WO2017182775A1 - Détection de microorganisme impliquant une filtration - Google Patents

Détection de microorganisme impliquant une filtration Download PDF

Info

Publication number
WO2017182775A1
WO2017182775A1 PCT/GB2017/051027 GB2017051027W WO2017182775A1 WO 2017182775 A1 WO2017182775 A1 WO 2017182775A1 GB 2017051027 W GB2017051027 W GB 2017051027W WO 2017182775 A1 WO2017182775 A1 WO 2017182775A1
Authority
WO
WIPO (PCT)
Prior art keywords
filter
microorganisms
nucleic acid
sample
μηι
Prior art date
Application number
PCT/GB2017/051027
Other languages
English (en)
Inventor
Daniel WRATTING
Conor MCGRATH
Matthew Crow
William Mullen
Helen BENNETT
Kevin Wood
Daniel HAMMETT
Original Assignee
Momentum Bioscience Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Momentum Bioscience Limited filed Critical Momentum Bioscience Limited
Publication of WO2017182775A1 publication Critical patent/WO2017182775A1/fr

Links

Classifications

    • 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
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • microorganism source of enzymatic activity The methods of the invention therefore enable determination of the absence and presence of microbial pathogens in samples such as un- purified blood, blood culture and other body fluids.
  • This invention also relates to devices, compositions of matter and kits comprising such reagents useful for carrying out the methods.
  • Measuring the presence and levels of certain molecules which are associated with cell viability is important in a number of contexts. For example, measuring levels of ATP is useful in mammalian cells for growth analysis and toxicology purposes. Culture approaches can be used to detect small numbers of bacteria but such techniques require several days to complete, especially when attempting to detect small numbers of bacteria and also when detecting slower growing microorganisms.
  • WO96/002665 describes a method for determining the presence and/or amount of microorganisms and/or their intracellular material present in a sample characterized in that the amount of adenylate kinase in the sample is estimated by mixing it with adenosine diphosphate (ADP), determining the amount of adenosine triphosphate (ATP) produced by the sample from this ADP, and relating the amount of ATP so produced to the presence/or amount of adenylate kinase and to microorganisms and/or their intracellular material, wherein the conversion of ADP to ATP is carried out in the presence of magnesium ions at a molar concentration sufficient to allow maximal conversion of ADP to ATP.
  • ADP adenosine diphosphate
  • ATP adenosine triphosphate
  • NAD-dependent ligases are described as a useful indicator of the presence of a microorganism in a sample.
  • Ligases are enzymes which catalyze ligation of nucleic acid molecules. The ligation reaction requires either ATP or NAD+ as co-factor depending upon the ligase concerned.
  • WO2011/130584 describes a method for detection of viable microorganisms based on detection of DNA or RNA polymerases in which a sample is contacted with a nucleic acid substrate that acts as a substrate for microbial polymerase, incubated under conditions suitable for polymerase activity from intact microorganisms and any resulting nucleic acid product is determined using a nucleic acid amplification technique such as quantitative polymerase chain reaction.
  • Such assays have been termed "ETGA assays", where ETGA stands for Enzymatic Template Generation and Amplification.
  • ETGA assays A problem with ETGA assays for viable microorganisms in crude samples is the presence of contaminating polymerase activity outside the microorganisms arising from host (e.g. human) cells and dead microorganisms. The ETGA assay is unable to distinguish microorganism
  • WO2010/119270 describes a method for removing DNA ligase activity outside intact microorganisms.
  • WO2011/070507 describes the selective lysis of animal cells using a non-ionic detergent and a buffer.
  • step b filtering the filtrate from step b through a second filter that retains
  • the first filter thus functions as a coarse filter to prevent large insoluble matter from passing, and thus impacting on the subsequent microorganism enzymatic activity detection methods.
  • the first filter prevents blockage of the second filter.
  • the initial filtration thus generates a filtrate.
  • This filtrate is then further filtered through a second filter.
  • the second filter has the functional requirement of retaining any
  • microorganisms contained in the filtrate within or upon the filter This permits
  • the filter/filtration body may be of a thickness sufficient to permit separation of the insoluble cellular material from the captured microorganisms.
  • a single filter/filtration body comprising a pore size on the inlet side of the filter/filtration body that prevents passage of insoluble cellular material and a pore size on the outlet side of the filter/filtration body that retains microorganisms within the filter/filtration body; or ii. a filter/filtration body comprising a laminate structure comprising a first layer that prevents passage of insoluble cellular material and a second layer that retains microorganisms within the filter/filtration body
  • a method of detecting the absence or presence of a (viable or recently viable) microorganism in a sample that may also contain non- microorganism cells comprises:
  • a single filter/filtration body comprising a pore size on the inlet side of the filter/filtration body that prevents passage of insoluble cellular material and a pore size on the outlet side of the filter/filtration body that retains microorganisms within the filter/filtration body; or ii. a filter/filtration body comprising a laminate structure comprising a first layer that prevents passage of insoluble cellular material and a second layer that retains microorganisms within the filter/filtration body
  • a method of detecting the absence or presence of a (viable or recently viable) microorganism in a sample that may also contain non-microorganism cells comprises:
  • a single filter/filtration body comprising a pore size on the inlet side of the filter/filtration body that prevents passage of insoluble cellular material and a pore size on the outlet side of the filter/filtration body that retains microorganisms within the filter/filtration body; or ii. a filter/filtration body comprising a laminate structure comprising a first layer that prevents passage of insoluble cellular material and a second layer that retains microorganisms within the filter/filtration body
  • step c lysis of the microorganisms retained within the filter/filtration body
  • step d incubating the lysate from step c with a nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the microorganisms; and e. specifically determining the absence or presence of a modified nucleic acid molecule resulting from the action of the nucleic acid modifying enzyme on the substrate nucleic acid molecule to indicate the absence or presence of the microorganism.
  • the filter/filtration body is a membrane.
  • Suitable membrane materials are discussed herein and include polysulfone membranes, such as polyethersulfone membranes (e.g. available from Merck Millipore).
  • Other membrane materials that may be utilised include polyvinylidene fluoride (PVDF) membranes (e.g. available from Merck Millipore) and glass fibre filters, optionally with a binder (e.g. available from Merck Millipore).
  • PVDF polyvinylidene fluoride
  • a binder e.g. available from Merck Millipore
  • Each may be provided in asymmetric form or as a laminate structure as discussed herein. Each may incorporate pores of the specific sizes as described in further detail herein.
  • Pore sizes are typically presented in terms of (maximum) "diameter”. However, the sizes herein generally refer to the maximum dimension of the pore to take account of the fact that different filters may contain non-circular pores. This dimension may represent the intended average of all pores in the filter, based on the usual manufacturing tolerances and the use of non-uniform materials, as would be readily appreciated by one skilled in the art.
  • Suitable pore sizes to prevent passage of insoluble cellular material may be, or may be on average, at least 1 ⁇ . They may be, or may be on average, between 1 ⁇ and 5 ⁇ , such as between 1 ⁇ and 2 ⁇ .
  • step b comprises use of a filter with a (minimum) pore size (on the inlet side) of at least 1 ⁇ .
  • the (minimum) pore size (on the inlet side) is between 1 ⁇ and 5 ⁇ , such as between 1 ⁇ and 2 ⁇ .
  • Suitable pore sizes to achieve retention of microorganisms on or within the filter may be, or may be on average no more than 0.5 ⁇ , no more than 0.45 ⁇ , no more than 0.25 ⁇ , no more than 0.22 ⁇ or no more than 0.2 ⁇ .
  • step c comprises use of a filter with a (maximum) pore size (on the outlet side) of no more than 0.5 ⁇ , no more than 0.45 ⁇ , no more than 0.25 ⁇ , no more than 0.22 ⁇ or no more than 0.2 ⁇ .
  • a particularly suitable membrane type tested herein is an asymmetric polysulfone membrane with a pore size of around 0.22 ⁇ (on the outlet side).
  • the duration of exposure to the high pH conditions is typically less than 20 minutes and may be not more than 10, 9, 8, 7, 6 or 5 minutes and may be around 5, 6, 7, 8, 9 or 10 minutes. In some embodiments the treatment is carried out for between around 2 and 15 minutes, such as around 5 minutes. By “around” is meant plus or minus 30 seconds.
  • any suitable reagent may be added to the second filter (containing the captured microorganisms) in order to provide high pH conditions.
  • the high pH conditions comprise contacting the sample with an alkali or a buffer.
  • NaOH or Na2C03 is used.
  • the concentration of the NaOH or Na2C03 is around 5mM or greater.
  • the buffer may have a pKa value above 9. Examples of suitable buffers include borate, carbonate and pyrophosphate buffers.
  • High pH is generally a pH of at least around 10, such as around 10, 11 , 12, 13 or 14.
  • Low pH is generally a pH of less than or equal to around 4, such as around 4, 3, 2, or 1.
  • around is meant 0.5 of a pH unit either side of the stated value. Altering the pH of the sample may be achieved using any suitable means, as would be readily appreciated by one skilled in the art. Microbial enzymes such as polymerases and ligases may be resistant to extremes of pH, whereas corresponding mammalian enzymes may be inactivated under the same pH conditions.
  • lysis of the microorganisms may be performed using a lysis reagent containing the substrate nucleic acid molecule.
  • a volume of lysis reagent is typically applied that will saturate the filter.
  • the volume applied to the filter is no more than 1.1 , 1.2, 1.3, 1.4 or 1.5 times the capacity of the filter. This helps to prevent enzymes released from the lysed microorganisms being washed out of the filter (to a significant degree).
  • the additional volume i.e. moderately greater than capacity
  • the additional volume may be beneficial to ensure previous solution contained within the filter is forced out. The additional volume may thus ensure saturation with a substantially undiluted lysis reagent.
  • any active process for applying the lysis reagent to the filter is stopped or otherwise controlled so as to prevent enzymes released from the lysed microorganisms being forced out of the filter (to a significant degree).
  • steps are advantageous because it is generally preferred that the incubation with the substrate nucleic acid molecule is performed in the filter.
  • the methods may involve pumping 250 ⁇ of lysis reagent onto the filter. The methods of the invention may thus incorporate the following steps:
  • PTO-AS oligo an antisense oligonucleotide comprising phosphorothioate nucleotides.
  • PTO-S1 oligo a sense oligonucleotide comprising
  • Detection of the amplification products may be by routine methods, such as, for example, gel electrophoresis but in some embodiments is carried out using real-time or end-point detection methods.
  • the method involves a step of lysis of the microorganisms retained within or upon the filter following filtration through the second filter in which the lysis reagent contains a nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the microorganisms.
  • step c incubating the lysate from step c with a nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the microorganisms; and e. specifically determining the absence or presence of a modified nucleic acid molecule resulting from the action of the nucleic acid modifying enzyme on the substrate nucleic acid molecule to indicate the absence or presence of the microorganism;
  • the microorganism that may be detected in the sample is a pathogenic microorganism, such as a pathogenic bacterium or fungus/yeast.
  • the bacterium may be any bacterium which is capable of causing infection or disease in a subject, preferably a human subject.
  • the bacteria comprises or consists essentially of or consists of any one or more of
  • the detection means comprises, or is, a nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the microorganisms.
  • BacT/ALERT blood broth (Sheep Blood in Alsever's; TCS Biosciences Cat# SB069 Lot# 30523900) was inoculated to approximately 1 x 10 7 cfu/mL for E. coli and S. aureus; and approximately 1 x 10 5 cfu/mL for C. albicans.
  • TVC plates were prepared for inoculated blood broth samples (Columbia Agar Base for E. coli and S. aureus; and Sabouraud Dextrose Agar for C. albicans) and 'No Spike Controls' (NSCs) (Columbia Agar Base). 1 mL blood broth was added to the mixing chamber of each SCP.
  • the ⁇ .22 ⁇ PES' filter was shortlisted because it had the best filtration performance of all filter configurations tested. '2 ⁇ GF + 0.22 ⁇ PVDF' was selected, despite performing similarly to ⁇ ⁇ GF + 0.22 ⁇ PVDF', because previous testing with a blood broth to lysis buffer ratio of 1 :4 showed that '2 ⁇ GF + 0.22 ⁇ PVDF' provided slightly lower filtration pressures (data not shown).
  • the '5 ⁇ PVDF + 0.22 ⁇ PES' dual filter configuration was shortlisted, despite performing worse than certain GF pre-filter configurations, because it was deemed important to compare both single and dual filter configurations in the context of the full Cognitor Minus HT test (see section 4.3).
  • Clinically-relevant microorganism species can be detected at approximately 10 3 cfu/mL.

Abstract

L'invention concerne un procédé de détection de l'absence ou de la présence d'un microorganisme effectué dans un échantillon qui peut également contenir des cellules ne provenant pas de microorganismes. Le procédé comprend la lyse sélective de cellules ne provenant pas de microorganismes présentes dans l'échantillon, de quelconques microorganismes présents dans l'échantillon étant en même temps laissés intacts. Le lysat est filtré sur un premier filtre qui empêche le passage de matière cellulaire insoluble à travers le filtre. Le filtrat est ensuite filtré sur un second filtre qui retient les microorganismes à l'intérieur du filtre ou sur le filtre. L'absence ou la présence de microorganismes retenus à l'intérieur du filtre ou sur le filtre peut ensuite être détectée. Les premier et second filtres sont avantageusement disposés dans une seule structure de filtration qui peut comprendre des pores asymétriques ou qui peut être une structure stratifiée. Des pores de plus grande taille du côté entrée empêchent le passage de matière insoluble (provenant de la lyse de cellules ne provenant pas de microorganismes) mais laissent intacts les microorganismes pour qu'ils passent du côté sortie du filtre. Le côté sortie du filtre a des plus petits pores permettant ainsi de piéger les microorganismes. L'invention concerne également des kits et produits associés.
PCT/GB2017/051027 2016-04-18 2017-04-12 Détection de microorganisme impliquant une filtration WO2017182775A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB201606750 2016-04-18
GB1606750.6 2016-04-18

Publications (1)

Publication Number Publication Date
WO2017182775A1 true WO2017182775A1 (fr) 2017-10-26

Family

ID=58579211

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2017/051027 WO2017182775A1 (fr) 2016-04-18 2017-04-12 Détection de microorganisme impliquant une filtration

Country Status (1)

Country Link
WO (1) WO2017182775A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021069903A1 (fr) 2019-10-08 2021-04-15 Momentum Bioscience Limited Capture de micro-organisme à partir d'une solution contenant un agent antimicrobien

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009007719A2 (fr) * 2007-07-09 2009-01-15 Iseao Technologies Limited Détection améliorée de micro-organismes
WO2010091080A2 (fr) * 2009-02-03 2010-08-12 Network Biosystems, Inc. Purification d'acide nucléique
WO2011070507A1 (fr) * 2009-12-08 2011-06-16 Koninklijke Philips Electronics N.V. Lyse sélective de cellules
US20120156716A1 (en) * 2010-12-17 2012-06-21 Biomerieux, Inc. Methods For The Isolation, Accumulation, Characterization And/Or Identification Of Microorganisms Using A Filtration And Sample Transfer Device
WO2013023198A2 (fr) * 2011-08-11 2013-02-14 Zybac, Llc Détection rapide et sensible de bactéries dans des produits sanguins, dans l'urine, et dans d'autres fluides
US20130309700A1 (en) * 2012-05-21 2013-11-21 Celsis International Limited Methods, devices, and systems of detecting microorganisms
WO2014193481A1 (fr) * 2013-05-31 2014-12-04 Rajesh Krishnamurthy Détection rapide de microbes
WO2016005768A1 (fr) * 2014-07-10 2016-01-14 Momentum Bioscience Limited Procédé et kit de détection de l'absence de micro-organismes

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009007719A2 (fr) * 2007-07-09 2009-01-15 Iseao Technologies Limited Détection améliorée de micro-organismes
WO2010091080A2 (fr) * 2009-02-03 2010-08-12 Network Biosystems, Inc. Purification d'acide nucléique
WO2011070507A1 (fr) * 2009-12-08 2011-06-16 Koninklijke Philips Electronics N.V. Lyse sélective de cellules
US20120156716A1 (en) * 2010-12-17 2012-06-21 Biomerieux, Inc. Methods For The Isolation, Accumulation, Characterization And/Or Identification Of Microorganisms Using A Filtration And Sample Transfer Device
WO2013023198A2 (fr) * 2011-08-11 2013-02-14 Zybac, Llc Détection rapide et sensible de bactéries dans des produits sanguins, dans l'urine, et dans d'autres fluides
US20130309700A1 (en) * 2012-05-21 2013-11-21 Celsis International Limited Methods, devices, and systems of detecting microorganisms
WO2014193481A1 (fr) * 2013-05-31 2014-12-04 Rajesh Krishnamurthy Détection rapide de microbes
WO2016005768A1 (fr) * 2014-07-10 2016-01-14 Momentum Bioscience Limited Procédé et kit de détection de l'absence de micro-organismes

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021069903A1 (fr) 2019-10-08 2021-04-15 Momentum Bioscience Limited Capture de micro-organisme à partir d'une solution contenant un agent antimicrobien
EP4283300A2 (fr) 2019-10-08 2023-11-29 Momentum Bioscience Limited Capture de micro-organisme à partir d'une solution contenant un agent antimicrobien

Similar Documents

Publication Publication Date Title
Hedman et al. Overcoming inhibition in real-time diagnostic PCR
KR100746372B1 (ko) 핵산 분자가 포함되는 직접 효소 반응 방법
AU2014293096B2 (en) Method and device for collection and amplification of circulating nucleic acids
EP3495507B1 (fr) Amplification et detection directes d'agents pathogènes viraux et bactériens
JP7476466B2 (ja) 微生物の分離及び検出
US11746389B2 (en) Method and kit of detecting the absence of micro-organisms
AU2007296460B2 (en) Removal of molecular assay interferences for nucleic acids employing buffered solutions of chaotropes
WO2011053871A2 (fr) Essai multi-amorces pour détection de mycoplasme
KR102648647B1 (ko) 짧은 호모폴리머릭 반복서열의 개선된 검출법
JP2016192967A (ja) 血液試料の微生物特異的フィルタ・インサイチュ分析の方法
WO2009016652A1 (fr) Système de tampon et procédé pour une amplification par réaction en chaîne par polymérase directe
JP6728608B2 (ja) 核酸分析用のサンプル調製方法
JP2014502510A5 (fr)
US20200377928A1 (en) Method of detecting minor bcr-abl1 gene
WO2017182775A1 (fr) Détection de microorganisme impliquant une filtration
US20210102245A1 (en) Direct nucleic acid analysis of environmental and biological samples
EP4283300A2 (fr) Capture de micro-organisme à partir d'une solution contenant un agent antimicrobien
EP3438280B1 (fr) Procédé de détection d'hémoplasme
Hosbrough A review of PCR inhibition and its mitigation in forensic DNA analysis
JP4187057B2 (ja) 核酸合成法
US9738939B2 (en) Method for differentiating between living and dead cells
JP2007151470A (ja) 試料を用いた核酸増幅方法
WO2016005767A1 (fr) Détection de micro-organismes viables
US20150337377A1 (en) Method of diagnosis of complement-mediated thrombotic microangiopathies
JP2005323617A (ja) 核酸合成法

Legal Events

Date Code Title Description
NENP Non-entry into the national phase

Ref country code: DE

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17718589

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 17718589

Country of ref document: EP

Kind code of ref document: A1