CH682156A5 - Listeria monocytogenes detection by enzymatic nucleic acid amplification - using oligo-nucleotide(s) derived from alpha-haemolysin and/or beta-haemo-lysin virulence factors in polymerase chain reactions - Google Patents

Listeria monocytogenes detection by enzymatic nucleic acid amplification - using oligo-nucleotide(s) derived from alpha-haemolysin and/or beta-haemo-lysin virulence factors in polymerase chain reactions Download PDF

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Publication number
CH682156A5
CH682156A5 CH2190/90A CH219090A CH682156A5 CH 682156 A5 CH682156 A5 CH 682156A5 CH 2190/90 A CH2190/90 A CH 2190/90A CH 219090 A CH219090 A CH 219090A CH 682156 A5 CH682156 A5 CH 682156A5
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Switzerland
Prior art keywords
nucleic acid
sequence
haemolysin
beta
alpha
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CH2190/90A
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German (de)
Inventor
Urs Candrian
Beda Furrer
Christiane Hoefelein
Juerg Luethy
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Urs Candrian
Beda Furrer
Christiane Hoefelein
Juerg Luethy
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Application filed by Urs Candrian, Beda Furrer, Christiane Hoefelein, Juerg Luethy filed Critical Urs Candrian
Priority to CH2190/90A priority Critical patent/CH682156A5/en
Publication of CH682156A5 publication Critical patent/CH682156A5/en

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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

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Detection method for Listeria (L.) monocytogenes (mono.) comprises using the enzymatic nucleic acid amplification of hly (alpha-haemolysin) and/or iap (beta-haemolysin) virulence factors. Pref. oligonucleotides (A) L01=5'-CGGAGGTTCCGCAAAAGATG-3'; L02=5'-CATCGACGGCAACCTCGGA-3'; L03=5'-CCATCTGTATAAGCTTTTGAAG-3'; and L04=5'-CCTCCAGAGTGATCGATGTT-3' are used for hly and oligonucleotides (B) AD03=5'-ACAAGCTGCACCTGTTGCAG-3'; AD07=5'-TGACAGCGTGTGTAGTAGTCA-3'; AD08=5'-GGCGCAGGTGTAGTTGCTTG-3' and AD09=5'-CTACACAAGCAACTACACCT-3' are used for iap. USE/ADVANTAGE - L. mono can be detected in food samples by the oligonucleotides A and B. C.f. the classic bacteriological methods, the method is very fast (1-2 days) and sensitive thereby reducing any risk during the handling of L. mono. and avoiding possible genetic changes during the cultivation, e.g. the loss of virulence features.

Description

       

  
 



  Das neue Verfahren hat gegenüber der klassischen, bakteriologischen Methodik zum Nachweis und zur Identifikation von Listeria monocytogenes eine Reihe gewichtiger Vorteile. Die bakterioIogische Methodik ist derart zeitaufwendig, dass in der Regel (z.B. bei Nahrungsmittelproben) ein Eingreifen (z.B. durch die autorisierten Behörden) zu spät kommt. Das neue Verfahren ist im Gegensatz dazu sehr schnell und kann je nach Bedlngungen in ein bis zwei Tagen durchgeführt werden. Ausserdem zeichnet es sich durch eine ausgezeichnete Sensitivität aus. Im Gegensatz zur klassischen Methodik können ferner nicht nur lebende, sondern auch lebensfähige, aber nicht kultivierbare und je nach Bedingungen auch tote Listeria monocytogenes erfasst werden. Zudem fallen die üblichen Anreicherungsschritte weg.

  Dadurch werden die Gefahr, die mit der Handhabung grosser Mengen eines pathogenen Keimes wie Listeria monocytogenes verbunden ist, und die Möglichkeit genetischer Veränderungen während der Kultivierung (z.B. Verlust von Virulenzeigenschaften) eliminiert. 



  Im folgenden wird anhand des Beispiels einer Milchprobe das Verfahren offengelegt. Aus einem Aliquot der zu untersuchenden Milchprobe werden mittels Zentrifugation vorhandene Bakterien und ZelIen gesammelt. Anschliessend wird das resultierende Pellet mit einem geeigneten Puffer mehrmals gewaschen. Die  Bakterien werden daraufhin mittels bekannter Methoden aufgeschlossen und die Nukleinsäuren freigesetzt. Zum rohen Aufschluss werden alle Basiskomponenten, die zur enzymatischen Amplifikation mittels der bekannten Methodik der PoIymerase-Kettenreaktion notwendig sind, hinzugefügt. Ausserdem werden geeignete Oligonukleotide aus der Gruppe L01, L02, L03 und L04 (alpha-Hämolysin) und/oder AD03, AD07, AD08 und AD09 (beta Hämolysin) hinzugefügt. Anschliessend wird die Polymerase-Kettenreaktion durchgeführt.

   Dabei entstehen, falls in der Probe Listeria monocytogenes vorhanden waren, Nukleinsäure-Fragmente, deren Länge typisch für das hly- beziehungsweise iap-Gen sind und die spezifisch für Listeria monocytogenes sind. Die Analyse der Fragmente kann mittels Polyacrylamid- oder Agarose- Gelelektrophorese erfolgen. 



  
 



  The new method has a number of important advantages over the classic bacteriological method for the detection and identification of Listeria monocytogenes. The bacteriological method is so time-consuming that intervention (e.g. by the authorized authorities) is usually too late (e.g. in the case of food samples). In contrast, the new procedure is very fast and can be carried out in one or two days, depending on the conditions. In addition, it is characterized by an excellent sensitivity. In contrast to the classic methodology, not only living, but also viable, but not cultivable and, depending on the conditions, dead Listeria monocytogenes can also be recorded. In addition, the usual enrichment steps are omitted.

  This eliminates the risk associated with handling large amounts of a pathogenic germ such as Listeria monocytogenes and the possibility of genetic changes during cultivation (e.g. loss of virulence).



  In the following, the method is disclosed using the example of a milk sample. Bacteria and cells are collected from an aliquot of the milk sample to be examined by centrifugation. The resulting pellet is then washed several times with a suitable buffer. The bacteria are then digested using known methods and the nucleic acids released. All basic components necessary for the enzymatic amplification using the known method of the polymerase chain reaction are added for the crude digestion. In addition, suitable oligonucleotides from the group L01, L02, L03 and L04 (alpha-hemolysin) and / or AD03, AD07, AD08 and AD09 (beta-hemolysin) are added. The polymerase chain reaction is then carried out.

   If Listeria monocytogenes were present in the sample, this results in nucleic acid fragments whose length is typical of the hly or iap gene and which are specific for Listeria monocytogenes. The fragments can be analyzed using polyacrylamide or agarose gel electrophoresis.

Claims (5)

1. Verfahren zum Nachweis und zur Identifikation von Listeria monocytogenes mittels enzymatischer Nucleinsäuren-Amplifikation, dadurch gekennzeichnet, dass die enzymatische Nucleinsäuren-Amplifikation in Gegenwart von Nucleinsäure-Sequenzen der hly- und/oder iap-Virulenzfaktoren durchgeführt wird.       1. A method for the detection and identification of Listeria monocytogenes by means of enzymatic nucleic acid amplification, characterized in that the enzymatic nucleic acid amplification is carried out in the presence of nucleic acid sequences of the hly and / or iap virulence factors. 2. Verfahren nach Patentanspruch 1, dadurch gekennzeichnet, dass als Nucleinsäure-Sequenzen des hly-Virulenzfaktors die Oligonukleotide L01, L02, L03 und L04 verwendet werden, wobei L01 durch die Sequenz 5 min -CGGAGGTTCCGCAAAAGATG-3 min , L02 durch die Sequenz 5 min -CATCGACGGCAACCTCGGA-3 min , L03 durch die Sequenz 5 min -CCATCTGTATAAGCTTTTGAAG-3 min und L04 durch die Sequenz 5 min -CCTCCAGAGTGATCGATGTT-3 min definiert sind. 2. The method according to claim 1, characterized in that the oligonucleotides L01, L02, L03 and L04 are used as nucleic acid sequences of the hly virulence factor, L01 by the sequence 5 min -CGGAGGTTCCGCAAAAGATG-3 min, L02 by the sequence 5 min -CATCGACGGCAACCTCGGA-3 min, L03 by the sequence 5 min -CCATCTGTATAAGCTTTTGAAG-3 min and L04 by the sequence 5 min -CCTCCAGAGTGATCGATGTT-3 min are defined. 3. 3rd Verfahren nach Patentanspruch 1, dadurch gekennzeichnet, dass als Nucleinsäure-Sequenzen des iap-Virulenzfaktors die Oligonukleotide AD03, AD07, AD08 und AD09 verwendet werden, wobei AD03 durch die Sequenz 5 min -ACAAGCTGCACCTGTTGCAG-3 min , AD07 durch die Sequenz 5 min -TGACAGCGTGTGTAGTAGCA-3 min , AD08 durch die Sequenz 5 min -GGCGCAGGTGTAGTTGCTTG-3 min und AD09 durch die Sequenz 5 min -CTACACAAGCAACTACACCT-3 min definiert sind. Method according to claim 1, characterized in that the oligonucleotides AD03, AD07, AD08 and AD09 are used as nucleic acid sequences of the iap virulence factor, AD03 by the sequence 5 min -ACAAGCTGCACCTGTTGCAG-3 min, AD07 by the sequence 5 min -TGACAGCTTGTGT -3 min, AD08 are defined by the sequence 5 min -GGCGCAGGTGTAGTTGCTTG-3 min and AD09 by the sequence 5 min -CTACACAAGCAACTACACCT-3 min. 4. Oligonukleotide zur Durchführung des Verfahrens nach Patentanspruch 1, dadurch gekennzeichnet, dass sie die Sequenzen L01, L02, L03 und L04 des hly-ViruIenzfaktors aufweisen, wobei L01 durch die Sequenz 5 min -CGGAGGTTCCGCAAAAGATG-3 min , L02 durch dle Sequenz 5 min -CATCGACGGCAACCTCGGA-3 min , L03 durch die Sequenz 5 min -CCATCTGTATAAGCTTTTGAAG-3 min und L04 durch die Sequenz 5 min -CCTCCAGAGTGATCGATGTT-3 min definiert sind. 4. oligonucleotides for performing the method according to claim 1, characterized in that they have the sequences L01, L02, L03 and L04 of the hly virulence factor, L01 by the sequence 5 min -CGGAGGTTCCGCAAAAGATG-3 min, L02 by the sequence 5 min -CATCGACGGCAACCTCGGA-3 min, L03 by the sequence 5 min -CCATCTGTATAAGCTTTTGAAG-3 min and L04 by the sequence 5 min -CCTCCAGAGTGATCGATGTT-3 min are defined. 5. 5. Oligonukleotide zur Durchführung des Verfahrens nach Patentanspruch 1, dadurch gekennzeichnet, dass sie die Sequenzen AD03, AD07, AD08 und AD09 des iap-Virulenzfaktors aufweisen, wobei AD03 durch die Sequenz 5 min -ACAAGCTGCACCTGTTGCAG-3 min , AD07 durch die Sequenz 5 min -TGACAGCGTGTGTAGTAGCA-3 min , AD08 durch die Sequenz 5 min -GGCGCAGGTGTAGTTGCTTG-3 min und AD09 durch die Sequenz 5 min -CTACACAAGCAACTACACCT-3 min definiert sind.  Oligonucleotides for performing the method according to claim 1, characterized in that they have the sequences AD03, AD07, AD08 and AD09 of the iap virulence factor, AD03 by the sequence 5 min -ACAAGCTGCACCTGTTGCAG-3 min, AD07 by the sequence 5 min -TGACAGCGTGTGTAGT -3 min, AD08 are defined by the sequence 5 min -GGCGCAGGTGTAGTTGCTTG-3 min and AD09 by the sequence 5 min -CTACACAAGCAACTACACCT-3 min.  
CH2190/90A 1990-06-28 1990-06-28 Listeria monocytogenes detection by enzymatic nucleic acid amplification - using oligo-nucleotide(s) derived from alpha-haemolysin and/or beta-haemo-lysin virulence factors in polymerase chain reactions CH682156A5 (en)

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CH2190/90A CH682156A5 (en) 1990-06-28 1990-06-28 Listeria monocytogenes detection by enzymatic nucleic acid amplification - using oligo-nucleotide(s) derived from alpha-haemolysin and/or beta-haemo-lysin virulence factors in polymerase chain reactions

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CH2190/90A CH682156A5 (en) 1990-06-28 1990-06-28 Listeria monocytogenes detection by enzymatic nucleic acid amplification - using oligo-nucleotide(s) derived from alpha-haemolysin and/or beta-haemo-lysin virulence factors in polymerase chain reactions

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0805214A2 (en) * 1996-04-30 1997-11-05 Universita' Degli Studi di Udine Method and relative primers to identify listeria monocytogenes in an organic substrate
WO1998020160A1 (en) * 1996-11-08 1998-05-14 E.I. Du Pont De Nemours And Company GENETIC MARKERS AND METHODS FOR THE DETECTION OF LISTERIA MONOCYTOGENES AND $i(LISTERIA SPP)
WO2001068900A2 (en) * 2000-03-15 2001-09-20 Vermicon Ag Method for specifically detecting microorganisms by polymerase chain reaction

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0805214A2 (en) * 1996-04-30 1997-11-05 Universita' Degli Studi di Udine Method and relative primers to identify listeria monocytogenes in an organic substrate
EP0805214A3 (en) * 1996-04-30 1998-04-22 Universita' Degli Studi di Udine Method and relative primers to identify listeria monocytogenes in an organic substrate
WO1998020160A1 (en) * 1996-11-08 1998-05-14 E.I. Du Pont De Nemours And Company GENETIC MARKERS AND METHODS FOR THE DETECTION OF LISTERIA MONOCYTOGENES AND $i(LISTERIA SPP)
WO2001068900A2 (en) * 2000-03-15 2001-09-20 Vermicon Ag Method for specifically detecting microorganisms by polymerase chain reaction
DE10012540A1 (en) * 2000-03-15 2001-09-27 Vermicon Ag Specifically detecting microorganisms in a sample, by polymerase chain reaction with reaction and competitor primers, useful for detecting subspecies of Listeria, in particular Listeria monocytogenes
WO2001068900A3 (en) * 2000-03-15 2002-08-22 Vermicon Ag Method for specifically detecting microorganisms by polymerase chain reaction
DE10012540B4 (en) * 2000-03-15 2004-09-23 Vermicon Ag Oligonucleotides and methods for the specific detection of microorganisms by polymerase chain reaction

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