Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-06-11
2004-09-28
Sitton, Jehanne (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S023100, C536S024300
Reexamination Certificate
active
06797468
ABSTRACT:
The invention provides oligonucleotides, method and kit for detecting
Listeria monocytogenes
by nucleic acid amplification and/or nucleic acid hybridization.
The genus Listeria consists of the six species
L. monocytogenes, L. grayi L. innocua, L. ivanovii, L. seligeri
and
L. welshimeri
. Among these, only strains of the species
L. monocytogenes
are pathogenic for humans, in particular for those with a weakened immune system and for the elderly and the newborn. The most common symptoms of listeriosis are septicemia, meningitis and miscarriages.
L. monocytogenes
infections are caused especially by consuming contaminated food, in particular milk products, meat, poultry and vegetables.
A large number of methods for detecting
L. monocytogenes
are known. Conventional detection methods for
L. monocytogenes
comprise preconcentrating and subsequently isolating colonies on selection media (Lovett et al., J. Food Protection 50 (1987), 188-192; McClain & Lee, J. Assoc. Off. Anal. Chem. 71 (1988), 660-664). Single colonies are examined for their morphology or for biochemical or serological properties. An analysis may take up to 6-8 days.
Since especially readily perishable food is frequently contaminated with
L. monocytogenes
, various high-speed methods for detecting
L. monocytogenes
have been developed. Such methods are based either on immunological methods or on the application of nucleic acid probes.
In this connection, detection may be carried out by direct hybridization of probes to microbe-specific DNA or RNA (see, for example, Datta, A. R. et al., Appl. Environ. Microbiol. 53 (1987), 2256-2259). The disadvantage of those methods is the low sensitivity, since at least 10
5
-10
6
copies of the target nucleic acid are required. This can be compensated by combination with an amplification of the target sequence, for example using the polymerase chain reaction (PCR). A plurality of PCR methods for detecting
L. monocytogenes
have been described in the literature [for a review see, for example, Jones, D. D. & Bej, A. K. in “PCR Technology, Current Innovations”, Griffin, H. G & Griffin, A. M., eds., (1994), 341-365]. See also U.S. Pat. Nos. 4,683,195; 4,683,202 and 4,965,188. Furthermore, the ligase chain reaction [WO publication 89/09835], “self-sustained sequence replication” [EP 329,822], “transcription based amplification system” [EP 310, 229] and Q&bgr; RNA replicase system [U.S. Pat. No. 4,957,858] may be employed for the amplification of nucleic acids.
Some test kits for detection by means of antibodies are already commercially available. Most of these tests, however, display only low sensitivity and specificity.
To detect specific microorganisms by means of nucleic acid hybridization or nucleic acid amplification, microbe-specific oligonucleotides are commonly used whose base sequence is characteristic for the DNA or RNA of a specific microorganism or of a group of microorganisms. When using said microbe-specific oligonucleotides (for example as primers or probes) in connection with the methods mentioned above, hybridization to the DNA/RNA or amplification of DNA/RNA can occur under suitable reaction conditions only if the DNA/RNA of the particular microorganisms to be detected is present.
The detection methods described for
L. monocytogenes
are based mainly on those target genes which play a role in the pathogenicity of
L. monocytogenes
. It is known that some of these genes are located on the chromosome next to each other in a virulence gene cluster. Since the listeriolysin gene (hlyA) has been recognized first as to be clearly necessary for the pathogenicity of
L. monocytogenes
(Cossart, P. et al., Infect. Immun. 57 (1989), 3629-3636), most of the genotypic detection methods are based on this gene. The hlyA gene, however, is also found with high homology in nonpathogenic listeria (i.e. in
L. seeligeri
and
L. ivanovii
). In said detection methods, the appearance of false-positive results cannot be completely dismissed, since single point mutations in the region of the binding sites of primers or probes may already be sufficient for this.
It was possible to show that the metalloprotease gene (mpl) which is located in the genome right next to the hlyA gene is only present in
L. monocytogenes
, and thus not in nonpathogenic listeria (Domann, E. et al., Infect. Immun. 59 (1991), 65-72).
The suitability in principle of the DNA region flanking the hlyA gene for detecting
L. monocytogenes
by means of hybridization or amplification has been described (Rossen, L. et al., Int. J. Food Microbiol. 14 (1991), 145-152); however, no oligonucleotide sequences for such detection methods have been published yet.
The sequence of the
L. monocytogenes
mpl gene is described in the EMBL database under accession number X54619 [Domann, E. et al., Infect. Immnun. 59 (1991), 65-72]. Furthermore, parts of the sequence of the
L. monocytogenes
mpl gene are listed in the EMBL database under accession number X60035 [Rasmussen, O.F. et al., Infect. Immun. 59 (1991), 3945-3951].
It was an object of the present invention to develop a detection method which is suitable for routine use and in which the probability of false-positive results appearing is as low as possible for the particular user, even under very variable experimental conditions.
In particular, oligonucleotide sequences are to be provided which can be employed in a detection method for the
L. monocytogenes
metalloprotease gene (mpl).
These objects are achieved by providing nucleic acid molecules of the sequences
(i) 5′-GAA AAA GCA TTT GAA GCC AT-3′ or
(ii) 5′-GCA ACT TCC GGC TCA GC-3′ or
(iii) 5′-TCG AAA AAG CAT TTG AAG CC-3′ or
(iv) 5′-GGT CAG AGT GAA GCT CAT GT-3′ or
(v) 5′-CTI TTC ACA TGA GCT TCA CTC TGA CCR A-3′ or
(vi) 5′-CTT TTT CTT TCA CTG GGT TTC CGA CAT-3 ′ or
(vii) 5′-GAT GAT TTC TTT TTC TTT CAC TGG ATT TCC AAT AT-3′ or
(viii) of the sequence complementary in each case to (i), (ii), (iii), (iv), (v), (vi), and (vii).
The oligonucleotides according to the invention may be defined as follows:
Oligonucleotide LM1: (sequence (i)=SEQ ID NO 1 corresponds to the position 2476 to 2495 of
L. monocytogenes
[according to Domann, E. et al. Infect. Immun. 59 (1991), 65-72).
Oligonucleotide LM 2: (sequence (ii)=SEQ ID NO 2) corresponds to the position 2608 to 2624 of
L. monocytogenes.
Oligonucleotide LM3: (sequence (iii)=SEQ ID NO 3) corresponds to the position 2474 to 2493 of
L. monocytogenes.
Oligonucleotide LM 4 (sequence (iv)=SEQ ID NO 4) corresponds to the position 2497 to 2516 of
L. monocytogenes.
Oligonucleotide LMR 1: (sequence (v)=SEQ ID NO 5) corresponds to the position 2495 to 2522 of
L. monocytogenes.
Oligonucleotide LMF 1: (sequence (vi)=SEQ ID NO 6) corresponds to the position 2525 to 2551 of
L. monocytogenes.
Oligonucleotide LMF 2 (sequence (vii) SEQ ID NO 7) corresponds to the position 2525 to 2559 of
L. monocytogenes.
In order to investigate the extent to which sequence variations of the mpl gene occur within the species
L. monocytogenes
, an internal fragment of 300 base pairs of 13
L. monocytogenes
strains of various serovars (2 strains of the serovars 1/2a, 1 strain of the serovar 1/2b, 1 strain of the serovar 1/2c, 1 strain of the serovar 3a, 1 strain of the serovar 3b, 1 strain of the serovar 3c, 1 strain of the serovar 4a, 1 strain of the serovar 4a/b, 1 strain of the serovar 4b, 1 strain of the serovar 4c, 1 strain of the serovar 4d, and 1 strain of the serovar 7) was sequenced. On the basis of sequence comparisons, it was surprisingly found that the oligonucleotides LM1, LM 2, LM3, LM 4, LMF 1, LMF 2, LMR 1 and also sequences complementary thereto lead to highly specific detections in detection methods for
L. monocytogenes
. The preferred probes in this connection are the oligonucleotides LM4, LMR1, LMF1 and LMF2 and the sequences complementary thereto.
The invention in particula
Berghof Kórnelia
Gasch Alexander
Scheu Pia
Biotecon Diagnostics GmbH
Frommer & Lawrence & Haug LLP
Santucci Ronald R.
Sitton Jehanne
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