GB2241242A - DNA probe for Neisseria meningitidis - Google Patents
DNA probe for Neisseria meningitidis Download PDFInfo
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- GB2241242A GB2241242A GB9000572A GB9000572A GB2241242A GB 2241242 A GB2241242 A GB 2241242A GB 9000572 A GB9000572 A GB 9000572A GB 9000572 A GB9000572 A GB 9000572A GB 2241242 A GB2241242 A GB 2241242A
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- United Kingdom
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- dna
- strains
- neisseria meningitidis
- plasmid
- pus210
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
Abstract
A specific fragment of DNA isolated from a repetitive element present in the genome of Neisseria meningitidis is disclosed. DNA probes based on a nucleotide sequence contained in this repetitive element may be used to obtain specific identification Neisseria meningitidis strains and greater differentiation of strains than with other typing methods, and also to detect Neisseria meningitidis in clinical samples. Plasmid pUS210 contains a clone from the repetitive element, and Escherichia coli containing it has been deposited with the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland under NCIMB number 40248 in accordance with the Budapest Treaty. The inserted fragment of neisserial DNA in plasmid pUS210 has an extent of 2.0 kb terminated at each end by sites of cleavage by restriction endonuclease Sau.3A. Mapping with other restriction endonucleases shows a single site for Sal.I at 0.18 kb from one end of the fragment, and a single site for Hin.dIII at 0.72 kb from the same end. No sites have been detected for Eco.Ri, Pst.I, Sph.I, and Sst.I.
Description
DETECTION AND IDENTIFICATION OF NEISSERIACEAE
This invention relates to the detection and identification of members of the
Neisseriaceae and to the new plasmid pUS210 having neisserial DNA which may be used for the detection, identification and genetic typing of Neisseria meningitidis and Neisserla gonorrhoea.
Neisseria mendngitidis is an important human pathogen, being the major causative agent of meningitis and septicaemia and causing a signifwcant degree of mortality in the United Kingdom and worldwide. Recent outbreaks, referred to as hyper-endemic disease, have occurred in geographically localized areas and caused great public anxiety.
Methods currently used to differentiate Nelsseria meningitidis strains employ antibodies to distinguish serogroups based on capsular polysaccharide, e6 e.g. groups A, B, C, 29E, W135 and Y, and serotypes and subtypes based on surface antigens.
Grouping and typing by these methods is inadequate to distinguish between strains found in various outbreaks of meningitis, and strains of Neisseria meningitidis isolated from outbreaks are frequently non-groupable or non-typeable. In particular, these methods have failed to differentiate certain strains associated with hyper-endemic disease, e.g. sulphonamide-resistant strains of serotype B15.16 responsible for prolonged outbreaks of meningitis recently in the United Kingdom,
Norway and elsewhere in northern Europe.
Other methods of typing Neisseria meningitidis strains include electrophoretic typing of certain enzymes, and fingerprinting of digests of the total genome with restriction endonuclease. These methods are slow and technically demanding, and they do not always differentiate strains associated with hyper-endemic disease from other strains usually found in the population. Moreover, serotyping and other methods such as electrophoretic typing do not necessarily reflect genetic identity.
This invention utilizes a specific fragment of deoxyribonucleic acid ("DNA") isolated from the genome of Nefsserfa meningitidis which contains an element present in the genome in multiple copies (the "repetitive element"). DNA probes based on a nucleotide sequence contained in this repetitive element may be used to differentiate pathogenic Nefsserfa species including Neisseria meningitidis and
Neisseria gonorrhoea. Such a probe may be used to obtain specific identificatibn of Neisseria meningftldfs strains and greater differentiation of strains than with other typing methods. The probe is therefore useful in epidemiological studies and specific control of meningitis, and it may also be used to detect Neisseria meningitidis in clinical samples.
DNA from Neisseria meningitidfs strain G3, isolated clinically from an outbreak of hyper-endemic disease in Gloucester, England and deposited with the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland under NCIMB number 40248, was cleaved with restriction endonuclease
Sau.3A to give a DNA library. The resulting digest of
DNA fragments was cloned into the well known plasmid vector pBR322, and plasmid DNA was isolated from the clones by standard procedures. DNA from randomly isolated clones was used in fingerprinting trials with various restriction endonuclease digests of genomic DNA from strains of Neisseria meningitidis.
Differentiation was poor, particularly within strains of B serogroup.
Clones containing DNA from a repetitive element in
Neisseria meningitidis were identified by screening the
DNA library for clones giving a relatively strong signal in colony hybridization with radio-labelled
Neisseria meningitidis DNA. Such screening identified one clone which hybridized with multiple fragments of the Neisseria meningitidis strain G3 used to construct the DNA library. Fingerprinting trials with this clone clearly differentiated serogroup B15.16 strains of
Neisseria meningitidis which had been isolated from many parts of the United Kingdom, and this allowed the typing of strains which could not be typed by conventional serotyping.
The plasmid containing this clone has been named "pUS210" and Escherichia coli containing it has been deposited with the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland under NCIMB number 40247 in accordance with the Budapest Treaty.
The present invention in one aspect is the new plasmid pUS210.
The inserted fragment of neisserial DNA in plasmid pUS210 has an extent of approximately 2.0 kb (where 1.0 kb is 1000 base-pairs of nucleotide) terminated at each end by sites of cleavage by restriction endonuclease Sau.3A. Mapping with other restriction endonucleases shows a single site for Sal.I at 0.18 kb from one end of the fragment, and a single site for Hin.dIII at 0.72 kb from the same end. No sites have been detected for
Eco.RI, Pst.I, Sph.I, and Sst.I.
It is possible that part of the repetitive element contained in the genome of Neisseria meningitidis strain G3 is not contained in plasmid pUS210. Such additional fragments of the repetitive element may be isolated by the following procedure. Genomic DNA of
Neisseria meningitidis strain G3 is partially digested with restriction endonuclease Bam.HI and cloned into the site for Bam.HI of the plasmid vector pBR322.
Clones containing DNA of the repetitive element and DNA homologous to the neisserial DNA of plasmid pSU210 are identified by hybridization with radio-labelled DNA of total Neisseria meningitidis strain G3 genome and radio-labelled DNA of the inserted fragment of neisserial DNA in plasmid pUS210.
The fragment of neisserial DNA is isolated from the plasmid pSU210 by standard procedures. From the ssam.HI digest of genomic DNA of Neisseria meningitidis G3, clones containing large fragments are selected for further study by mapping with restriction endonucleases and by hybridization to both the Neisseria meningitidis
DNA and the pSU210 DNA. Fragments of the repetitive element which are not present in pSU210 are identified as those which hybridize relatively strongly to
Neisseria meningitidis DNA but not to the inserted neisserial DNA of plasmid pSU2 10.
The present invention in another aspect is a nucleotide sequence which has greater than 70% homology to a sequence of 30 base-pairs contained in the repetitive element which is present in Neisseria meningitidis strain G3 and is contained partly or wholly in plasmid pSU210. The invention in this aspect is in particular a nucleotide sequence which has a greater than 70 > h homology to a sequence of 30 base-pairs contained in the neisserial DNA of plasmid pSU910.
In a further aspect, the invention provides probes containing such a nucleotide sequence, which may be used in methods of identifying and differentiating strains of Neisseria meningitidis and Neisseria gonorrhoea, and of detecting members of the Neisseriaceae in clinical samples or microbiological cultures e.g. for diagnosis of disease.
By standard procedures of genetic manipulation, a bacterium, virus or eukaryotic cell containing a nucleotide sequence according to the invention may be produced as the result of genetic modification.
Protein encoded by a nucleotide sequence according to the invention may be produced by such a genetically modified organism by standard procedures, and the invention further provides a diagnostic or immunological reagent comprising or obtained by use of such a protein.
The specific description which follows is given by way of example. Standard procedures were followed, as described for example in "Molecular Cloning, a
Laboratory Manual", second edition, by J. Sambrook, E. F. Fritsch and T. Maniatis, published by Cold Spring Harbor Laboratory Press, 1989.
Figure 1 is a photograph of an autoradiograph obtained by exposure of the membrane obtained when DNA from various strains of Nefsseria meningitidis digested with Eco.RI was probed with pU5210 as described later.
-Figure 2 is a photograph similarly obtained when DNA from strains of
Neisseria meningitidis isolated from the areas of hyper-endemic disease at
Gloucester and Plymouth was probed with pUS210.
Figure 3 is a photograph similarly obtained when DNA from other members of the Neisseriaceae was probed with pUS210.
Isolation of DNA
Strains of Neisseria meningitidis, including sulphonamide-resistant strains of serotype B15.16 isolated from patients and contacts, other serogroup B and C strains of different serotypes, and non-groupable and non-typeable strains, were cultured on chocolate agar at 37'C for 48 h with 5X COz, and harvested in a medium comprising 5 ml sucrose (10% v/v) 50 mM tris(2-hydroxyethyl)amine hydrochloride pH 8O, and 10 mM ethylenediamine-tetraacetic acid. Lysozyme was added to a concentration of. 10 MgXml and the mixture was incubated on ice for 30 min.Sodium dodecyl sulphate and Proteinase K were then added to final concentrations of 1 v/v and 10 pg/ml respectively and the solution was incubated at 55 C for 1 h. After incubation the solution was extracted once with phenol saturated with 10 mM tris(hydroxyethyl)amine hydrochloride pH 8O containing 1 mM ethylenediamine-tetraacetic acid and then twice with 50:50 phenol chloroform. DNA was precipitated with ethanol, washed in 70X ethanol, dried, and resuspended in 10 mM tris(hydroxyethyl)amine hydrochloride pH 8O containing 1 mM ethylenediamine-tetraacetic acid. Average yield was 750 pg DNA from l.g of
Neisseria meningitidis.
Restriction endonuclease cleavage
DNA was cleaved with commercially available restriction endonucleases in accordance with the manufacturers' instructions. Ribonuclease (5 pg/ml) and spermidine (4 mM) were included in the mixtures. Typically, 5 Mg DNA was cleaved with 50 units of restriction endonuclease in a total volume of 50 pl for 4 h at 37 C. Aliquots of digests corresponding to 1 pg of digested DNA were electrophoresed through 1 agarose and then used for Southern blots, and the remaining digest was stored.
Southern blotting and hybridization
Southern blotting was on nylon membranes. Probe DNA free of vector sequences was isolated using "Geneclean" (Bio 101 Inc.). Random primed probe labelling was with 32P deoxycytosine triphosphate (Amersham - International). Hybridizations were performed in 6x SSC, 5x Denhardt's solution, 0 5% sodium dodecyl sulphate and 100 pg/ml herring testes DNA at 65 C overnight. Membranes were washed at 65'C in 6x SSC containing 01X sodium dodecyl sulphate for 30 min followed by 0.1X SSC containing 01X sodium dodecyl sulphate for 30 min.
A stock solution of 20x SSC was made up as follows. 3 M Sodium chloride and 0 3 M sodium citrate were dissolved in distilled water to a final volume of one litre and autoclaved after the pH had been adjusted to 7 0.
A stock solution of SOX Denhardt's solution was made up as follows. 0 5 g "Ficoll" (mw 40,00.0), 0 5 g polyvinylpyrrolidone and 0 5 g bovine serum albumin were dissolved in sterile deionized distilled water to a final volume of one litre which was dispensed into aliquots and stored at -20C.
rsolation of DNA probes
A neisserial DNA library was constructed from Neisseria meningitidis strain G3 partially digested with restriction endonuclease Sau.3A, and cloned into the Barn. HI site of dephosphorylated pBR322. The ligation mixture was transformed into
Escherichia coli strain PLK-F ("Stratagene"), providing a methylcytosine-restrict ion background suitable for cloning neisserial DNA. Randomly isolated clones were tested for their ability to differentiate strains of Neisseria ineningitidis in hybridization experiments. Clones containing repetitive DNA were isolated by screening the library using colony hybridization with radio-labelled Neisseria meningitidis DNA.Clones giving a relatively strong signal were isolated as presumptive clones of repetitive DNA. The clones were further investigated by isolation and purification of plasmid DNA by standard procedures and use of this
DNA as hybridization probe. Flasmid DNA, or inserted neisserial DNA isolated from plasmid DNA by standard procedures, was radio-labelled and used to probe digests of DNA from Neisseria meningitidis strains. DNA from randomly isolated clones and
DNA from clones identified as containing repetitive DNA were tested for their ability to differentiate Neisseria meningitidis strains in hybridization experiments. Confirmation that clones possessed repetitive DNA was provided by testing the ability of such clones to hybridize to multiple fragments in Southern blots of Neisseria meningitidis DNA digested with restriction endonuclease.
Fingerprinting using randomly isolated genomic clones
Three random clones, totalling 8 kb genomic DNA, were used as probes in
Southern blots against four separate digests, made with restriction endonucleases
Cla.I, Ta q. I, Fst.I and Hpa.I respectively, of genomic DNA from Neisseria mentngitddis strains. Although such probes frequently differentiated between strains of groups B and C, differentiation within group B strains was poor. No differentiation was observed within strains of serotype B15.16, whether isolated from patients or their contacts. In addition, no differences were found between serotype B15.16 strains isolated from areas of hyper-endemic disease and those from the rest of the United Kingdom.
One of the random clones showed background hybridization to multiple fragments which were highly polymorphic in all cases. This hybridization, present after high-stringency washing, reflects hybridization to short repetitive sequences within the Neisseria meningitidis genome. Previous studies with Neisseria gonorrhoea, which shares greater than 96X DNA homology with Neisseria meningitidis, have revealed short repetitive sequences which produce similar results in Southern blots. Although such sequences can provide useful information, the signals obtained are poor and the banding patterns are too complex for typing. For this reason improved probes were sought based on larger repetitive sequences capable of producing clear banding patterns which were simple to determine.
Fingerprinting using a specific repetitive DNA probe
Screening for repetitive DNA identified a clone of about2 kb, present in plasmid pUS2 10, which hybridized with multiple fragments of Neisseria meningitidis strain G3 used in constructing the DNA library. Hybridization experiments using a pUS210 probe with Neisseria meningitidis DNA gave clearly different banding patterns for strains of serotype B15.16 isolated from many parts of the United
Kingdom. Furthermore, typing of non-groupable serotype 15 strains revealed that such strains may be either similar to or distinct from serotype B15.16 strains.
Thus this probe allows the typing of such strains which cannot be typed by conventional serotyping. The use of this probe therefore provides a significant advantage in differentiation compared with other probes and a considerable advance on serotyping.
The value of this probe was clearly demonstrated in examining strains from two areas of hyper-endemic disease in the United Kingdom, the first from the
Gloucester area and the second from the Plymouth area. In both cases, and in contrast to strains from elsewhere in the United Kingdom, all strains examined from diseased patients were identical. In addition, the strains isolated from patients in Gloucester were identical to those isolated from patients in Plymouth.
This indicates that the probe is capable of identifying a strain of Neisseria meningitidis which is responsible for hyper-endemic disease in the United Kingdom.
Examination of other strains indicates that this strain is presently rare elsewhere in the United Kingdom. The identification of this strain, which may have increased virulence compared with other strains in the United Kingdom, is extremely important in controlling meningococcal disease in the United Kingdom.
The probe also identifies a group of related strains which may be responsible for hyper-endemic disease in Norway. The use of the pSU210 probe is presently the only method available for identifying the strains responsible for hyper-endemic meningococcal disease.
Preliminary data indicate that the pSU210 probe also hybridizes to multiple polymorphic banding patterns in DNA from Neisseria gonorrhoea and other members of the Neisseriaceae. The probe may therefore be useful in typing strains of
Neisseria gonorrhoea and other members of the Neisseriaceae.
The pSU210 probe may also be useful in the detection of Neisseria menJngitidis for diagnosis of meningococcal disease.
Hybridization of pUS,710 to meningococcal DNA
Radio-labelled pUS210 was hybridized, by Southern blotting and hybridization, to fragments of Neisseria menfngitidis DNA digested with restriction endonuclease.
As shown in Figure 1, approximately five strongly hybridizing bands were obtained from Neisseria meningitidis strain G3, the strain from which the DNA library was prepared (lane 1 in Figure 1). Other strains of serotype B15.16 gave between about five and about nine strongly hybridizing bands, as shown in Figure 1.
When DNA from Neisseria meningitidis strain G3 was digested with various restriction endonucleases, the approximate number of strongly hybridizing bands that were obtained is shown in the following table.
Restriction endonuclease Number of bands
Eco.RI 5 Cla.I 9
Hin.dIII 8
Hpa.I 5 Pst.I 5
These data suggest that the genome of Neisseria meningitidis strain G3 contains four or five copies of the repetitive element present in the fragment of neisserial DNA in plasmid pSU210, and that the repetitive element has a single site for the restriction endonucleases Hin.dIII and Cls.I but no site for Eco.RI, HpaI or Fst.I. However, it is possible that parts of the repetitive element that are not contained in the clone pUS210 may have sites for the latter restriction endonucleases.
Dffferentfation of Neisseria meningitidis strains
DNA from a number of Neisseria meningitfdis strains was digested with restriction endonuclease Eco.RI and probed with pUS210 by Southern blotting. The result, shown in Figure 1, shows that the strains give a number of clearly different banding patterns. Further analysis of the data shows that epidemiologically linked strains give similar or identical banding patterns. This could be used to identify carriers and contacts of patients.
Identification of strains causing hyper-endemic disease
DNA from Neisseria meningitidis strains isolated from the areas of hyperendemic disease at Gloucester and Plymouth were digested with restriction endonuclease EcoRI and probed with pUS210 by Southern blotting. The result, shown in Figure 2, shows that strains from these foci of hyper-endemic disease give identical banding patterns, in contrast to the results shown in Figure 1 for strains from elsewhere in the United Kingdom. The neisserial DNA cloned into plasmid pUS210 can therefore be used to identify the strain responsible for these outbreaks of hyper-endemic disease. The availability of a method of identifying the strain allows disease due to the strain to be monitored and control measures to be introduced.
Differentiation and detection of other Nefsseriaceae
DNA samples from Neisseria gonorrhoea, Neisseria pharyngis and Neisseria flavescens were digested with restriction endonuclease Eco.RI, Southern blotted, and probed with pUS210. The results, in Figure 3, show that the strains of
Neisseria gonorrhoea give polymorphic banding patterns. In addition, the banding patterns obtained for Neisseria gonorrhoea and the other members of the
Neisseriaceae were distinct from those obtained with Neisseria meningitidis strains (see Figures 1 and 2) and from each other. The pUS210 probe can therefore be used to detect and to differentiate specifically strains of other members of the Neisseriaceae.
Claims (11)
1. Plasmid pUS210.
2. A nucleotide sequence which has greater than 70% homology to a sequence of 30 base-pairs contained in the repetitive element which is present in Neisseria meningitidis strain G3 and is contained partly or wholly in plasmid pUS210.
3. A nucleotide sequence which has greater than 70% homology to a sequence of 30 base-pairs contained in the neisserial DNA of plasmid pUS210.
4. A probe for detecting or identifying members of the Neisseriaceae which contains a nucleotide sequence as claimed in claim 2 or claim 3.
5. A diagnostic reagent for detecting members of the Neisseriaceae comprising or obtained by use of a nucleotide sequence as claimed in claim 2 or claim 3.
6. A method of identifying and differentiating strains of Neisserfs meningitidis which comprises the use of a probe as claimed in claim 4.
7. A method of identifying and differentiating strains of Neisseria gonorrhoea which comprises the use of a probe as claimed in claim 4.
8. A method of detecting members of the Neisseriaceae in clinical samples or microbiological cultures which comprises the use of a probe as claimed in claim 4.
9. A bacterium, virus or eukaryote cell which contains as the result of genetic modification a nucleotide sequence as claimed in claim 2 or claim 3.
10. Protein encoded by a nucleotide sequence as claimed in claim 2 or claim 3 and produced by a genetically modified organism as claimed in claim 8.
11. An immunological reagent comprising or obtained by use of a protein as claimed in claim 9.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9000572A GB2241242A (en) | 1990-01-10 | 1990-01-10 | DNA probe for Neisseria meningitidis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9000572A GB2241242A (en) | 1990-01-10 | 1990-01-10 | DNA probe for Neisseria meningitidis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| GB9000572D0 GB9000572D0 (en) | 1990-03-14 |
| GB2241242A true GB2241242A (en) | 1991-08-28 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| GB9000572A Withdrawn GB2241242A (en) | 1990-01-10 | 1990-01-10 | DNA probe for Neisseria meningitidis |
Country Status (1)
| Country | Link |
|---|---|
| GB (1) | GB2241242A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1993003186A1 (en) * | 1991-07-31 | 1993-02-18 | F.Hoffmann-La Roche Ag | Methods and reagents for detection of bacteria in cerebrospinal fluid |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0173339A2 (en) * | 1984-08-30 | 1986-03-05 | Enzo Biochem, Inc. | Composition and method for the dectection of the presence of a polynucleotide sequence of interest |
-
1990
- 1990-01-10 GB GB9000572A patent/GB2241242A/en not_active Withdrawn
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0173339A2 (en) * | 1984-08-30 | 1986-03-05 | Enzo Biochem, Inc. | Composition and method for the dectection of the presence of a polynucleotide sequence of interest |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1993003186A1 (en) * | 1991-07-31 | 1993-02-18 | F.Hoffmann-La Roche Ag | Methods and reagents for detection of bacteria in cerebrospinal fluid |
Also Published As
| Publication number | Publication date |
|---|---|
| GB9000572D0 (en) | 1990-03-14 |
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| WAP | Application withdrawn, taken to be withdrawn or refused ** after publication under section 16(1) |