WO2015100123A1 - Détection et quantification d'acides nucléiques pour évaluer la biomasse microbienne dans des défauts de papier et feutres pour machine à papier - Google Patents

Détection et quantification d'acides nucléiques pour évaluer la biomasse microbienne dans des défauts de papier et feutres pour machine à papier Download PDF

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WO2015100123A1
WO2015100123A1 PCT/US2014/071128 US2014071128W WO2015100123A1 WO 2015100123 A1 WO2015100123 A1 WO 2015100123A1 US 2014071128 W US2014071128 W US 2014071128W WO 2015100123 A1 WO2015100123 A1 WO 2015100123A1
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Prior art keywords
organisms
item
defect
dna
organism
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PCT/US2014/071128
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English (en)
Inventor
Laura E. Rice
Liliya LUND
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Nalco Company
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Priority claimed from US14/138,526 external-priority patent/US9290802B2/en
Application filed by Nalco Company filed Critical Nalco Company
Publication of WO2015100123A1 publication Critical patent/WO2015100123A1/fr

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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
    • C12Q1/6893—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for protozoa
    • 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
    • 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
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates generally to compositions of matter, apparatuses and methods useful in detecting and identifying microorganisms causing or present in machine felts and on paper defects.
  • paper is produced in a continuous manner from a fibrous suspension (pulp furnish) generally made of water and cellulose fibers.
  • pulp furnish generally made of water and cellulose fibers.
  • a typical paper manufacturing process consists of 3 stages: forming, pressing, and drying.
  • dilute pulp furnish is directed on a wire or between 2 wires. The majority of the water is drained from the pulp furnish, through the wire, creating a wet paper web.
  • the pressing stage the paper web comes in contact with one or generally more porous Machine Felts that are used to extract much of the remaining water from the web.
  • the pickup felt is the first felt that the wet paper web contacts which is used to remove the paper web from the wire, via a suction pickup roll positioned behind the felt, and then to transport the paper web to the rest of the press section.
  • the paper web then generally passes through one or more presses each consisting of rotating press rolls and/or stationary elements such as press shoes that are positioned in close proximity to each other forming, what is commonly referred to as, a press nip.
  • a press nip In each nip the paper web comes in contact with either one or two Machine Felts where water is forced from the paper web and into the press felt via pressure and/or vacuum.
  • single- felted press nips the paper web is in contact with the press roll on one side and the felt on the other.
  • double-felted press nips the paper web passes between the two felts. After the press section, the paper web is dried to remove the remaining water, usually by weaving through a series of steam heated dryer cans.
  • Machine felts often consist of wool or nylon base fabric generally made of from 1 to 4 individual layers of filaments arranged in a weave pattern. An extruded polymeric membrane or mesh can also be included as one or more of the base fabric layers. Batt fibers, of smaller diameter than the base fabric filaments, are needled into the base on both sides giving the felt a thick, blanket-like appearance.
  • Machine Felts are designed to quickly take in water from the paper web in the nip and hold the water so that it does not re-absorb back into the sheet as the paper and felt exit the press nip. Machine Felts are normally a belt passing through an endless loop that circulates continuously between sheet contact stages and return stages. Water pulled into the felt from the paper web at the nip is generally removed from the felt by vacuum during the felt return stage at, what is frequently referred to as, the uhle box.
  • Papermaking systems utilize several raw materials that introduce
  • microorganisms into the machine system. This includes virgin wood fiber, recycled fiber, freshwater, starch, dyes, and other chemical additives. Microorganisms proliferate in many or all of the warm, nutrient rich environments present within papermaking systems and diverse microbial communities result. Inadequate control of microbial growth allows for the formation of surface deposits that slough, leading to filter or nozzle plugging and defects (e.g. spots or holes) or breaks in the sheet. Microorganisms can also proliferate in the felts and machine fabrics, negatively impacting water removal and machine or operational efficiency.
  • Microbial growth in papermaking systems can be quite harmful and costly.
  • the growth of microorganisms on equipment surfaces can lead to the formation of deposits that slough and contribute to sheet defects and holes.
  • Contaminated shower water treatments or process water can lead to the growth of microbes on felts which commonly result in the formation of plugs on the felts.
  • These plugs in turn cause a number of problems most notably the impairment of water removal from paper web.
  • microbial growth can result in an excessive and costly need for multiple boil-outs and cleanings of felts or other papermaking equipment.
  • microorganisms occurs because this can result in a treatment which further degrades the quality of the paper, further impacts process equipment, and/or may not even control the underlying microbial infestation. Moreover incorrectly distinguishing between biologically caused problems and mechanical or chemical caused problems can further result in inadequate, wasteful, and possibly counter-productive efforts.
  • microorganisms are present in a papermaking system. These methods however are particularly deficient when applied to paper sheets or felts. Some of the prior art methods such as US Patents 8,012,758, 7,981,679, and 7,949,432 detect various effects in the fluids of the papermaking system produced by living microbiological organisms. Other methods such as US 5,281,537 rely on obtaining a sample of living microorganism contaminant and growing more of it so as to perform various analyses. In the context of paper sheets and felts however these methods are particularly inadequate as by the time samples of the felt or paper are taken they no longer contain sufficient (or any) live organisms to culture or any of the chemical products that they produce.
  • spectroscopy often produce false positives or negatives because they detect materials that may have non-biological origins (such as chemical additives or contamination).
  • At least one embodiment of the invention is directed towards a method of identifying a microorganism infestation in a papermaking process.
  • the method comprises the steps of: 1) noting a defect on an item associated with a papermaking process, 2) conducting at least one DNA Analysis on at least one sample taken from the item, the DNA Analysis may include but is not limited to qPCR, PCR, digital PCR, ion semiconductor sequencing, pyrosequencing, sequencing by synthesis, sequencing by litigation, chain terminating sequencing, and any combination thereof.
  • the item may be a felt.
  • the defect may be one or more plugs in the felt.
  • the item may be a paper sheet produced by the papermaking process and the defect may be one or more holes, discoloration, streaks, spots, translucent spots, and any combination thereof on the paper sheet.
  • the method may further comprise the step of recording the identified organism into a format which can be stored and/or transmitted.
  • the method may further comprise the step of conducting a biocidal program associated with remedying the identified organism.
  • the DNA analysis may be a qPCR analysis.
  • the threshold of the DNA analysis may be 10 4 cells per ml or 10 4 cells per gram.
  • the item may be so desiccated that there are no living organisms on the item that may have caused the defect.
  • the conditions of the item may differ so much from the fluids the item encounters during the papermaking process that the organisms which inhabit the items differ from those in the fluids and determining the inhabitants of the fluids will produce an incorrect identification of the organisms on the item causing the defect.
  • the method may further comprise the step of applying sufficient kinds of primers to samples of the item such that the presence of any organisms above the threshold can be determined.
  • the method may further comprise the step of identifying the defect as being non-biologically based if the DNA analysis does not indicate that any organisms exceed the threshold.
  • the method may further comprise the step of applying a remedy for non-biological chemical contamination to the papermaking process.
  • the DNA analysis may determine the quantity of organisms infesting the sample.
  • the item may have passed through a heat or dryer section of the papermaking process before the defect is noted and therefore the organisms which caused the defect may have been killed.
  • FIG. 1 contains three graphs illustrating the results of samples the invention was applied to.
  • FIG. 2 illustrates a graph of the total bacterial load of samples the invention was applied to.
  • FIG. 3 is a graph of the total bacterial load of samples the invention was applied to.
  • FIG. 4 illustrates pie charts denoting microbial diversity varied in DNA samples collected from machine felts from two different paper mills.
  • DNA Based Analysis means an method of analyzing DNA, including but not limited to qPCR, PCR, digital PCR, ion semiconductor sequencing, pyrosequencing, sequencing by synthesis, sequencing by litigation, chain terminating sequencing, and any combination thereof.
  • Ion Torrent Semiconductor Sequencing means a method of DNA sequencing based on the detection of hydrogen ions that are released during the polymerization of DNA. This is a method of "sequencing by synthesis", during which a complementary strand is built based on the sequence of a template strand, a representative example of this is described at the website:
  • Polyrosequencing means a method of DNA sequencing (determining the order of nucleotides in DNA) based on the “sequencing bysynthesis” principle. It differs from Sanger sequencing, in that it relies on the detection of pyrophosphate release on nucleotide incorporation, rather than chain termination with
  • the desired DNA sequence is able to be determined by light emitted upon incorporation of the next complementary nucleotide by the fact that only one out of four of the possible A/T/C/G nucleotides are added and available at a time so that only one letter can be incorporated on the single stranded template (which is the sequence to be determined).
  • a representative example can be found in the Article Pyrosequencing Sheds Light on DNA Sequencing, by Mostafa Ronaghi, Genome Research, 11:3-11 (2001) which can be found at
  • Sequence by Synthesis means a technique used to determine the series of base pairs in DNA, also known as DNA sequencing. This sequencing method is based on reversible dye-terminators that enable the identification of single bases as they are introduced into DNA strands. Representative examples can be found at the websites: http ://nxseq .bitesizebio .com/articles/sequencing-b y- synthesis-explaining- the-illumina- sequencin g-technolo g y/ and
  • Chromatation sequencing means a method which requires a single- stranded DNA template, a DNA primer, aDNA polymerase, normal
  • deoxynucleosidetriphosphates dNTPs
  • dideoxyNTPs modified nucleotides
  • the ddNTPs may be radioactively or fluorescently labelled for detection in automated sequencing machines.
  • Chain termination sequencing may involve the synthesis of new strands of DNA complementary to a single-stranded template (step I).
  • the template DNA is supplied with a mixture of all four deoxynucleotides, four dideoxynucleotides— each labeled with a different color fluorescent tag, and
  • DNA polymerase (step II). As all four deoxynucleotides are present, chain elongation proceeds until, by chance, DNA polymerase inserts a dideoxynucleotide. The result is a new set of DNA chains all of different lengths (step III). The fragments are then separated by size using gel electrophoresis (step IV). As each labeled DNA fragment passes a detector at the bottom of the gel, the color is recorded. The DNA sequence is then reconstructed from the pattern of colors representing each nucleotide sequence (step V).
  • Digital polymerase chain reaction or (digital PCR, DigitalPCR, dPCR, or dePCR) means a refinement of conventional polymerase chain reaction methods that can be used to directly quantify and clonally amplify nucleic acids including DNA, cDNA or RNA.
  • the key difference between dPCR and traditional PCR lies in the method of measuring nucleic acids amounts, with the former being a more precise method than PCR.
  • PCR carries out one reaction per single sample.
  • dPCR also carries out a single reaction within a sample, however the sample is separated into a large number of partitions and the reaction is carried out in each partition
  • DGGE Denaturing Gradient Gel Electrophoresis
  • Defect means an unwanted attribute of an item associated with a papermaking process. It includes but is not limited to one or more plugs on a felt, and such attributes of paper sheet as holes, discoloration, streaks, spots, translucent spots, and any combination thereof.
  • Felt means a belt made of interweaved wool or any other fiber used in a papermaking process which functions as a conveyer of materials wherein the interweaved fibers define a plurality of lumens through which water or other fluids may pass. Felts may also provide cushioning between press rolls and may also be a medium used to remove water from papermaking materials. Felts include but are not limited to bottom felts, bottom board felts, cylinder tissue wet felts, drier felts, endless felts, pickup felts, suction pickup felts, Harper top felts, and top felts.
  • Paper Product or Paper Sheet means any formed fibrous structure end product of a papermaking process traditionally, but not necessarily, comprising cellulose fibers. Examples of such end products include but are not limited to facial tissue, bath tissue, table napkins, copy paper, printer paper, writing paper, notebook paper, newspaper, paper board, poster paper, bond paper, cardboard, and the like.
  • Papermaking Process means one or more processes for converting raw materials into paper products and which includes but is not limited one or more of such steps as pulping, digesting, refining, drying, calandering, pressing, crepeing, dewatering, and bleaching.
  • PCR Analysis means polymerase chain reaction analysis.
  • Plug means a solid, semisolid, viscous, and/or other deposit of material positioned within the lumens of a felt. Plugs may inhibit the flow of material through the lumens, and/or may impair any other functionality of a felt.
  • Primer means a composition of matter, typically a short strand of nucleotides, known to be complementary to specific sections of DNA and serve as a starting point for synthesis of a nucleotide chain complementary to DNA adjacent to the specific section of DNA.
  • Probe means a composition of matter constructed and arranged to bind to a targeted section of DNA and which can be readily detected when so bound and thereby be used to indicate the presence or absence of the targeted section of DNA.
  • qPCR Analysis means quantitative and/or qualitative polymerase chain reaction analysis.
  • Microorganisms means any organism small enough to insinuate itself within, adjacent to, on top of, or attached to equipment used in a papermaking process, it includes but is not limited to those organisms so small that they cannot be seen without the aid of a microscope, collections or colonies of such small organisms that can be seen by the naked eye but which comprise a number of individual organisms that are too small to be seen by the naked eye, as well as one or more organisms that can be seen by the naked eye, it includes but is not limited to any organism whose presence, in some way impairs the papermaking process such as forming plugs within felts and/or causing defects within paper sheets.
  • a highly sensitive and rapid detection method for microorganisms located in paper sheets and machine felts.
  • the method includes analysis of DNA present in samples extracts.
  • the samples themselves are fragments of a felt or a sheet of paper. These samples are highly desiccated and contain little or no live samples of the contaminating microorganisms.
  • Some prior art methods of utilizing DNA analysis include WO 2005/042082 which describes an in situ method utilizing probes to determine the presence or absence of a microorganism. In situ methods however are not applicable to paper sheets or felts as they are dried out when sampled. Also the in situ method involves applying the probes during cell division of the microorganisms which is not possible on paper sheets or felts with little or no more living organisms on them.
  • the DNA based analysis involves the use of probes.
  • the DNA based analysis involves the use of PCR primers to detect the presence or absence of microorganisms.
  • US Patent 5,928,875 describes the use of PCR primers to detect the presence or absence of spore forming bacteria.
  • the primer is targeted towards a part of a DNA strand which is highly conserved among a group of organisms. As a result, detecting the presence of that particular part of DNA is definitive proof of the presence a specific organism.
  • PCR analysis is of particular use in analyzing felts and paper sheets due to the difficultly of correctly identifying its contaminating microorganisms because they lack viable organisms for traditional plating methods or ATP measurements.
  • the PCR analysis involves utilizing one or more of the methods described in the Article Primer Directed Enzymatic Amplification of DNA with a Thermostable DNA Polymerase, by Randall Saiki et al., Science, Volume 239, pp. 487-491 (1988). In at least one embodiment the PCR analysis involves utilizing one or more of the methods described in the Article Specific Synthesis of DNA in Vitro via a Polymerase-Catalyzed Chain Reaction, by Kary Mullis et al., Methods In Enzymology, Volume 155, pp. 335-350 (1987).
  • the PCR analysis is a qPCR analysis as described in Trade Brochure qPCR guide, prefaced by Jo Vandesompele, (as downloaded from website http://www.eurogentec.com/file-browser.html on January 19, 2012).
  • the method is a quantitative qPCR analysis.
  • the method is a qualitative qPCR analysis. As illustrated in FIG.
  • the polymerase chain reaction is a method for targeting sequences of nucleic acid (DNA or RNA) and increasing the copy number of the target sequence to obtain useful quantities of nucleic acid for down-stream analysis
  • This method can be applied to the detection of microorganisms in a variety of samples that include, but are not limited to, machine felts, sheet defects, machine deposits, etc.
  • Quantitative PCR utilizes the same methodology as PCR, but it includes a real-time quantitative component.
  • primers are used to target a DNA sequence of interest based on the identity of the organism or function of a specific gene.
  • Some form of detection such as fluorescence may be used to detect the resulting DNA or 'DNA amplicon'.
  • the change in fluorescence is directly proportional to the change in the quantity of target DNA.
  • the number of cycles required to reach the pre-determined fluorescence threshold is compared to a standard that corresponds to the specific DNA target.
  • a standard is typically the target gene that is pure and of known quantity at concentrations that span several logs.
  • the number of copies of target DNA present in the sample is calculated using the standard curve.
  • the copy number per sample is then used to determine the number of cells per sample.
  • a primer set is used which targets DNA sequences from bacteria using a conservative approach to quantify total bacteria.
  • a primer set is used which targets primary biofilm-forming bacteria, including Meiothermus, Pseudoxanthomonas , and Deinococcus.
  • a primer set is used to target an adaptive biofilm-former which belongs to the Sphingomonadacea family of bacteria.
  • the adaptive biofilm-former exhibited higher tolerance to oxidant-based biocontrol programs compared to other biofilm and planktonic microorganisms.
  • the primer is used to distinguish between fungal and bacterial infestations.
  • Machine felts commonly pass in and out of shower streams and liquid basins containing various microorganisms from which live samples can be easily obtained.
  • the dynamic state of the felt often means that the population of organisms inhabiting the felt will differ from those present within the shower streams and liquid basin it contacts.
  • a typical analysis of the shower streams and liquid basins will not correctly identify what microorganisms are present within the felt.
  • a PCR analysis of a felt sample which takes into account the sorts of organisms which are known to be able to inhabit felts however allows for a truly accurate analysis of felt contaminations.
  • the DNA based analysis of the sample involves discounting the possibility of the presence of microorganisms known to not inhabit machine felts and/or end product paper sheets.
  • the method involves limiting the primers used to those associated with organisms known to inhabit machine felts and/or end product paper sheets.
  • the method involves distinguishing between DNA at the biological kingdom level. Biological life can be categorized according to five kingdoms: Monera, Protist, Plant, Animal, and Fungus. These organisms have hugely differing DNA and a protocol which focuses on identifying the organism's DNA at the kingdom level is vastly simpler than more specific determinations. Because with felts, the organisms from different kingdoms are often best treated differently, such a simple form of identification can be used to accurately identify the specific regimen best targeted to the particular contaminant.
  • more than one primer is used to identify organisms that have more than one uniquely recognizable nucleotide sequence.
  • the PCR analysis is used to detect genome sequences associated with enzymes unique to or nearly unique to specific organisms.
  • the method involves detecting a defect and then utilizing the PCR analysis to properly associate the source of the defect. In at least one embodiment the method determines if the defect is totally biologically based, totally non-biologically chemical based, or resulting from a combination of non- biologically chemical, mechanical, and biologically based sources.
  • the defect is one or more plugs on a felt.
  • the defect is a paper sheet having at least one or more of: a hole, a hole with a discolored halo around at least a portion of it, a streak of discoloration, a spot, a translucent spot, and any combination thereof.
  • a threshold level is methodology used to discount false positives.
  • PCR analysis detects traces of organisms that while present are not causes of a particular defect.
  • the method involves discounting the presence of any organism detected at a concentration lower than a pre-determined level known for one or more particular organisms.
  • the method involves discounting the presence of any organism detected at level lower than 10 4 cells per gram (of the defect).
  • the method involves discounting the presence of any organism detected at level lower than 10 4 cells per ml.
  • results of the analysis are used to augment the biocontrol program by determining how much, what kind, and how often, one or more biocidal compositions are added to one or more locations within a
  • any and all of the above and below embodiments are applied to a process water system or industrial system other than a papermaking process.
  • the method is able to detect microoganisms that would not otherwise be detected by prior art methods. For example in cases where foulant is caused by an infestation of anaerobic or sulfate reducing organisms, methods such as ORP detection would not correctly identify the foulant source as biological and would therefore incorrectly suggest applying an chemical not an anti- bilogical approach. Utilizing the DNA approach would however always correctly indicate a biological infestation because all life contains DNA.
  • a method is used for assessing microbial diversity. This can include problematic microorganisms found in machine deposits, sheet defects, finished products, felts, etc.
  • the method is based on analysis of nucleic acids in sample extracts. More specifically, it utilizes PCR such as but not limited to qPCR for the detection of total organisms such as bacteria; Sphingomonas species; Erythrobacter species; Pseudomonas species; Burkholderia species;
  • Haliscomenobacter species Saprospira species; Schlegelella species; Leptothrix species; Sphaerotilus natans; Bacillus species; Anoxybacillus species; members of the Cytophaga-Flavobacterium-Bacteroides phylum; green nonsulfur bacteria, including Herpetosiphon, members of the Deinococcus-Thermus phylum, including Meiothermus species; catalase-producing bacteria, amylase-producing bacteria, urease-producing bacteria, fungi, etc.
  • These techniques utilize primers and standards pairs that allow for detection and quantification of target organisms based on conserved sequences.
  • the primers target regions in the microbial genome that are highly conserved through evolution, while primers for specific phyla or genera target more variable regions of the genome.
  • the diversity index can also be expressed quantitatively as the relative abundance of several target organisms.
  • the diversity index for any part of a process can be measured at times when machines or processes are running well, thus creating a baseline.
  • the diversity index measured at times of poor machine or process performance can then be compared to the baseline to look for fluctuations in microbial populations and to determine which bacterial groups are responsible for problems in the process.
  • the diversity index can also be quantified for ease of comparison using the Shannon diversity index calculation to compare monitoring data among sample locations or relative to a baseline. Treatment strategies and feed points can then be altered accordingly to combat the problem.
  • RNA Ribonucleic acid
  • mRNA messenger RNA
  • the quantitative (real time) polymerase chain reaction method can be applied to detect messenger ribosomal nucleic acids (mRNA).
  • mRNA messenger ribosomal nucleic acids
  • mRNA is transcribed DNA that is sent to the ribosome to serve as a blueprint for protein synthesis in a process known as translation.
  • mRNA is produced only by living cells.
  • RNA from living cells can be isolated with the use of commercially available kits. Detection of mRNA requires an extra step in the quantitative polymerase chain reaction.
  • Reverse transcriptase is added to the reaction cocktail to transcribe mRNA into its complementary DNA (cDNA). Two sets of primers are required for this experiment. The first targets specific mRNA, while the second is used to amplify the resulting cDNA produced by the reverse transcriptase reaction.
  • cDNA complementary DNA
  • a coated free sheet mill experienced persistent deposition in one of the machine headboxes, which was believed to be the cause of defects in the final product. Microorganisms were assumed to be the underlying cause of the problem. However, traditional monitoring techniques (e.g. standard plate counts and ATP levels) did not indicate elevated levels of microbial activity.
  • Sheet defects from this mill were analyzed using the same qPCR-based approach. It is impossible to determine bacterial content in defects using traditional plating and ATP methods because many of the bacteria that may have been present in the defect are killed by the high temperatures of the dryer section. Chemical analysis does not provide a definitive answer about bacteria present in the sheet as it relies on ninhydrin staining. This approach is non-specific and prone to false positive and false negative results. DNA analysis of holes and sheet defects from this mill detected very low bacterial density ( Figure 2, Samples 1-5). Primary and adaptive biofilm-formers were not detected in the sheet defects analyzed from this mill. Therefore, bacterial slime was not likely contributing to defects and quality issues at this mill.
  • a coated free sheet mill utilized a competitive oxidant-based biocontrol program for several years. Control of microbial growth was perceived as adequate; however, there was an opportunity to further reduce sheet breaks for improved process efficiency.
  • the program was implemented and optimized in several phases. Bacterial density throughout the process remained low and a reduction in sheet breaks was documented. The average number of breaks per day decreased from an average of 1.2 breaks per day to 0.42 breaks per day.
  • PCR and qPCR methods provide more accurate information regarding microbial growth and biofilm formation in industrial water systems. These strategies allow for rapid analysis of the contribution of microorganisms to deposit formation and can be used to rapidly determine whether or not deposits containing microorganisms are contributing to defects. Quantitative qPCR techniques allow for rapid analysis of sheet defects to determine the contribution of microorganisms to quality issues. This new approach has been demonstrated to allow for a more proactive diagnosis of problems leading to improved machine efficiency and product quality.

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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des procédés et des compositions permettant d'identifier la présence de micro-organismes spécifiques dans une partie particulière d'un processus de fabrication de papier. Le procédé consiste à obtenir un échantillon du processus de sorte qu'il ne reste que peu ou aucun exemple vivant du micro-organisme. Cependant, étant donné que l'ADN provenant des microbes est encore présent, une analyse permettant d'identifier des parties d'ADN propre au microbe particulier décèlera correctement le micro-organisme présent. Ceci permet une analyse des infestations présentes sur les feutres ou les feuilles de papier qui généralement ne comportent plus de nombreux micro-organismes vivants lorsque des échantillons sont prélevés à des fins d'analyse.
PCT/US2014/071128 2013-12-23 2014-12-18 Détection et quantification d'acides nucléiques pour évaluer la biomasse microbienne dans des défauts de papier et feutres pour machine à papier WO2015100123A1 (fr)

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US14/138,526 US9290802B2 (en) 2012-01-24 2013-12-23 Detection and quantification of nucleic acid to assess microbial biomass in paper defects and machine felts
US14/138,526 2013-12-23

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EP3283639A4 (fr) * 2015-04-15 2018-11-21 Ecolab USA Inc. Procédé pour la détermination de la diversité et de la viabilité des seuils utilisés pour évaluer des micro-organismes dans des échantillons de traitement
CN110409225A (zh) * 2019-07-30 2019-11-05 云南卓印科技有限公司 一种防伪纸及其制备方法、验证方法和用途

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