide. These primers yielded a 103-by product with all positive specimens. Lane: 1, 50-by ladder of DNA molecular weight markers (Pharmacia Biotech) ; 2, 3, 4, E. coli; 5, Klebsiella pneumoniae; 6, 7, 8, E. coli; 9, 10, 11, negative blood culture specimens; 12, Klebsiella pneumoniae; 13, 14, E. coli;
15, negative control with the benzyl alcohol-guanidine hydrochloride method without DNA;
76, negative control with master mix without DNA.
DETAILED DESCRIPTION OF THE INVENTION
The present invention concerns the detection of the Enferobacteriaceae species. The tested species and their origin are listed in Table 1.
TABLE 1. Gram-negative bacilli used as control in this study Isolates Origin Enterobacteriaceae E. coli *ATCC 25922 Klebsiella pneumoniae ATCC 13883 Salmonella typhimurium ATCC 14028 Citrobacter freundii ATCC 8090 Enterobacter cloacae ATCC 23355 Proteus mirabilis ATCC 7002 Providencia rettgeri ATCC 872292 Serratia marcescens ATCC 8100 Shigella sonnei ATCC 25931 Yersinia enterocolitica ATCC 862196 Pseudomonas aeruginosa ATCC 27853 *ATCC, American Type Culture Collection.
Several oligonucleotides have been designed on the basis of rffT gene as shown in Figure 1 b. These oligonucleotides are listed below:
- RFFT11 having the nucleotide sequence of SEQ ID NO. 1;
- RFFT20 having the nucleotide sequence of SEQ ID NO. 2;
- RFFT17 having the nucleotide sequence of SEQ ID NO. 3;
- RFFT21 having the nucleotide sequence of SEQ ID NO. 4;
- RFFT22 having the nucleotide sequence of SEQ ID NO. 5;
- RFFT7 having the nucleotide sequence of SEQ ID NO. 6;
RFFT8 having the nucleotide sequence of SEQ ID NO. 7; and RFFT18 having the nucleotide sequence of SEQ ID NO. 8.
Based on rfe gene, other oligonucleotides have been designed as shown in Figure 1a.
All these oligonucleotides have been used as oligonucleotide primers for PCR
detection of Enterobacteriaceae species. These oligonucleotides listed in Table 2.
TABLE 2. Oligonucleotide primers Primer Sequence of Position of Size of designate oligonucleotide primers amplicon (bp) amplicon (bp) Template primers, external rfe ECA1 (+strand) 5' GGTGTTCGGCAAGCT TTATCTCAG-3' (643-666) 763 ECA2 (-strand) 5' GGTTAAATTGGGGCTGCCACCACG-3' (1405-1382) Nested primers Rfe KI (+strand) 5' CTGGGTTATATCTTTGGCTCC-3' (671-691) 197 K6 (-strand) 5' ATTGCGAGGCTGGTTTGCC-3' (867-849) KA7 (+strand) 5' GCGGCCATTAATGCGTTCAAC-3' (746-766) 122 Template primers, external rffT
RFFT17 (+strand) 5' GGTAAGCGTCGGCATCTTCTT-3' (32089-32109) 178 RFFT18 (-strand) 5' AAACAGCCACGCTTTGCTGT-3' (32266-32247) Nested primers rffT
RFFT7 (+strand) 5' CGGCTTAACTCCTACAGTCAG-3' (32135-32155) 103 RFFT8 (-strand) 5' GAAAGTAGACCACCAGCATCG-3' (32237-32217) RFFT22 (+strand) 5' GCTGTTCCGGCTTAACTCCTA-3' (32128-32148) 110 Template primers used alone (non nested) RFFT11 (+strand) 5' GCAAACGCGTTGCTGATGTAC-3' (31995-32015) 243 RFFT20 (+strand) 5' TGGAGACCAATC TTACGTGGG-3' (32046-32066) 192 RFFT21 (+strand) 5' TGCACAACGGCTTTTTGCTG-3' (32112-32131 ) 126 RFFT8 (-strand) 5' GAAAGTAGACCACCAGCATCG-3' (32237-32217) As it can be seen in Figure 1 b, the primers based on rftT gene are located in a region of rffT gene between position +31995 and position +32266. Their length vary between 20 and 21 nucleic acids. As it is well known in the field, a primer for PCR
may be shorter or longer than 20 or 21 nucleic acids. Thus, it should be understood that primers being longer or shorter than those shown in Figure 1 b, are also part of the present invention as well as primers comprising the nucleotide sequence shown in ll SEQ ID NO. 1, 2, 3, 4, 5, 6, 7 or 8, or a substantial part thereof. It is also well known in the field that variations of one nucleic acid or a few nucleic acids may not interfere with the capacity of hybridization of the oligonucleotide primer to DNA.
Therefore, a substantial part of said nucleotide sequence also encompasses a primer having one or a few nucleic acids different from the nucleotide sequence.
All the above-described oligonucleotide primers directed to the rfff gene are found useful for the PCR detection of Enterobacteriaceae species, and methods for PCR
detection of Enterobacteriaceae species using these primers are achieved by the present invention.
As shown in Table 2, RFFT11 (SEQ ID NO. 1), RFFT20 (SEQ ID N0. 2), RFFT17 (SEQ ID NO. 3), RFFT21 (SEQ ID NO. 4), RFFT22 (SEQ ID NO. 5), and RFFT7 (SEQ
ID NO. 6) can be used as forward oligonucleotide primers; and RFFT8 (SEQ ID
NO. 7) and RFFT18 (SEQ ID NO. 8) can be used as reverse oligonucleotide primers.
In a preferred embodiment of the invention, RFFT7 (SEQ ID NO. 6) and RFFT8 (SEQ
ID NO. 7) are respectively used as forward and reverse primers.
In another preferred embodiment of the invention, RFFT22 (SEQ ID NO. 5) and RFFT8 (SEQ ID NO. 7) are respectively used as forward and reverse primers.
In a further preferred embodiment of the invention, RFFT17 (SEQ ID NO. 3) and RFFT18 (SEQ ID NO. 8) are respectively used as forward and reverse primers.
A PCR or a nested PCR may performed to detect Enterobacteriaceae species.
In another further preferred embodiment of the invention, a nested PCR is achieved for the detection of Enterobacteriaceae species. In such a nested PCR, several combinations of primers can be used. It is a preferred combination to select (SEQ ID NO. 3) and RFFT18 (SEQ ID NO. 8) as template forward and reverse primers for a first amplification, and RFFT7 (SEQ ID N0. 6) and RFFT8 (SEQ ID
NO. 7) as nested forward and reverse primers for a second amplification.
Alternatively, RFFT7 (SEQ ID NO. 6) can be replaced by RFFT22 (SEQ ID NO. 5) in the above-mentioned preferred combination.
Detection of Enterobacteriaceae species has been achieved in blood samples and urine samples as described herein below.
Among the Enterobacteriaceae species, Escherichia coli, Klebsiella pneumoniae, Enterobacter sp., Serratia sp., Salmonella sp., Shigella sp., and Citrobacter sp. have been successfully detected accordingly to the present invention as described herein below.
A diagnostic assay for diagnosing Enterobacteriaceae species in a sample is also part of the present invention. The assay comprises:
- means for extracting DNA from the sample;
- a solution for detecting Enterobacteriaceae species DNA from the extracted DNA
by PCR, the solution comprising a PCR master mixture and a pair of primers including a forward primer and a reverse primer selected from the above-mentioned primers according to the invention and where the reverse primer is in a downstream position from the forward primer.
In order to produce highly specific and general PCR detection assays for E.
coli and Enterobacteriaceae, one set of template primers has been evaluated for each of the targeted genes (rfe and n~T). The primer pairs ECA1/ECA2 for rfe and rffT17/rffT18 for rffT were used for the amplication of E. coli genomic DNA. They both presented a DNA band at 763 by and 178 by respectively (Fig. 2, lanes 2 and 3). Then, the specificity of several primer pairs with corresponding template pairs has been examined. As expected, all primer sets presented the expected specific bands (see Figure 2, lines 5, 6, 8, 9, 10 and 11 ).
Sensitivity of amplification has been tested and the results are shown in Figure 3.
Using serial dilutions of bacterial E. coli suspensions, detection of 190 bacterial cells has been achieved under the PCR conditions for nested PCR with the rfe primers.
The template primers were ECAI/ECA2 and the nested primers, KA7 and K6 while with template primers RFFT17/RFFT18 used in combination with the nested primers pair RFFT22/RFFTB, a detection level of the 120 bacterial cells was obtained (see Figure 3).
Specificity of the assay has been tested and the results are shown in Figure 4 and compiled in Table 3. To assess species specificity of the rfe specific primer sets, 60 strains of Enterobacteriaceae which included 44 E. coli were used for the n'e primers pairs. Although primers from the rfe region were highly specific for E. coli bacterial strains detecting 44 out of 44 strains tested and none of the 16 other Enterobacteriaceae species was detected. With the objective of developing a general Enterobacteriaceae diagnostic assay and due to the very specific detection of E. coli only, evaluation of these primers was not pursued.
The research then focused on the evaluation of rffT region primer pairs. Each pair of primers was tested against a limited number of species, including Enterobacteriaceae:
E. coli, Klebsiella sp., Enterobacter sp., Salmonella sp., Enterococcus sp., Serratia sp., Shigella sp. and Citrobacter sp, and non-Enterobacteriaceae:
Stapylocococcus sp., Pseudomonas sp., to determine the pairs presenting the larger spectrum of Enterobacteriaceae sp. detection with the highest specificity (see Table 3).
Table 3. Species of Enterobacteriaceae detected by different primer pairs Isolates Set of primers E . coli + + + + + +
Klebsiella + + + +
sp.
Salmonella + + + + + + a sp.
Enterobacter +a sp.
Serratia sp. + + + _ _ Shigella sp. + + + + + +
Citrobacter -l+ - NT NT - +
sp.
A positive sign confirms the presence of a band with the expected size different for each primer pair.
NT; Not tested a A strong non-specific band was also observed.
The primer pairs RFFT7/RFFT8 had the best profile. All others pairs presented a restricted pattern with slight variations. For example, the primers detected a specific 178-by band, which was particularly strong with Citrobacter sp., in contrast with the other primer pairs studied. Moreover, some primer pairs occasionally presented a non-specific band which was also observed with primer pair RFFT7/RFFT8 using DNA from Enterobacfer sp., Citrobacter sp. and Yersinia sp..
Nonetheless, RFFT7/RFFT8 primer pairs is still presenting the best detection profile, giving a strong and unequivocal 103 by signal with 6 out of 10 Enterobacteriaceae tested (see Figure 4).
It has been found that additional purification of bacterial genomic DNA by Qiagen columns did not increase the number of bacteria detected. Furthermore, non-Enterobacteriaceae bacterial species such as Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus sp., were also studied and did not yield the desired fragment. Of a total of 116 bacterial species tested and comprising 95 Enterobacteriaceae, 10 Staphylococcus sp., 10 Pseudomonas sp. and 1 Enterococcus sp., 54 Enterobacteriaceae were positively detected. None of the non-Enterobacterial species could be detected with these primers and there were no false positives demonstrating 100% specificity for Enterobacteriaceae. Moreover, Klebsiella sp., Salmonella sp. and Shigella sp. genomic DNA was always amplified, suggesting 100% detectability of these bacteria species (Table 4).
TABLE 4. Microorganisms detected by PCR with the primers RFFT7 and RFFT8 Microrganisms Total no. No. of isolatesNo. of isolates of isolates detected not detected Enterobacteriaceae sp.
E. coli 10 10 0 Klebsiella sp. 10 10 0 Salmonella sp. 10 10 0 Shigella sp. 8 8 0 Enterobacter sp. 10 7 3 Serratia sp. 10 5 5 Citrobacter sp. 10 4 6 Proteus sp. 10 0 10 Providencia sp. 10 0 10 Yersinia sp. 7 0 7 Non-enterobacteriaceae sp Staphylococcus 10 0 10 sp.
Pseudomona sp. 10 0 10 Enterococcus sp. 1 0 1 Detection of Enterobacteriaceae in patient urine was assessed, and the results are shown in Figure 5 and compiled in Table 5. The primer pair RFFT7/RFFTB, was evaluated for the detection of Enterobacteriaceae sp. in clinical urine samples. A
group of 56 urine specimens (51 from Enterobacteriaceae specimens and 5 non-Enterobacteriaceae) was diagnosed by a conventional method (Kloos W.E and Bannerman T.L Staphylococcus and Micrococcus. In Murray P.R., Baron E.J., Pfaller M.A., Tenover F.C. and Yolken R.H. (ed), Manual of Clinical Microbiology 1995, pp.
282-298). The PCR technique allowed the detection only of samples which were from enterobacteriaceae specimens. Moreover using these primers and a non-nested PCR, DNA from 48 of these 51 specimens (94.1 %) could be detected by an approach consisting of a longer PCR program for a total of 50 cycles. A 103 by product was present only with positive specimens of Enterobacteriaceae (fig. 5). There was no false positive amplification with Pseudomonas sp., Gram-positive cocci and 2 negative urine specimens (Table 5).
TABLE 5. Clinical isolates detected by PCR with the primer pair RFFT7/RFFTB in urine specimens with > 100,000 CFU/mL a Microorganisms Total no. of No. of isolates No. of isolates isolates detected not detected Enterobacteriaceae E. coli. 39 39 0 Klebsiella sp. 8 8 0 Enterobacter sp. 2 1 1 Proteus sp. 2 0 2 Non-enferobacteriaceae Pseudomonas sp. 1 0 1 Enterococcus sp 3 0 3 Staphylococcus sp. 1 0 1 a Colony-forming units (CFU) determined by the 0.001 ml loop quantitative culture method.
Detection of Enterobacteriaceae in blood culture specimens with RFFT7/RFFT8 were assessed, and the results are shown in Figure 6. A total of 13 blood cultures were studied by DNA extraction with the benzyl alcohol-guanidine hydrochloride method.
Two sets of primers: RFFT7/RFFT8 and RFFT17/RFFT18 were evaluated for the detection of bacteria. For each pair of primers, only the longer PCR program was used. Out of 10 positive blood cultures there were 8 E. coli and 2 Klebsiella pneumoniae. Ten were found to contain Enterobacterial-specific PCR fragments with 100% sensitivity with RFFT7/RFFT8 while the sensitivity was 90% (9/10) with RFFT17/RFFT18 which did not detect a Klebsiella pneumoniae strain. No false positives were obtained from the 3 blood culture-negative specimens (see Figure 6).
DESCRIPTION OF THE TECHNIQUES USED IN THE INVENTION
Growth and characterization of bacteria The bacterial strains analyzed were either obtained from the American Type Culture Collection or isolated in a clinical microbiology laboratory. Gram-negative bacilli were identified with the Vitek system (Merieux, France). Cocci were characterized by the conventional method of Kloos and Bannerman (Supra). The bacteria were cultured on blood agar plates and incubated at 37°C for 18-24 h. The colonies obtained were inoculated in trypticase soy broth (TSB) and incubated at the same temperature for 18-24 h. Viable counts were achieved by plating serial dilutions of the broth culture.
Preparation of bacterial genomic DNA
DNA from bacterial suspension cultures in TSB was extracted according to the method of LeBouguenec et al. (Le Bouguenec C. et al. Rapid and Specific Detection of the pap, afa, and sfa Adhesin-Encoding Operons in Uropathogenic Escherichia coli Strains by Polymerase Chain Reaction. J. Clin. Microbiol. 1992, 30, pp. 1189-1193).
Briefly, bacteria contained in 1 ml of broth culture were collected by centrifugation at 3,OOOrpm for 3 min. The pellet was re-suspended in 200 pl of sterile distilled water, boiled for 15 min and again centrifuged at 14,OOOg for 3 min. The supernatant was carefully transferred to a sterile tube and maintained at -20°C.
DNA extraction from urine specimens Patient urine specimens with 100,000 CFU/ml or more were obtained from the hospital microbiology laboratory and stored at -20°C. DNA was extracted using QiAamp tissue method according to the manufacturer's protocol (QIAGEN, Mississauga, Ontario, Canada) with minor modifications. Briefly, 1 ml of urine was centrifuged at 6,OOOg for 10 min. The supernatant was discarded, and the remaining pellet was lysed by adding 180 ul of lysis buffer (buffer ATL, provide by manufacturer) and detergent. 20 pl of proteinase K (20 mg/ml) was also added, followed by incubation at 55°C for 2 h. Subsequently, 200 pl of AL buffer (provide by manufacturer) was added, vortexed vigorously, incubated at 70°C for 10 min and then at 95°C for 15 min, followed by mixing with 210 pl of sterile 100%
ethanol. This suspension was applied on a Qiagen column and centrifuged for 1 min in a 2-ml collection tube at 6,OOOg. The column was successively washed with AW buffer (provided by manufacturer), for 1 and 3 min, and the filtrates were discarded.
Two hundred pl of preheated AL buffer (at 70° C) was added to the column.
DNA was recovered by 1 min centrifugation and stored in a sterile microfuge tube at -20°C.
Bacterial DNA extraction from blood cultures Bacterial strains were recovered from blood cultures using BACTEC 9240 instrumentation (Becton Dickinson, Sparks, MD) and standard 10 aerobic/F and lytic/10 anaerobic F bottles. For maximum recovery, a 10-ml aliquot of patient blood was inoculated in each bottle and incubated at 35°C with agitation. A
sensor located at the bottom of each bottle responded by increasing fluorescence proportionally to the elevation of C02 concentration resulting from organism metabolism (Rohner P. et al. Comparative Evaluation of BACTEC Aerobic Plus/F and Septi-Chek Release Blood Culture Media. J. Clin. Microbiol. 1996, 34, pp.126-129). When a positive signal was obtained, a sample was taken from the bottle with a sterile syringe and Gram-staining was performed to confirm the presence of viable microorganisms. If Gram-staining showed Gram-negative bacteria, subcultures were prepared on MacConkey agar plates and incubated aerobically at 37°C.
Subcultures were also produced on blood and/or chocolate agar plates which were incubated at 37°C in 5% CO2. The anaerobic subcultures on blood aaar were supplemented with vitamin and incubated overnight. For aerobic bacteria, after overnight incubation, if colonies were observed on blood agar plates, identification was undertaken by conventional methods (Farmer, J.J. Enterobacferiaceae Introduction and Identification. In Murray P.R. et al. Manual of Clinical Microbiology 1995, pp. 438-464; Kloos W.E and Bannerman T.L Staphylococcus and Micrococcus.
In Murray P.R. et al., Manual of Clinical Microbiology 1995, pp. 282-298). The Gram-negative rods were identified biochemically with the Vitek system.
DNA was extracted only in samples taken from positive aerobic bottles when positive growth was observed, or after 5 days when the culture bottles were discarded.
Five hundred pl aliquots of the broth medium containing erythrocytes were removed from the aerobic bottles under sterile conditions, put in conical tubes and kept at -20°C until processed.
The benzyl alcohol-guanidine hydrochloride method (Fredricks D.N. and Relman D.A.
Improved Amplification of Microbial DNA from Blood Cultures by Removal of the PCR
inhibitors Sodium Polyanetholsulfonate. J. Clin. Microbiol. 1998, 36, pp. 2810-2816) was used for DNA extraction, with minor modifications. A 100-pl aliquot of the blood broth mixture containing bacteria was lysed by brief mixing with the same amount of lysis buffer (5 M guanidine hydrochloride-100 mM Tris, pH 8.0 in sterile water). This was followed by the addition of 400 pl of water and 800 pl of 99% benzyl alcohol (Sigma Chemical Company) and extensive vortexing. The suspension was centrifuged for 5 min at 6,OOOg, and 400 NI of the supernatant transferred to a microfuge tube for precipitation with 40 pl of 3.0 M sodium acetate and 440 pl of isopropanol.
The DNA
precipitate was recovered by centrifugation at 6,OOOg for 30 min, washed with 70%
ethanol, air-dried, and dissolved in 100 pl of 10 mM Tris-0.1 mM EDTA, pH 8.5.
Oligonucleotide primers for PCR detection E. coli genome organization in the ECA operon is known and its complete sequence is available. Based on this knowledge, several primers were designed from the rfelrff group of genes located on E. coli genome positions 84.5 to 86.5 min (Daniels D.L. et al. Analysis of the Escherichia coli genome: DNA sequence of the region from 84.5 to 86.5 minutes. Science 1992, 257 (5071), pp. 771-778; Blattner F.R. et al. The Complete Genome Sequence of Escherichia coli K-12. Science. 1997, 277, pp.
1453-1462). Since nucleotide sequence of the E. coli rfe gene have already been published, we took advantage of this sequence to design several sets of primers (see table 2) for the amplification of the E coli DNA (Meier-Dieter U. et al.
Nucleotide Sequence of the Escherichia coli rfe Gene Involved in the Synthesis of Enterobacterial Common Antigen. J. Biol. Chem. 1992 , 267, pp. 746-753). Their specific positions on the sequence are presented in figure 1a. The sequence of rftl' gene was available by genebank (accession number : M87049) and the positions of the primers are shown in figure 1 b.
The template primers ECA1/ECA2 and RFFT17/RFFT18 were used in combination with nested primers for PCR-based detection of E. coli and Enterobacteriaceae strains respectively.
PCR Amplification of bacterial DNA
Reactions were performed in a total volume of 55 ul. Forty-five pl of a master mixture containing 5 pl of 10 X PCR buffer (500 mM KCI, 15 mM MgCl2 and 100 mM Tris-HCI), 8 pl of 1.25 pM of dNTP stock solution, 2.5 pl of each of the forward and reverse primers (stock 1 pg/ml) and 0.25 pl (1.25 units) of TAQ polymerase (Pharmacia, Baie d'Urfe, Quebec, Canada) and 26.75 pl of sterile distilled water were mixed with 10 ul of bacterial DNA samples. The negative controls contained all ingredients of the master mixture without any DNA samples. For urine specimens, the final concentration of the primers was 0.45 pM, and 5 units of TAQ polymerase were used.
For the nested PCR and blood culture specimens, the concentration of the primers and Taq polymerase was respectively 0.9 pM and 2.5 units. A 3 pl aliquot of template PCR reaction product was used for the nested PCR. Amplifications were performed with a Perkin Elmer 9600 cycler under the following conditions: cycles of 2 min denaturation at 94°C and 1 min annealing at 60°C, followed by 2 min of polymerization at 72°C (this cycle was repeated 25 times). For nested PCR, the first amplification (template) cycle was as described previously, while the nested PCR consisted of 25 cycles of shorter duration: 30 sec denaturation at 94°C, 15 sec, annealing at 60°C and sec polymerization at 72°C. A 10 NL aliquot from the PCR reaction product was electrophoresed on 1.5% agarose mini-gel, stained with ethidium bromide and photographed with a Polaroid camera.
For the study of urine specimens, a non-nested PCR was chosen to avoid contamination, which can occur with the nested PCR, particularly with the transfer of DNA into another tube after the first amplification. The non-nested approach consisted of the successive use of the longer PCR program followed by the shorter one for a total of 50 cycles.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: CORPORATION DU CENTRE DE RECHERCHE DU CENTRE
HOSPITALIER DE L'UNIVERSITE DE MONTREAL
(B) STREET: 3850 Saint-Urbain (C) CITY: Montreal (E) COUNTRY: Canada (F) POSTAL CODE: H2W 1T8 (ii) TITLE OF THE INVENTION: Oligonucleotide primers for the specific PCR detection of enterobacteriaceae species DNA using RFE-RFF gene templates (iii) NUMBER OF SEQUENCES: 8 (iv) CORRESPONDENCE ADDRESS:
(A) NAME: ROBIC
(B) STREET: 55 Saint-Jacques (C) CITY: Montreal (E) COUNTRY: Canada (F) POSTAL CODE: H2Y 3X2 (v) COMPUTER-READABLE FORM:
(A) TYPE OF SUPPORT: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: WordPad (vi) CURRENT APPLICATION DATA:
(A) APPLICATION DATA: 2,286,512 (B) FILING DATE: 27 October 1999 (viii) PATENT AGENT INFORMATION
(A) NAME: ROBIC
(B) REFERENCE NUMBER: 26740-0005 (2) INFORMATION FOR SEQ ID N0: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 nucleic acids (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (iii) ANTISENS: NO
(ix) FEATURE:
(A) NAME/KEY: RFFT11 (B) LOCATION: 31995..32015 (xi) SEQUENCE DESCRIPTION: SEQ ID N0: l:
(2) INFORMATION FOR SEQ ID N0: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 nucleic acids (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (iii) ANTISENS: NO
(ix) FEATURE:
(A) NAME/KEY: RFFT20 (B) LOCATION: 32046..32066 (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 2:
(2) INFORMATION FOR SEQ ID N0: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 nucleic acids (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (iii) ANTISENS: NO
(ix) FEATURE:
(A) NAME/KEY: RFFT17 (B) LOCATION: 32089..32109 (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 3:
(2) INFORMATION FOR SEQ ID N0: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 nucleic acids (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (iii) ANTISENS: NO
(ix) FEATURE:
(A) NAME/KEY: RFFT21 (B) LOCATION: 32112..32131 (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 4;
(2) INFORMATION FOR SEQ ID N0: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 nucleic acids (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (iii) ANTISENS: NO
(ix) FEATURE:
(A) NAME/KEY: RFFT22 (B) LOCATION: 32128..32148 (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 5:
(2) INFORMATION FOR SEQ ID N0: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 nucleic acids (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (iii) ANTISENS: NO
(ix) FEATURE:
(A) NAME/KEY: RFFT7 (B) LOCATION: 32135..32155 (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 6:
(2) INFORMATION FOR SEQ ID N0: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 nucleic acids (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (iii) ANTISENS: YES
(ix) FEATURE:
(A) NAME/KEY: RFFT8 (B) LOCATION: 32237..32217 (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 7:
(2) INFORMATION FOR SEQ ID N0: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 nucleic acids (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (iii) ANTISENS: YES
(ix) FEATURE:
(A) NAME/KEY: RFFT18 (B) LOCATION: 32266..32247 (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 8:
The present invention relates to novel PCR oligonucleotide primers for the detection of Enterobacteriaceae species. More particularly, these oligonucleotide primers are directed to rfff gene of Enterobacteriaceae species. The present invention also relates to uses of these oligonucleotide primers for PCR detection of Enterobacteriaceae species and methods for detecting Enterobacteriaceae species by PCR using these oligonucleotide primers.
BACKGROUND OF THE INVENTION
Enterobacteriaceae are gram-negative bacilli usually found in the gastrointestinal tract and are frequently associated with septicemia, meningitis, pneumonia, peritonitis and urinary tract infections. This family of bacteria includes Escherichia coli, Klebsiella pneumoniae, Enterobacter sp., Serratia sp., Salmonella sp., Shigella sp., Citrobacter sp., Yersinia sp., Proteus sp., and Providencia sp. (Farmer, J.J.
Enterobacteriaceae Introduction and Identification. In Murray P.R. et aL,Manual of Clinical Microbiology 1995, pp. 438-464). A specific common feature of this family (Kuhn, H.M. et al., ECA, the Enterobacterial Common Antigen. FEMS Microbiology Reviews. 1988, 54 , pp.
195-222) is the synthesis of enterobacterial common antigen (ECA).
ECA, a glycophospholipid surface antigen of the enterobacteriaceae family, is composed of carbohydrates linked to L-glycerophosphatidyl residues. The carbohydrates comprise N-acetyl-D-glucosamine (GIcNAc), N-acetyl-D-mannosaminuronic (ManNAcA), 4-acetamido-4 and 6-dideoxy-D-galactose (Fuc4NAc). These amino-sugars constitute linear polysaccharide chains of repetitive trisaccharide units. The polysaccharide chains and the lipid component are respectively responsible for serological specificity and anchorage of ECA to the external membrane of enterobacteriaceae (Meier-Dieter U. et al., Nucleotide Sequence of the Escherichia coli rfe Gene Involved in the Synthesis of Enterobacterial Common Antigen. J. Biol. Chem. 1992 , 267 , pp. 746-753; and Ohta M.O. et al., Cloning and expression of the rfe-rff Gene Cluster of Escherichia coli. Mol.
Microbiol.
1991, 5, pp. 1853-1862). ECA has immunologic properties (Peter, H. et al.
Monoclonal Antibodies to Enterobacterial Common Antigen and to Escherichia coli Lipopolysaccharide Outer Core: Demonstration of an Antigen Determinant Shared by Enterobacterial Common Antigen and E. coli K5 Capsular Polysaccharide. Infect.
Immun. 1995, 50, pp. 459-466). A protective action has not been demonstrated in humans but its immunologic properties have been used for diagnostic purposes.
The most frequent diagnostic methods of ECA detection rely on enzyme immunoassay and the passive hemagglutination test (Hubner I. et al. Rapid Determination of Members of the Family Enterobacteriaceae in Drinking Water by an Immunological Assay Using a Monoclonal Antibody Against Enterobacterial Common Antigen. Appl. Environ. Microb. 1992, 58, pp. 3187-3191; and Bayardelle P. and Ranger S. Rapid Detection of Enterobacteriaceae in Urine with Passive Hemagglutination Test. American Society for Microbiology, 95th Meeting.
Washington.
May 1995). The Sensitivity of the enzyme immunoassay is low for Enterobacteriaceae and varies according to the ECA concentration of the species. The detection limit is about 3,9X105 colony-forming units per ml (CFU/ml) of water and is higher for other species (1,3X10' CFU/ml). Although specific, the test presents false positive in 0.3%
of samples. Passive hemagglutination can be used for urine samples with 100,000 CFU/ml but gave false positive results at 4.6%.
Although ECA genes have been well characterized (Meier-Dieter U. et al. J.
Biol.
Chem. 1992, 267, pp. 746-753; Ohta M.O. et al. Mol. Microbiol. 1991, 5, pp.
1853-1862; Marolda C.L. and Valvano M.A. Genetic Analysis of the dTDP-Rhamnose Biosynthesis Region of the Escherichia coli VW187 (07:KI) rfb Gene Cluster:
Identification of Fonctional Homologs of rfb8 and rfbA in the rff Cluster and Correct Location of the rffE Gene. J. Bacteriol. 1995, 177, pp. 5539-5546; Alexander D.C. and Valvano M.A. Role of the rfe Gene in the Biosynthesis of the Escherichia coli 07-Specific Lipopolysaccharide and Other O-Specific Polysaccharides Containing N-Acetylglucosamine. J. Bacteriol. 1994, 176, pp. 7079-7084; Whitfield C. et al.
Modulation of the Surface Architecture of Gram-negative Bacteria by the Action of Surface Polymer: Lipid A-core Ligase and by Determinants of Polymer Chain Length.
Mol. Microbiol. 1997, 23, pp. 629-638; Amor P.A. and Whitfield. Molecular and Functional Analysis of Genes Required for Expression of Group IB K Antigens in Escherichia coli: Characterization of the his-region Containing Gene Clusters for Multiple Cell-surface Polysaccharides. Mol. Microbiol. 1997, 26 pp.145-161) and new nomenclature implemented (Reeves P.R. et al. Bacterial Polysaccharide Synthesis and Gene Nomenclature. Trends in Microbiology. 1996, 4, pp. 495-503), no PCR-based diagnostic test has yet been developed. Some previous PCR primers designed to determine the function of certain genes are species-specific (Marolda C.L.
and Valvano M.A. J. Bacteriol. 1995, 177, pp. 5539-5546). In the new nomenclature, rfe and rff gene cluster is now identified wec gene cluster.
The detection of pathogen-specific genomic DNA was attempted several years ago from diverse infectious agents in clinical specimens (Song J.H et al.
Detection of Salmonella typhi in the Blood of Patients with Typhoid Fever by Polymerase Chain Reaction. J.Clin. Microbiol. 1993, 31, pp. 1439-1443; Zhang Y. et al.
Detection of Streptococcus pneumoniae in Whole Blood by PCR. J. Clin. Microbiol. 1995, 33, pp.
596-601; Greisen,K. et al. PCR Primers and Probes for the 16S rRNA Gene of Most Species of Pathogenic Bacteria, Including Bacteria found in Cerebrospinal fluid. J.
Clin. Microbiol. 1994, 32, pp. 335-351; Whelen A.C. and Persing D.H. The Role of Nucleic Acid Amplification and Detection in the Clinical Microbiology Laboratory.
Annual Review of Microbiology. 1996, 50, pp. 349-373; Hashimoto Y. et al.
Development of Nested PCR Based on the Viab Sequence To Detect Salmonella typhi. J. Clin. Microbiol. 1995, 33, pp. 775-777). Broad-range PCR primers for rRNA have been used to amplify either gram-positive or gram-negative bacteria but this technique calls for a combination of PCR and Southern blot hybridization which is time-consuming and does not allow specific identification of Enterobacteriaceae species (Greisen,K., M. et al. PCR Primers and Probes for the 16S rRNA Gene of Most Species of Pathogenic Bacteria, Including Bacteria Found in Cerebrospinal fluid.
a J. Clin. Microbiol. 1994, 32, pp. 335-351; Song J.H et al. Detection of Salmonella typhi in the Blood of Patients with Typhoid Fever by Polymerase Chain Reaction.
J.Clin. Microbiol. 1993, 31, pp. 1439-1443).
It has been hypothesized that n'e and rff genes, implicated in the synthesis of ECA, represent good targets for the broad spectrum detection of Enterobacteriaceae.
The rfe gene is essential for ECA synthesis although its precise function has not been determined. It appears to encode for a transferase which catalyzes the synthesis of GIcNAc-pyrophosphorylundecaprol (lipid I), the essential first intermediate lipid involved in ECA synthesis, while the rftT gene encodes for a transferase which reassembles Fuc4NAc and ManNAcA, thus completing the repetitive trisaccharide unit of ECA.
It is an object of the present invention to develop a fast PCR-based diagnostic test and to evaluate its sensitivity and specificity as well as its applicability to the routine detection of enterobacteriaceae in biological fluids such as patient blood and urine.
SUMMARY OF THE INVENTION
A first aspect of the invention provides an oligonucleotide selected from the group consisting of:
- RFFT11 having the nucleotide sequence of SEQ ID NO. 1;
- RFFT20 having the nucleotide sequence of SEQ ID NO. 2;
- RFFT17 having the nucleotide sequence of SEQ ID NO. 3;
- RFFT21 having the nucleotide sequence of SEQ ID NO. 4;
- RFFT22 having the nucleotide sequence of SEQ ID NO. 5;
- RFFT7 having the nucleotide sequence of SEQ ID NO. 6;
- RFFT8 having the nucleotide sequence of SEQ ID NO. 7; and RFFT18 having the nucleotide sequence of SEQ ID NO. 8.
A second aspect of the invention provides an oligonucleotide primer selected from the group consisting of RFFT11 (SEQ ID NO. 1); RFFT20 (SEQ ID NO. 2); RFFT17 (SEQ
r ID NO. 3); RFFT21 (SEQ ID N0.4); RFFT22 (SEQ ID NO. 5); RFFT7 (SEQ ID
NO. 6); RFFT8 (SEQ ID NO. 7); and RFFT18 (SEQ ID NO. 8).
A third aspect of the invention provides an oligonucleotide primer comprising the nucleotide sequence of the oligonucleotide selected from the above-defined group or a substantial part thereof.
A fourth aspect of the invention provides an oligonucleotide primer having a nucleotide sequence of about 21 nucleotides being complementary or identical to a sequence of rfff gene of enterobacteriaceae species where the sequence of rfff gene is located between position +31995 and position +32266 of the rfff gene.
A fifth aspect of the invention provides a use of a pair of oligonucleotide primers including a forward primer and a reverse primer for PCR detection of enterobacteriaceae species, wherein the forward primer is selected from the group consisting of RFFT11 (SEQ ID N0. 1); RFFT20 (SEQ ID N0. 2); RFFT17 (SEQ ID
NO. 3); RFFT21 (SEQ ID N0.4); RFFT22 (SEQ ID N0.5); and RFFT7 (SEQ ID
NO. 6); and the reverse primer is selected from the group consisting of RFFT8 (SEQ
ID NO. 7); and RFFT18 (SEQ ID NO. 8).
A sixth aspect of the invention provides a use of an oligonucleotide primer as provided by the above-mentioned third or fourth aspect of the invention, for PCR
detection of enterobacteriaceae species.
A seventh aspect of the invention provides a method of detection of Enterobacteriaceae species comprising the steps of:
a) extracting DNA from a sample;
b) amplifying a fragment of the extracted DNA by PCR using a forward oligonucleotide primer selected from the group consisting of RFFT11 (SEQ ID
NO. 1);
RFFT20 (SEQ ID NO. 2); RFFT17 (SEQ ID NO. 3); RFFT21 (SEQ ID NO. 4); RFFT22 (SEQ ID NO. 5); and RFFT7 (SEQ ID NO. 6); and a reverse oligonucleotide primer selected from the group consisting of RFFT8 (SEQ ID NO. 7); and RFFT18 (SEQ ID
NO. 8) and c) detecting the amplified fragment of the DNA.
An eighth aspect of the invention provides a method of detection of enterobacteriaceae species by nested PCR comprising the steps of:
a) extracting DNA from a sample;
b) amplifying a fragment of the extracted DNA by PCR using RFFT17 (SEQ ID
NO. 3) as forward oligonucleotide primer and RFFT18 (SEQ ID NO. 8) as reverse oligonucleotide primer;
b-1 ) amplifying a portion of the fragment amplified in step (b), by PCR using RFFT7 (SEQ ID NO. 6) as forward primer and RFFT8 (SEQ ID NO. 7) as reverse primer; and c) detecting the portion of the DNA amplified in step (b-1 ).
A ninth aspect of the invention provides a method of detection of Enterobacteriaceae species comprising the steps of:
a) extracting DNA from a sample;
b) amplifying a fragment of the extracted DNA by PCR using a forward oligonucleotide primer comprising the nucleotide sequence of the oligonucleotide selected from the group consisting of RFFT11 (SEQ ID N0. 1); RFFT20 (SEQ ID
NO. 2); RFFT17 (SEQ ID NO. 3); RFFT21 (SEQ ID NO. 4); RFFT22 (SEQ ID NO. 5);
and RFFT7 (SEQ ID N0.6) or a substantial part thereof; and a reverse oligonucleotide primer comprising the nucleotide sequence of the oligonucleotide selected from the group consisting of RFFT8 (SEQ ID NO. 7); and RFFT18 (SEQ ID
NO. 8) or a substantial part thereof; and c) detecting the amplified fragment of the DNA.
A tenth aspect of the invention provides a diagnostic assay for diagnosing Enterobacteriaceae species in a sample. The assay comprising:
- means for extracting DNA from the sample;
- a solution for detecting Enterobacteriaceae species DNA from the extracted DNA
by PCR, the solution comprising a PCR master mixture and a pair of primers including a forward primer and a reverse primer selected from the primers above-mentioned in the second, third or fourth aspect of the invention where the reverse primer is in a downstream position from the forward primer.
The present invention provides a simple and rapid technique of detection of Enterobacteriaceae species.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 a shows the nucleotide sequence of the n'e gene and the respective position of the oligonucleotide primers ECA1, ECA2, K1, K6 and KA7.
Figure 1 b shows the nucleotide sequence of the rffTgene and the respective position of the oligonucleotide primers RFFT11, RFFT20, RFFT17, RFFT21, RFFT22, RFFT7, RFFT8 and RFFT18.
Figure 2 represents a gel of electrophoresis showing the specificities of the primer sets tested with E. coli DNA strain ATCC 25922. Agarose 1.5% gel electrophoresis run with 10 pl of PCR product and stained with ethydium bromide. Lanes: 1, 250 by ladder of DNA molecular weight markers (Pharmacia Biotech) ; 2, primers ECA1/ECA2 (763 bp) ; 3, primers rffTl7/rffTl8 (178 bp) ; 4, 50 by ladder; 5, primers KA7/K6 (122 bp); 6, primers KI/K6 (197 bp); 7, 50 by ladder; 8, primers rffT7/rffT8 (103 bp); 9, primers n'fTll/rfff8 (243 bp); 10, primers rffT20/rffT8 (192 bp);
11, primers rffT21/rffT8 (126bp); 12, primers rffT22/rffT8 (110 bp); 13 negative control with master mix without DNA.
Figure 3 represents a gel of electrophoresis showing sensitivity of nested PCR
evaluated by serial dilution of E. coli. Template primer pairs were studied with the primers RFFT17 /RFFT18 and ECA1/ECA2. Panel A: agarose (1.5%) gel electrophoresis run with 10 NI of PCR product and stained with ethydium bromide. A
g 110-by PCR product was detected with dilutions of E. coli. and the rffT7/rffT8 primer pairs. Lane: 1, 50 by ladder of DNA molecular weight markers; 2, 40,000 bacteria; 3, 7.000 bacteria; 4, 120 bacteria; 5, 8 bacteria; 6, negative control (blank Qiagen without DNA); 7, negative control (master mix without DNA).
Figure 4 represents a gel of electrophoresis showing the specificities of primers RFFT7 and RFFT8 to detect several Entobacteriaceae species. Agarose (1.5%) gel electrophoresis run with 10 pl of PCR product and stained with ethydium bromide. The primers RFFT7 and RFFT8 yield a 103-by product. Lane: 1, E. coli; 2, Klebsiella pneumoniae; 3, Salmonella typhimurium; 4, Citrobacter freundii; 5, Enterobacter cloacae; 6, Proteus mirabilis; 7, Providencia rettgeri; 8, Serratia marcescens; 9, Shigella sonnei; 10, Yersinia enterocolitica; 11, Pseudomonas aeruginosa; 12, positive control of E. coli using ECA1iECA2 primers; 13, 14, negative controls (master mix without DNA); 15, 50 by ladder of DNA molecular weight markers (Pharmacia Biotech).
Figure 5 represents a gel of electrophoresis showing the detection of Enterobacteriaceae species in urine samples with the primers RFFT7/RFFTB.
Agarose (1.5%) gel electrophoresis run with 10 ul of PCR product and stained with ethydium bromide. A 103-by product was present only with positive specimens with enterobacteriaceae. Lane: 1., 50-by ladder of DNA molecular weight markers (Pharmacia Biotech): 2, Klebsiella pneumoniae; 3-6, E, coli; 7, Klebsiella pneumoniae;
8, 9, E. coli; 10, 11, 12, Klebsiella pneumoniae; 13, 14 Enterococcus; 15, negative control (blank Qiagen without DNA); 16, negative control (master mix without DNA).
Figure 6 represents a gel of electrophoresis showing the detection of Enterobacteriaceae species in blood samples with the primers RFFT7 and RFFTB.
Agarose (1.5%) gel electrophoresis run with 10 pl of PCR product and stained with ethydium bromide. These primers yielded a 103-by product with all positive specimens. Lane: 1, 50-by ladder of DNA molecular weight markers (Pharmacia Biotech) ; 2, 3, 4, E. coli; 5, Klebsiella pneumoniae; 6, 7, 8, E. coli; 9, 10, 11, negative blood culture specimens; 12, Klebsiella pneumoniae; 13, 14, E. coli;
15, negative control with the benzyl alcohol-guanidine hydrochloride method without DNA;
76, negative control with master mix without DNA.
DETAILED DESCRIPTION OF THE INVENTION
The present invention concerns the detection of the Enferobacteriaceae species. The tested species and their origin are listed in Table 1.
TABLE 1. Gram-negative bacilli used as control in this study Isolates Origin Enterobacteriaceae E. coli *ATCC 25922 Klebsiella pneumoniae ATCC 13883 Salmonella typhimurium ATCC 14028 Citrobacter freundii ATCC 8090 Enterobacter cloacae ATCC 23355 Proteus mirabilis ATCC 7002 Providencia rettgeri ATCC 872292 Serratia marcescens ATCC 8100 Shigella sonnei ATCC 25931 Yersinia enterocolitica ATCC 862196 Pseudomonas aeruginosa ATCC 27853 *ATCC, American Type Culture Collection.
Several oligonucleotides have been designed on the basis of rffT gene as shown in Figure 1 b. These oligonucleotides are listed below:
- RFFT11 having the nucleotide sequence of SEQ ID NO. 1;
- RFFT20 having the nucleotide sequence of SEQ ID NO. 2;
- RFFT17 having the nucleotide sequence of SEQ ID NO. 3;
- RFFT21 having the nucleotide sequence of SEQ ID NO. 4;
- RFFT22 having the nucleotide sequence of SEQ ID NO. 5;
- RFFT7 having the nucleotide sequence of SEQ ID NO. 6;
RFFT8 having the nucleotide sequence of SEQ ID NO. 7; and RFFT18 having the nucleotide sequence of SEQ ID NO. 8.
Based on rfe gene, other oligonucleotides have been designed as shown in Figure 1a.
All these oligonucleotides have been used as oligonucleotide primers for PCR
detection of Enterobacteriaceae species. These oligonucleotides listed in Table 2.
TABLE 2. Oligonucleotide primers Primer Sequence of Position of Size of designate oligonucleotide primers amplicon (bp) amplicon (bp) Template primers, external rfe ECA1 (+strand) 5' GGTGTTCGGCAAGCT TTATCTCAG-3' (643-666) 763 ECA2 (-strand) 5' GGTTAAATTGGGGCTGCCACCACG-3' (1405-1382) Nested primers Rfe KI (+strand) 5' CTGGGTTATATCTTTGGCTCC-3' (671-691) 197 K6 (-strand) 5' ATTGCGAGGCTGGTTTGCC-3' (867-849) KA7 (+strand) 5' GCGGCCATTAATGCGTTCAAC-3' (746-766) 122 Template primers, external rffT
RFFT17 (+strand) 5' GGTAAGCGTCGGCATCTTCTT-3' (32089-32109) 178 RFFT18 (-strand) 5' AAACAGCCACGCTTTGCTGT-3' (32266-32247) Nested primers rffT
RFFT7 (+strand) 5' CGGCTTAACTCCTACAGTCAG-3' (32135-32155) 103 RFFT8 (-strand) 5' GAAAGTAGACCACCAGCATCG-3' (32237-32217) RFFT22 (+strand) 5' GCTGTTCCGGCTTAACTCCTA-3' (32128-32148) 110 Template primers used alone (non nested) RFFT11 (+strand) 5' GCAAACGCGTTGCTGATGTAC-3' (31995-32015) 243 RFFT20 (+strand) 5' TGGAGACCAATC TTACGTGGG-3' (32046-32066) 192 RFFT21 (+strand) 5' TGCACAACGGCTTTTTGCTG-3' (32112-32131 ) 126 RFFT8 (-strand) 5' GAAAGTAGACCACCAGCATCG-3' (32237-32217) As it can be seen in Figure 1 b, the primers based on rftT gene are located in a region of rffT gene between position +31995 and position +32266. Their length vary between 20 and 21 nucleic acids. As it is well known in the field, a primer for PCR
may be shorter or longer than 20 or 21 nucleic acids. Thus, it should be understood that primers being longer or shorter than those shown in Figure 1 b, are also part of the present invention as well as primers comprising the nucleotide sequence shown in ll SEQ ID NO. 1, 2, 3, 4, 5, 6, 7 or 8, or a substantial part thereof. It is also well known in the field that variations of one nucleic acid or a few nucleic acids may not interfere with the capacity of hybridization of the oligonucleotide primer to DNA.
Therefore, a substantial part of said nucleotide sequence also encompasses a primer having one or a few nucleic acids different from the nucleotide sequence.
All the above-described oligonucleotide primers directed to the rfff gene are found useful for the PCR detection of Enterobacteriaceae species, and methods for PCR
detection of Enterobacteriaceae species using these primers are achieved by the present invention.
As shown in Table 2, RFFT11 (SEQ ID NO. 1), RFFT20 (SEQ ID N0. 2), RFFT17 (SEQ ID NO. 3), RFFT21 (SEQ ID NO. 4), RFFT22 (SEQ ID NO. 5), and RFFT7 (SEQ
ID NO. 6) can be used as forward oligonucleotide primers; and RFFT8 (SEQ ID
NO. 7) and RFFT18 (SEQ ID NO. 8) can be used as reverse oligonucleotide primers.
In a preferred embodiment of the invention, RFFT7 (SEQ ID NO. 6) and RFFT8 (SEQ
ID NO. 7) are respectively used as forward and reverse primers.
In another preferred embodiment of the invention, RFFT22 (SEQ ID NO. 5) and RFFT8 (SEQ ID NO. 7) are respectively used as forward and reverse primers.
In a further preferred embodiment of the invention, RFFT17 (SEQ ID NO. 3) and RFFT18 (SEQ ID NO. 8) are respectively used as forward and reverse primers.
A PCR or a nested PCR may performed to detect Enterobacteriaceae species.
In another further preferred embodiment of the invention, a nested PCR is achieved for the detection of Enterobacteriaceae species. In such a nested PCR, several combinations of primers can be used. It is a preferred combination to select (SEQ ID NO. 3) and RFFT18 (SEQ ID NO. 8) as template forward and reverse primers for a first amplification, and RFFT7 (SEQ ID N0. 6) and RFFT8 (SEQ ID
NO. 7) as nested forward and reverse primers for a second amplification.
Alternatively, RFFT7 (SEQ ID NO. 6) can be replaced by RFFT22 (SEQ ID NO. 5) in the above-mentioned preferred combination.
Detection of Enterobacteriaceae species has been achieved in blood samples and urine samples as described herein below.
Among the Enterobacteriaceae species, Escherichia coli, Klebsiella pneumoniae, Enterobacter sp., Serratia sp., Salmonella sp., Shigella sp., and Citrobacter sp. have been successfully detected accordingly to the present invention as described herein below.
A diagnostic assay for diagnosing Enterobacteriaceae species in a sample is also part of the present invention. The assay comprises:
- means for extracting DNA from the sample;
- a solution for detecting Enterobacteriaceae species DNA from the extracted DNA
by PCR, the solution comprising a PCR master mixture and a pair of primers including a forward primer and a reverse primer selected from the above-mentioned primers according to the invention and where the reverse primer is in a downstream position from the forward primer.
In order to produce highly specific and general PCR detection assays for E.
coli and Enterobacteriaceae, one set of template primers has been evaluated for each of the targeted genes (rfe and n~T). The primer pairs ECA1/ECA2 for rfe and rffT17/rffT18 for rffT were used for the amplication of E. coli genomic DNA. They both presented a DNA band at 763 by and 178 by respectively (Fig. 2, lanes 2 and 3). Then, the specificity of several primer pairs with corresponding template pairs has been examined. As expected, all primer sets presented the expected specific bands (see Figure 2, lines 5, 6, 8, 9, 10 and 11 ).
Sensitivity of amplification has been tested and the results are shown in Figure 3.
Using serial dilutions of bacterial E. coli suspensions, detection of 190 bacterial cells has been achieved under the PCR conditions for nested PCR with the rfe primers.
The template primers were ECAI/ECA2 and the nested primers, KA7 and K6 while with template primers RFFT17/RFFT18 used in combination with the nested primers pair RFFT22/RFFTB, a detection level of the 120 bacterial cells was obtained (see Figure 3).
Specificity of the assay has been tested and the results are shown in Figure 4 and compiled in Table 3. To assess species specificity of the rfe specific primer sets, 60 strains of Enterobacteriaceae which included 44 E. coli were used for the n'e primers pairs. Although primers from the rfe region were highly specific for E. coli bacterial strains detecting 44 out of 44 strains tested and none of the 16 other Enterobacteriaceae species was detected. With the objective of developing a general Enterobacteriaceae diagnostic assay and due to the very specific detection of E. coli only, evaluation of these primers was not pursued.
The research then focused on the evaluation of rffT region primer pairs. Each pair of primers was tested against a limited number of species, including Enterobacteriaceae:
E. coli, Klebsiella sp., Enterobacter sp., Salmonella sp., Enterococcus sp., Serratia sp., Shigella sp. and Citrobacter sp, and non-Enterobacteriaceae:
Stapylocococcus sp., Pseudomonas sp., to determine the pairs presenting the larger spectrum of Enterobacteriaceae sp. detection with the highest specificity (see Table 3).
Table 3. Species of Enterobacteriaceae detected by different primer pairs Isolates Set of primers E . coli + + + + + +
Klebsiella + + + +
sp.
Salmonella + + + + + + a sp.
Enterobacter +a sp.
Serratia sp. + + + _ _ Shigella sp. + + + + + +
Citrobacter -l+ - NT NT - +
sp.
A positive sign confirms the presence of a band with the expected size different for each primer pair.
NT; Not tested a A strong non-specific band was also observed.
The primer pairs RFFT7/RFFT8 had the best profile. All others pairs presented a restricted pattern with slight variations. For example, the primers detected a specific 178-by band, which was particularly strong with Citrobacter sp., in contrast with the other primer pairs studied. Moreover, some primer pairs occasionally presented a non-specific band which was also observed with primer pair RFFT7/RFFT8 using DNA from Enterobacfer sp., Citrobacter sp. and Yersinia sp..
Nonetheless, RFFT7/RFFT8 primer pairs is still presenting the best detection profile, giving a strong and unequivocal 103 by signal with 6 out of 10 Enterobacteriaceae tested (see Figure 4).
It has been found that additional purification of bacterial genomic DNA by Qiagen columns did not increase the number of bacteria detected. Furthermore, non-Enterobacteriaceae bacterial species such as Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus sp., were also studied and did not yield the desired fragment. Of a total of 116 bacterial species tested and comprising 95 Enterobacteriaceae, 10 Staphylococcus sp., 10 Pseudomonas sp. and 1 Enterococcus sp., 54 Enterobacteriaceae were positively detected. None of the non-Enterobacterial species could be detected with these primers and there were no false positives demonstrating 100% specificity for Enterobacteriaceae. Moreover, Klebsiella sp., Salmonella sp. and Shigella sp. genomic DNA was always amplified, suggesting 100% detectability of these bacteria species (Table 4).
TABLE 4. Microorganisms detected by PCR with the primers RFFT7 and RFFT8 Microrganisms Total no. No. of isolatesNo. of isolates of isolates detected not detected Enterobacteriaceae sp.
E. coli 10 10 0 Klebsiella sp. 10 10 0 Salmonella sp. 10 10 0 Shigella sp. 8 8 0 Enterobacter sp. 10 7 3 Serratia sp. 10 5 5 Citrobacter sp. 10 4 6 Proteus sp. 10 0 10 Providencia sp. 10 0 10 Yersinia sp. 7 0 7 Non-enterobacteriaceae sp Staphylococcus 10 0 10 sp.
Pseudomona sp. 10 0 10 Enterococcus sp. 1 0 1 Detection of Enterobacteriaceae in patient urine was assessed, and the results are shown in Figure 5 and compiled in Table 5. The primer pair RFFT7/RFFTB, was evaluated for the detection of Enterobacteriaceae sp. in clinical urine samples. A
group of 56 urine specimens (51 from Enterobacteriaceae specimens and 5 non-Enterobacteriaceae) was diagnosed by a conventional method (Kloos W.E and Bannerman T.L Staphylococcus and Micrococcus. In Murray P.R., Baron E.J., Pfaller M.A., Tenover F.C. and Yolken R.H. (ed), Manual of Clinical Microbiology 1995, pp.
282-298). The PCR technique allowed the detection only of samples which were from enterobacteriaceae specimens. Moreover using these primers and a non-nested PCR, DNA from 48 of these 51 specimens (94.1 %) could be detected by an approach consisting of a longer PCR program for a total of 50 cycles. A 103 by product was present only with positive specimens of Enterobacteriaceae (fig. 5). There was no false positive amplification with Pseudomonas sp., Gram-positive cocci and 2 negative urine specimens (Table 5).
TABLE 5. Clinical isolates detected by PCR with the primer pair RFFT7/RFFTB in urine specimens with > 100,000 CFU/mL a Microorganisms Total no. of No. of isolates No. of isolates isolates detected not detected Enterobacteriaceae E. coli. 39 39 0 Klebsiella sp. 8 8 0 Enterobacter sp. 2 1 1 Proteus sp. 2 0 2 Non-enferobacteriaceae Pseudomonas sp. 1 0 1 Enterococcus sp 3 0 3 Staphylococcus sp. 1 0 1 a Colony-forming units (CFU) determined by the 0.001 ml loop quantitative culture method.
Detection of Enterobacteriaceae in blood culture specimens with RFFT7/RFFT8 were assessed, and the results are shown in Figure 6. A total of 13 blood cultures were studied by DNA extraction with the benzyl alcohol-guanidine hydrochloride method.
Two sets of primers: RFFT7/RFFT8 and RFFT17/RFFT18 were evaluated for the detection of bacteria. For each pair of primers, only the longer PCR program was used. Out of 10 positive blood cultures there were 8 E. coli and 2 Klebsiella pneumoniae. Ten were found to contain Enterobacterial-specific PCR fragments with 100% sensitivity with RFFT7/RFFT8 while the sensitivity was 90% (9/10) with RFFT17/RFFT18 which did not detect a Klebsiella pneumoniae strain. No false positives were obtained from the 3 blood culture-negative specimens (see Figure 6).
DESCRIPTION OF THE TECHNIQUES USED IN THE INVENTION
Growth and characterization of bacteria The bacterial strains analyzed were either obtained from the American Type Culture Collection or isolated in a clinical microbiology laboratory. Gram-negative bacilli were identified with the Vitek system (Merieux, France). Cocci were characterized by the conventional method of Kloos and Bannerman (Supra). The bacteria were cultured on blood agar plates and incubated at 37°C for 18-24 h. The colonies obtained were inoculated in trypticase soy broth (TSB) and incubated at the same temperature for 18-24 h. Viable counts were achieved by plating serial dilutions of the broth culture.
Preparation of bacterial genomic DNA
DNA from bacterial suspension cultures in TSB was extracted according to the method of LeBouguenec et al. (Le Bouguenec C. et al. Rapid and Specific Detection of the pap, afa, and sfa Adhesin-Encoding Operons in Uropathogenic Escherichia coli Strains by Polymerase Chain Reaction. J. Clin. Microbiol. 1992, 30, pp. 1189-1193).
Briefly, bacteria contained in 1 ml of broth culture were collected by centrifugation at 3,OOOrpm for 3 min. The pellet was re-suspended in 200 pl of sterile distilled water, boiled for 15 min and again centrifuged at 14,OOOg for 3 min. The supernatant was carefully transferred to a sterile tube and maintained at -20°C.
DNA extraction from urine specimens Patient urine specimens with 100,000 CFU/ml or more were obtained from the hospital microbiology laboratory and stored at -20°C. DNA was extracted using QiAamp tissue method according to the manufacturer's protocol (QIAGEN, Mississauga, Ontario, Canada) with minor modifications. Briefly, 1 ml of urine was centrifuged at 6,OOOg for 10 min. The supernatant was discarded, and the remaining pellet was lysed by adding 180 ul of lysis buffer (buffer ATL, provide by manufacturer) and detergent. 20 pl of proteinase K (20 mg/ml) was also added, followed by incubation at 55°C for 2 h. Subsequently, 200 pl of AL buffer (provide by manufacturer) was added, vortexed vigorously, incubated at 70°C for 10 min and then at 95°C for 15 min, followed by mixing with 210 pl of sterile 100%
ethanol. This suspension was applied on a Qiagen column and centrifuged for 1 min in a 2-ml collection tube at 6,OOOg. The column was successively washed with AW buffer (provided by manufacturer), for 1 and 3 min, and the filtrates were discarded.
Two hundred pl of preheated AL buffer (at 70° C) was added to the column.
DNA was recovered by 1 min centrifugation and stored in a sterile microfuge tube at -20°C.
Bacterial DNA extraction from blood cultures Bacterial strains were recovered from blood cultures using BACTEC 9240 instrumentation (Becton Dickinson, Sparks, MD) and standard 10 aerobic/F and lytic/10 anaerobic F bottles. For maximum recovery, a 10-ml aliquot of patient blood was inoculated in each bottle and incubated at 35°C with agitation. A
sensor located at the bottom of each bottle responded by increasing fluorescence proportionally to the elevation of C02 concentration resulting from organism metabolism (Rohner P. et al. Comparative Evaluation of BACTEC Aerobic Plus/F and Septi-Chek Release Blood Culture Media. J. Clin. Microbiol. 1996, 34, pp.126-129). When a positive signal was obtained, a sample was taken from the bottle with a sterile syringe and Gram-staining was performed to confirm the presence of viable microorganisms. If Gram-staining showed Gram-negative bacteria, subcultures were prepared on MacConkey agar plates and incubated aerobically at 37°C.
Subcultures were also produced on blood and/or chocolate agar plates which were incubated at 37°C in 5% CO2. The anaerobic subcultures on blood aaar were supplemented with vitamin and incubated overnight. For aerobic bacteria, after overnight incubation, if colonies were observed on blood agar plates, identification was undertaken by conventional methods (Farmer, J.J. Enterobacferiaceae Introduction and Identification. In Murray P.R. et al. Manual of Clinical Microbiology 1995, pp. 438-464; Kloos W.E and Bannerman T.L Staphylococcus and Micrococcus.
In Murray P.R. et al., Manual of Clinical Microbiology 1995, pp. 282-298). The Gram-negative rods were identified biochemically with the Vitek system.
DNA was extracted only in samples taken from positive aerobic bottles when positive growth was observed, or after 5 days when the culture bottles were discarded.
Five hundred pl aliquots of the broth medium containing erythrocytes were removed from the aerobic bottles under sterile conditions, put in conical tubes and kept at -20°C until processed.
The benzyl alcohol-guanidine hydrochloride method (Fredricks D.N. and Relman D.A.
Improved Amplification of Microbial DNA from Blood Cultures by Removal of the PCR
inhibitors Sodium Polyanetholsulfonate. J. Clin. Microbiol. 1998, 36, pp. 2810-2816) was used for DNA extraction, with minor modifications. A 100-pl aliquot of the blood broth mixture containing bacteria was lysed by brief mixing with the same amount of lysis buffer (5 M guanidine hydrochloride-100 mM Tris, pH 8.0 in sterile water). This was followed by the addition of 400 pl of water and 800 pl of 99% benzyl alcohol (Sigma Chemical Company) and extensive vortexing. The suspension was centrifuged for 5 min at 6,OOOg, and 400 NI of the supernatant transferred to a microfuge tube for precipitation with 40 pl of 3.0 M sodium acetate and 440 pl of isopropanol.
The DNA
precipitate was recovered by centrifugation at 6,OOOg for 30 min, washed with 70%
ethanol, air-dried, and dissolved in 100 pl of 10 mM Tris-0.1 mM EDTA, pH 8.5.
Oligonucleotide primers for PCR detection E. coli genome organization in the ECA operon is known and its complete sequence is available. Based on this knowledge, several primers were designed from the rfelrff group of genes located on E. coli genome positions 84.5 to 86.5 min (Daniels D.L. et al. Analysis of the Escherichia coli genome: DNA sequence of the region from 84.5 to 86.5 minutes. Science 1992, 257 (5071), pp. 771-778; Blattner F.R. et al. The Complete Genome Sequence of Escherichia coli K-12. Science. 1997, 277, pp.
1453-1462). Since nucleotide sequence of the E. coli rfe gene have already been published, we took advantage of this sequence to design several sets of primers (see table 2) for the amplification of the E coli DNA (Meier-Dieter U. et al.
Nucleotide Sequence of the Escherichia coli rfe Gene Involved in the Synthesis of Enterobacterial Common Antigen. J. Biol. Chem. 1992 , 267, pp. 746-753). Their specific positions on the sequence are presented in figure 1a. The sequence of rftl' gene was available by genebank (accession number : M87049) and the positions of the primers are shown in figure 1 b.
The template primers ECA1/ECA2 and RFFT17/RFFT18 were used in combination with nested primers for PCR-based detection of E. coli and Enterobacteriaceae strains respectively.
PCR Amplification of bacterial DNA
Reactions were performed in a total volume of 55 ul. Forty-five pl of a master mixture containing 5 pl of 10 X PCR buffer (500 mM KCI, 15 mM MgCl2 and 100 mM Tris-HCI), 8 pl of 1.25 pM of dNTP stock solution, 2.5 pl of each of the forward and reverse primers (stock 1 pg/ml) and 0.25 pl (1.25 units) of TAQ polymerase (Pharmacia, Baie d'Urfe, Quebec, Canada) and 26.75 pl of sterile distilled water were mixed with 10 ul of bacterial DNA samples. The negative controls contained all ingredients of the master mixture without any DNA samples. For urine specimens, the final concentration of the primers was 0.45 pM, and 5 units of TAQ polymerase were used.
For the nested PCR and blood culture specimens, the concentration of the primers and Taq polymerase was respectively 0.9 pM and 2.5 units. A 3 pl aliquot of template PCR reaction product was used for the nested PCR. Amplifications were performed with a Perkin Elmer 9600 cycler under the following conditions: cycles of 2 min denaturation at 94°C and 1 min annealing at 60°C, followed by 2 min of polymerization at 72°C (this cycle was repeated 25 times). For nested PCR, the first amplification (template) cycle was as described previously, while the nested PCR consisted of 25 cycles of shorter duration: 30 sec denaturation at 94°C, 15 sec, annealing at 60°C and sec polymerization at 72°C. A 10 NL aliquot from the PCR reaction product was electrophoresed on 1.5% agarose mini-gel, stained with ethidium bromide and photographed with a Polaroid camera.
For the study of urine specimens, a non-nested PCR was chosen to avoid contamination, which can occur with the nested PCR, particularly with the transfer of DNA into another tube after the first amplification. The non-nested approach consisted of the successive use of the longer PCR program followed by the shorter one for a total of 50 cycles.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: CORPORATION DU CENTRE DE RECHERCHE DU CENTRE
HOSPITALIER DE L'UNIVERSITE DE MONTREAL
(B) STREET: 3850 Saint-Urbain (C) CITY: Montreal (E) COUNTRY: Canada (F) POSTAL CODE: H2W 1T8 (ii) TITLE OF THE INVENTION: Oligonucleotide primers for the specific PCR detection of enterobacteriaceae species DNA using RFE-RFF gene templates (iii) NUMBER OF SEQUENCES: 8 (iv) CORRESPONDENCE ADDRESS:
(A) NAME: ROBIC
(B) STREET: 55 Saint-Jacques (C) CITY: Montreal (E) COUNTRY: Canada (F) POSTAL CODE: H2Y 3X2 (v) COMPUTER-READABLE FORM:
(A) TYPE OF SUPPORT: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: WordPad (vi) CURRENT APPLICATION DATA:
(A) APPLICATION DATA: 2,286,512 (B) FILING DATE: 27 October 1999 (viii) PATENT AGENT INFORMATION
(A) NAME: ROBIC
(B) REFERENCE NUMBER: 26740-0005 (2) INFORMATION FOR SEQ ID N0: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 nucleic acids (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (iii) ANTISENS: NO
(ix) FEATURE:
(A) NAME/KEY: RFFT11 (B) LOCATION: 31995..32015 (xi) SEQUENCE DESCRIPTION: SEQ ID N0: l:
(2) INFORMATION FOR SEQ ID N0: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 nucleic acids (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (iii) ANTISENS: NO
(ix) FEATURE:
(A) NAME/KEY: RFFT20 (B) LOCATION: 32046..32066 (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 2:
(2) INFORMATION FOR SEQ ID N0: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 nucleic acids (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (iii) ANTISENS: NO
(ix) FEATURE:
(A) NAME/KEY: RFFT17 (B) LOCATION: 32089..32109 (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 3:
(2) INFORMATION FOR SEQ ID N0: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 nucleic acids (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (iii) ANTISENS: NO
(ix) FEATURE:
(A) NAME/KEY: RFFT21 (B) LOCATION: 32112..32131 (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 4;
(2) INFORMATION FOR SEQ ID N0: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 nucleic acids (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (iii) ANTISENS: NO
(ix) FEATURE:
(A) NAME/KEY: RFFT22 (B) LOCATION: 32128..32148 (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 5:
(2) INFORMATION FOR SEQ ID N0: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 nucleic acids (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (iii) ANTISENS: NO
(ix) FEATURE:
(A) NAME/KEY: RFFT7 (B) LOCATION: 32135..32155 (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 6:
(2) INFORMATION FOR SEQ ID N0: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 nucleic acids (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (iii) ANTISENS: YES
(ix) FEATURE:
(A) NAME/KEY: RFFT8 (B) LOCATION: 32237..32217 (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 7:
(2) INFORMATION FOR SEQ ID N0: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 nucleic acids (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (iii) ANTISENS: YES
(ix) FEATURE:
(A) NAME/KEY: RFFT18 (B) LOCATION: 32266..32247 (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 8:
Claims (23)
1. An oligonucleotide selected from the group consisting of:
- RFFT11 having the nucleotide sequence of SEQ ID NO. 1;
- RFFT20 having the nucleotide sequence of SEQ ID NO. 2;
- RFFT17 having the nucleotide sequence of SEQ ID NO. 3;
- RFFT21 having the nucleotide sequence of SEQ ID NO. 4;
- RFFT22 having the nucleotide sequence of SEQ ID NO. 5;
- RFFT7 having the nucleotide sequence of SEQ ID NO. 6;
- RFFT8 having the nucleotide sequence of SEQ ID NO. 7; and - RFFT18 having the nucleotide sequence of SEQ ID NO. 8.
- RFFT11 having the nucleotide sequence of SEQ ID NO. 1;
- RFFT20 having the nucleotide sequence of SEQ ID NO. 2;
- RFFT17 having the nucleotide sequence of SEQ ID NO. 3;
- RFFT21 having the nucleotide sequence of SEQ ID NO. 4;
- RFFT22 having the nucleotide sequence of SEQ ID NO. 5;
- RFFT7 having the nucleotide sequence of SEQ ID NO. 6;
- RFFT8 having the nucleotide sequence of SEQ ID NO. 7; and - RFFT18 having the nucleotide sequence of SEQ ID NO. 8.
2. An oligonucleotide primer having the nucleotide sequence of the oligonucleotide of claim 1.
3. The oligonucleotide primer according to claim 2, wherein the primer is selected from the group of RFFT17 (SEQ ID NO. 3); RFFT22 (SEQ ID NO.5); RFFT7 (SEQ ID
NO. 6); RFFT8 (SEQ ID NO. 7); and RFFT18 (SEQ ID NO. 8).
NO. 6); RFFT8 (SEQ ID NO. 7); and RFFT18 (SEQ ID NO. 8).
4. An oligonucleotide primer comprising the nucleotide sequence of the oligonucleotide of claim 1 or a substantial part thereof.
5. An oligonucleotide primer having a nucleotide sequence being complementary or identical to a sequence of rffT gene of Enterobacteriaceae species where the sequence of rffT gene is located between position +31995 and position +32266.
6. An oligonucleotide primer of claim 5, wherein the nucleotide sequence is of about 21 nucleotides.
7. Use of a pair of oligonucleotide primers including a forward primer and a reverse primer for PCR detection of Enterobacteriaceae species from a sample, wherein the forward primer is selected from the group consisting of RFFT11 (SEQ ID
NO. 1); RFFT20 (SEQ ID NO. 2); RFFT17 (SEQ ID NO. 3); RFFT21 (SEQ ID NO. 4);
RFFT22 (SEQ ID NO. 5); and RFFT7 (SEQ ID NO. 6); and the reverse primer is selected form the group consisting of RFFT8 (SEQ ID NO. 7); and RFFT18 (SEQ ID
NO. 8).
NO. 1); RFFT20 (SEQ ID NO. 2); RFFT17 (SEQ ID NO. 3); RFFT21 (SEQ ID NO. 4);
RFFT22 (SEQ ID NO. 5); and RFFT7 (SEQ ID NO. 6); and the reverse primer is selected form the group consisting of RFFT8 (SEQ ID NO. 7); and RFFT18 (SEQ ID
NO. 8).
8. Use of claim 7, wherein the forward primer is RFFT22 (SEQ ID NO. 5) and the reverse primer is RFFT 8 (SEQ ID NO. 7).
9. Use of claim 7, wherein the forward primer is RFFT7 (SEQ ID NO. 6) and the reverse primer is RFFT8 (SEQ ID NO. 7).
10. Use of claim 7, wherein the forward primer is RFFT17 (SEQ ID NO. 3) and the reverse primer is RFFT18 (SEQ ID NO. 8).
11. Use of an oligonucleotide primer according to claims 4, 5 or 6, for PCR
detection of Enterobacteriaceae species.
detection of Enterobacteriaceae species.
12. Use of any one of claims 7 to 11, wherein the sample is a blood sample or a urine sample.
13. Use of any one of claims 7 to 12, wherein the Enterobacteriaceae species is selected from the group consisting of Escherichia coli, Klebsiella pneumoniae, Enterobacter sp., Serratia sp., Salmonella sp., Shigella sp., and Citrobacter sp.
14. Method of detection of Enterobacteriaceae species comprising the steps of:
a) extracting DNA from a sample;
b) amplifying a fragment of the extracted DNA by PCR using a forward oligonucleotide primer selected from the group consisting of RFFT11 (SEQ ID
NO. 1);
RFFT20 (SEQ ID NO. 2); RFFT17 (SEQ ID NO. 3); RFFT21 (SEQ ID NO. 4); RFFT22 (SEQ ID NO. 5); and RFFT7 (SEQ ID NO. 6); and a reverse oligonucleotide primer selected from the group consisting of RFFT8 (SEQ ID NO. 7); and RFFT18 (SEQ ID
NO. 8) and c) detecting the amplified fragment of the DNA.
a) extracting DNA from a sample;
b) amplifying a fragment of the extracted DNA by PCR using a forward oligonucleotide primer selected from the group consisting of RFFT11 (SEQ ID
NO. 1);
RFFT20 (SEQ ID NO. 2); RFFT17 (SEQ ID NO. 3); RFFT21 (SEQ ID NO. 4); RFFT22 (SEQ ID NO. 5); and RFFT7 (SEQ ID NO. 6); and a reverse oligonucleotide primer selected from the group consisting of RFFT8 (SEQ ID NO. 7); and RFFT18 (SEQ ID
NO. 8) and c) detecting the amplified fragment of the DNA.
15. Method of claim 14, wherein the forward primer is RFFT7 (SEQ ID NO. 6) and the reverse primer is RFFT8 (SEQ ID NO. 7).
16. Method of claim 14, wherein the forward primer is RFFT 22 (SEQ ID NO. 5) and the reverse primer is RFFT8 (SEQ ID NO. 7).
17. Method of claim 14, wherein the forward primer is RFFT17 (SEQ ID NO. 3) and the reverse primer is RFFT18 (SEQ ID NO. 8).
18. Method of claim 17, wherein step (b) is followed by the step of:
b-1) amplifying a portion of the fragment amplified in step (b), by PCR using a forward primer being RFFT7 (SEQ ID NO. 6) and a reverse primer being RFFT8 (SEQ
ID NO. 7).;
and wherein step (c) is detecting the portion amplified in step (b-1).
b-1) amplifying a portion of the fragment amplified in step (b), by PCR using a forward primer being RFFT7 (SEQ ID NO. 6) and a reverse primer being RFFT8 (SEQ
ID NO. 7).;
and wherein step (c) is detecting the portion amplified in step (b-1).
19. Method of detection of Enterobacteriaceae species comprising the steps of:
a) extracting DNA from a sample;
b) amplifying a fragment of the extracted DNA by PCR using a forward oligonucleotide primer comprising the nucleotide sequence of the oligonucleotide selected from the group consisting of RFFT11 (SEQ ID NO. 1); RFFT20 (SEQ ID
NO. 2); RFFT17 (SEQ ID NO. 3); RFFT21 (SEQ ID NO. 4); RFFT22 (SEQ ID NO. 5);
and RFFT7 (SEQ ID NO.6) or a substantial part thereof; and a reverse oligonucleotide primer comprising the nucleotide sequence of the oligonucleotide selected from the group consisting of RFFT8 (SEQ ID NO. 7); and RFFT18 (SEQ ID
NO. 8) or a substantial part thereof; and c) detecting the amplified fragment of the DNA.
a) extracting DNA from a sample;
b) amplifying a fragment of the extracted DNA by PCR using a forward oligonucleotide primer comprising the nucleotide sequence of the oligonucleotide selected from the group consisting of RFFT11 (SEQ ID NO. 1); RFFT20 (SEQ ID
NO. 2); RFFT17 (SEQ ID NO. 3); RFFT21 (SEQ ID NO. 4); RFFT22 (SEQ ID NO. 5);
and RFFT7 (SEQ ID NO.6) or a substantial part thereof; and a reverse oligonucleotide primer comprising the nucleotide sequence of the oligonucleotide selected from the group consisting of RFFT8 (SEQ ID NO. 7); and RFFT18 (SEQ ID
NO. 8) or a substantial part thereof; and c) detecting the amplified fragment of the DNA.
20. Method of detection of Enterobacteriaceae species comprising the steps of:
a) extracting DNA from a sample;
b) amplifying a fragment of the extracted DNA by PCR using a forward oligonucleotide primer having a nucleotide sequence substantially identical to a 5' - 3' sequence of rffT gene of Enterobacteriaceae species; and a reverse primer having a nucleotide sequence substantially identical to a complementary sequence of the 5'-3' sequence of the rffT gene of Enterobacteriaceae species wherein the forward primer and the reverse primer or the 5'-3' sequence are complementary sequences of the rffT
gene located between position +31995 and position +32266; and c) detecting the amplified fragment of the DNA.
a) extracting DNA from a sample;
b) amplifying a fragment of the extracted DNA by PCR using a forward oligonucleotide primer having a nucleotide sequence substantially identical to a 5' - 3' sequence of rffT gene of Enterobacteriaceae species; and a reverse primer having a nucleotide sequence substantially identical to a complementary sequence of the 5'-3' sequence of the rffT gene of Enterobacteriaceae species wherein the forward primer and the reverse primer or the 5'-3' sequence are complementary sequences of the rffT
gene located between position +31995 and position +32266; and c) detecting the amplified fragment of the DNA.
21. Method of claim 20, wherein the primers have a nucleotide sequence of about 21 nucleotides.
22. Diagnostic assay for diagnosing Enterobacteriaceae species in a sample, the assay comprising:
- means for extracting DNA from the sample;
- a solution for detecting Enterobacteriaceae species DNA from the extracted DNA by PCR, the solution comprising a PCR master mixture and a pair of primers including a forward primer and a reverse primer as defined in claims 7, 8, 9 or 10.
- means for extracting DNA from the sample;
- a solution for detecting Enterobacteriaceae species DNA from the extracted DNA by PCR, the solution comprising a PCR master mixture and a pair of primers including a forward primer and a reverse primer as defined in claims 7, 8, 9 or 10.
23. Diagnostic assay for diagnosing Enterobacteriaceae species in a sample, the assay comprising:
- means for extracting DNA from the sample;
- a solution for detecting Enterobacteriaceae species DNA from the extracted DNA by PCR, the solution comprising a PCR master mixture and a pair of primers including a forward primer as defined in claims 4, 5, or 6, and a reverse primer as defined in claims 4, 5, or 6 and being in a downstream position from the forward primer.
- means for extracting DNA from the sample;
- a solution for detecting Enterobacteriaceae species DNA from the extracted DNA by PCR, the solution comprising a PCR master mixture and a pair of primers including a forward primer as defined in claims 4, 5, or 6, and a reverse primer as defined in claims 4, 5, or 6 and being in a downstream position from the forward primer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002286512A CA2286512A1 (en) | 1999-10-27 | 1999-10-27 | Oligonucleotide primers for the specific pcr detection of enterobacteriaceae species dna using rfe-rff gene templates |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002286512A CA2286512A1 (en) | 1999-10-27 | 1999-10-27 | Oligonucleotide primers for the specific pcr detection of enterobacteriaceae species dna using rfe-rff gene templates |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2286512A1 true CA2286512A1 (en) | 2001-04-27 |
Family
ID=4164396
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002286512A Abandoned CA2286512A1 (en) | 1999-10-27 | 1999-10-27 | Oligonucleotide primers for the specific pcr detection of enterobacteriaceae species dna using rfe-rff gene templates |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2286512A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003052143A2 (en) * | 2001-12-19 | 2003-06-26 | Angles D Auriac Marc B | New primers for the detection and identification of bacterial indicator groups and virulence factors |
-
1999
- 1999-10-27 CA CA002286512A patent/CA2286512A1/en not_active Abandoned
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003052143A2 (en) * | 2001-12-19 | 2003-06-26 | Angles D Auriac Marc B | New primers for the detection and identification of bacterial indicator groups and virulence factors |
| WO2003052143A3 (en) * | 2001-12-19 | 2003-10-02 | D Auriac Marc B Angles | New primers for the detection and identification of bacterial indicator groups and virulence factors |
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