US20120129181A1 - Detection of bacterial (mollicutes) contamination - Google Patents

Detection of bacterial (mollicutes) contamination Download PDF

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US20120129181A1
US20120129181A1 US13/364,050 US201213364050A US2012129181A1 US 20120129181 A1 US20120129181 A1 US 20120129181A1 US 201213364050 A US201213364050 A US 201213364050A US 2012129181 A1 US2012129181 A1 US 2012129181A1
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pcr
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Christian Birkner
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Roche Diagnostics Operations Inc
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Priority to US14/549,647 priority Critical patent/US20150086990A1/en
Priority to US15/626,641 priority patent/US11078545B2/en
Priority to US17/360,121 priority patent/US20210324453A1/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
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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
    • C12Q2527/00—Reactions demanding special reaction conditions
    • C12Q2527/125—Specific component of sample, medium or buffer
    • 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
    • C12Q2527/00—Reactions demanding special reaction conditions
    • C12Q2527/137—Concentration of a component of medium
    • 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
    • C12Q2549/00—Reactions characterised by the features used to influence the efficiency or specificity
    • C12Q2549/10—Reactions characterised by the features used to influence the efficiency or specificity the purpose being that of reducing false positive or false negative signals
    • C12Q2549/125—Reactions characterised by the features used to influence the efficiency or specificity the purpose being that of reducing false positive or false negative signals using sterilising/blocking agents, e.g. albumin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present disclosure relates to contamination within biological samples. More specifically, the instant disclosure relates to a system and method for detecting bacterial contamination within biological samples.
  • Mycoplasma is a genus of bacteria belonging to the class of Mollicutes which lack a cell wall. Without a cell wall, mycoplasma bacteria are unaffected by many common antibiotics such as penicillin or other beta-lactam antibiotics that target prokaryotic cell wall synthesis. There are over 100 recognized species of the genus Mycoplasma , one of several genera in the Mollicutes. Mollicutes are parasites or commensals of humans, other animals (including insects), and plants (although the genus Mycoplasma is by definition restricted to vertebrate hosts).
  • Cholesterol is required for the growth of species of the genus Mycoplasma , as well as certain other genera of mollicutes.
  • the optimum growth temperature of such species is often the temperature of their host, if the host is warm-bodied (e.g., 37° C. in humans), or ambient temperature if the host is unable to regulate its own internal temperature.
  • 16S ribosomal RNA sequences, as well as gene content strongly suggest that mollicutes, including the genus mycoplasmas, are closely related to either the Lactobacillus or the Clostridium branch of the phylogenetic tree (Firmicutes sensu stricto).
  • Mycoplasma species are often found in research laboratories as contaminants in cell culture.
  • Mycoplasma cell culture contamination can occur due to contamination from individuals or contaminated cell culture medium ingredients, for example.
  • Mycoplasma cells are physically small—less than 1 ⁇ m—and are difficult to detect with a conventional microscope.
  • Mycoplasmas may induce cellular changes, including chromosome aberrations, changes in metabolism and cell growth. Severe Mycoplasma infections have the potential to destroy a cell line.
  • Mycoplasmas are also involved as pathogens in a number of diseases.
  • mycoplasma pneumoniae is the major causative agent of community-acquired pneumonia.
  • Spiroplasma is another genus of bacteria belonging to the class of Mollicutes. Species of the genus Spiroplasma have been linked with the transmissible spongiform encephalopathies (TSEs) scrapie in sheep, chronic wasting disease (CWD) in deer, and Creutzfeldt—Jakob disease in humans ( Spiroplasma species isolates from both scrapie-affected sheep brain and CWD-affected deer brain which was inoculated intra-cranially into sheep and goat, respectively, induced spongiform encephalopathy resembling natural TSE in these animals).
  • TSEs transmissible spongiform encephalopathies
  • CWD chronic wasting disease
  • Creutzfeldt—Jakob disease Spiroplasma species isolates from both scrapie-affected sheep brain and CWD-affected deer brain which was inoculated intra-cranially into sheep and goat, respectively, induced spongiform encephalopathy re
  • Spiroplasma bacteria have also been shown to grow in embryonated eggs and can be passaged in such a culture system.
  • Embryonated eggs play a major role in the production of vaccines.
  • human vaccines against influenza have been available for almost 60 years and, until recently, were prepared almost entirely from viruses grown in the allantoic cavity of 9 to 11 day old embryonated chicken eggs.
  • the present disclosure provides an improved PCR-based amplification of a target sequence by suppressing non-specific amplification products.
  • This improvement concerns the use of a primer pair optimized to amplify a nucleic acid of a contaminant in the background of genomic DNA of a first organism.
  • PCR polymerase chain reaction
  • an improved method for determining the presence or absence of a bacterial contaminant in a liquid sample with biological material includes the steps of processing the sample and purifying the nucleic acids from the processed sample, forming a composition (reaction mixture) for a PCR-based amplification reaction, performing PCR, and detecting the presences or absence of an amplified target sequence.
  • the composition includes a first primer according to SEQ ID NO: 1, a second primer according to SEQ ID NO:2, and the purified nucleic acid of the processing (and purifying) step or a measured fraction thereof as a template.
  • the presence of an amplified target sequence indicates the presence of the bacterial contaminant in the sample, and the absence of said amplified target sequence indicates the absence of the bacterial contaminant in the sample.
  • a predetermined amount of DNA from CHO cells is added to at least one of the sample, the processed sample, the purified nucleic acids, or the reaction mixture.
  • the added DNA from CHO cells reduces unspecific amplification in the step of detecting.
  • compositions comprising amniotic fluid from embryonated eggs and DNA from CHO cells.
  • a composition which comprises purified nucleic acids from a sample and DNA from CHO cells (which is free of prokaryotic DNA).
  • the sample from which the purified DNA is obtained is selected from the group consisting of amniotic fluid, a suspension of eukaryotic cells, and a supernatant from a suspension of eukaryotic cells.
  • a process for amplifying DNA of a prokaryotic contaminant in DNA isolated from a sample selected from the group consisting of amniotic fluid, a suspension of eukaryotic cells, and a supernatant from a suspension of eukaryotic cells is provided.
  • Such processes, according to the instant disclosure, include the use of a reaction mixture and/or composition as disclosed herein.
  • kits comprising a lysis reagent, purified DNA from CHO cells at a predefined concentration, a first primer according to SEQ ID NO:1 and a second primer according to SEQ ID NO:2.
  • the DNA from the CHO cells is free of prokaryotic DNA.
  • some or all of the components are provided in separate containers.
  • an improved method for performing a PCR is provided.
  • a specific amplification product having a size in the range of about 100 base pairs (bp) to about 1500 bp is formed by DNA polymerase-catalyzed extension of a pair of oligonucleotide primers.
  • the improved method includes the steps of providing a pair of oligonucleotide primers; providing genomic DNA of a first eukaryotic organism; providing a third template comprising genomic DNA of a second eukaryotic organism which is suspected to contain genomic DNA of said one or more prokaryotic organism; mixing the pair of primers into a reaction mixture and also mixing into the relation mixture the third template and a measured amount of the genomic DNA of the first eukaryotic organism, and performing PCR under conditions such that the formation of non-specific PCR amplification product is suppressed.
  • each of the primers hybridize to the target sequence comprised in the genomic complement of the 16S-rRNA of one or more prokaryotic organism(s).
  • the pair of oligonucleotide primers is capable of forming a specific amplification product from a first template and the PCR is conducted under predetermined conditions in a reaction mixture having a predetermined composition.
  • the first template comprises genomic DNA of a first eukaryotic organism and genomic DNA of the one or more prokaryotic organism(s).
  • the pair of oligonucleotide primers (during PCR) does not form an amplification product from a second template under the same conditions in the reaction mixture.
  • the second template genomic DNA of the one or more prokaryotic organism(s) is absent but genomic DNA of the first eukaryotic organism is comprised.
  • FIG. 1 is a gel showing PCR amplification products (of GAPDH-specific primers) obtained from a MDCK cell culture without the addition of CHO DNA.
  • FIG. 2 is a gel showing PCR amplification products (of GAPDH-specific primers) obtained from a MDCK cell culture with the addition of CHO DNA.
  • FIG. 3 is a gel showing PCR amplification products (of primers according to SEQ ID NOs: 1 and 2) obtained from Adipose Stem Cells spiked with various combinations of Mycoplasma orale and CHO DNA.
  • FIG. 4 is another gel showing PCR amplification products (of primers according to SEQ ID NOs: 1 and 2) obtained from Adipose Stem Cells spiked with various combinations of Mycoplasma orale and CHO DNA.
  • FIG. 5 is a gel showing PCR amplification products (of primers according to SEQ ID NOs: 1 and 2) obtained from buffer spiked with various combinations fo Acholeplasma laidlawii DNA and CHO DNA.
  • FIG. 6 is another gel showing PCR amplification products (of primers according to SEQ ID NOs: 1 and 2) obtained from buffer spiked with various combinations of Acholeplasma laidlawii DNA and CHO DNA.
  • FIG. 7 is a gel showing PCR amplification products (of primers according to SEQ ID NOs: 1 and 2) obtained from samples from Vero cells spiked with various combinations of Acholeplasma laidlawii DNA, Mycoplasma orale DNA, and CHO DNA.
  • FIG. 8 is another gel showing PCR amplification products (of primers according to SEQ ID NOs: 1 and 2) obtained from samples from Vero cells spiked with various combinations of Acholeplasma laidlawii DNA, Mycoplasma orale DNA, and CHO DNA.
  • FIG. 9 is a gel showing PCR amplification products (of primers according to SEQ ID NOs: 1 and 2) obtained from samples from embryonated chicken eggs inoculated with various combinations of Acholeplasma laidlawii and CHO DNA.
  • FIG. 10 is another gel showing PCR amplification products (of primers according to SEQ ID NOs: 1 and 2) obtained from samples from embryonated chicken eggs inoculated with various combinations of Acholeplasma laidlawii and CHO DNA.
  • FIG. 11 is yet another gel showing PCR amplification products (of primers according to SEQ ID NOs: 1 and 2) obtained from samples from embryonated chicken eggs inoculated with various combinations of Acholeplasma laidlawii and CHO DNA.
  • FIG. 12 is another gel showing PCR amplification products (of primers according to SEQ ID NOs: 1 and 2) obtained from samples from embryonated chicken eggs inoculated with various combinations of Acholeplasma laidlawii and CHO DNA.
  • FIG. 13 is a gel showing PCR amplification products (of primers according to SEQ ID NOs: 1 and 2) obtained from buffer spiked with various combinations of Acholeplasma laidlawii DNA and calf thymus DNA.
  • FIG. 14 is a gel showing PCR amplification products (of primers according to SEQ ID NOs: 1 and 2) obtained from samples from embryonated chicken eggs inoculated with various combinations of Acholeplasma laidlawii and calf thymus DNA.
  • SEQ ID NO.: 1 is a universal primer (forward) for the detection of Mycoplasma and related species.
  • SEQ ID NO.: 2 is a universal primer (reverse) for the detection of Mycoplasma and related species.
  • SEQ ID NO.: 3 is a forward primer specific for a target sequence in the Glyceraldehyde 3-phosphate dehydrogenase gene (control sequence).
  • SEQ ID NO.: 4 is a reverse primer specific for a target sequence in the Glyceraldehyde 3-phosphate dehydrogenase gene (control sequence).
  • a microorganism means one microorganism or more than one microorganism.
  • the range When designating a range of numerical values such as a concentration range, the range may be indicated by the word “between”, followed by a first value n 1 , the word “and”, and a second value n 2 .
  • the designated range can be indicated by the expression “in the range of n 1 to n 2 ”. If not stated otherwise, when a designated range is indicated, the lower boundary of the designated range is understood as being the value either equal to, or higher than the first value. The higher boundary of the designated range is understood as being either the value equal to, or lower than the second value”. Thus, a value x in the designated range is given by n 1 ⁇ x ⁇ n 2 .
  • n in combination with a numerical value n indicates a value x in the interval given by the numerical value ⁇ 5% of the value, i.e. n ⁇ 0.05*n ⁇ x ⁇ n+0.05*n.
  • sample refers to a complex sample, such as a biological sample, i.e., a sample with biological material.
  • the sample may contain a plurality of organic and inorganic compounds which are desired to be separated from nucleic acids comprised in the biological material.
  • sample may also encompass an aqueous solution containing nucleic acids derived from other origins, e.g. from chemical or enzymatic reaction mixtures, or from a previous purification of biological sample material.
  • biological sample from which nucleic acids are purified, may encompass samples comprising viruses or bacterial cells, as well as isolated cells from multicellular organisms such as human and animal cells, as well as primary cultures of tissue and cultures of cell lines, as well as supernatants and rinses thereof.
  • the present disclosure also encompasses biological samples such as a fluid from the human or animal body.
  • the sample can be whole blood, blood serum, blood plasma, cerebral fluid, sputum, stool, biopsy specimens, bone marrow, oral rinses, tissues, urine, or mixtures thereof.
  • the sample may be a “liquid sample”, i.e. the sample is in a fluidic state and the sample fluid comprises water and a biological material.
  • the biological material may comprise cells or cellular components.
  • the liquid sample is selected from the group consisting of a cell culture supernatant, a suspension of cultured cells, and an amniotic fluid.
  • the amniotic fluid is from embryonated avian eggs.
  • processing or “processed,” in combination with “liquid sample,” is that the sample is treated by adding one or more compounds, and mixing the one or more compounds with the liquid sample, thereby resulting in a “processed sample”.
  • An exemplary compound which can be used for such treatment may be selected from the group consisting of a detergent, a surfactant, an organic solvent, a chaotropic agent, a protease, and a nuclease inhibiting agent.
  • a “chaotropic agent,” according to the present disclosure includes any chemical substance which disturbs the ordered structure of liquid water.
  • a chaotropic agent as referred to herein, may also facilitates unfolding, extension, and dissociation of proteins.
  • An exemplary processed sample according to the instant disclosure includes a lysate.
  • a “lysate” or a “lysed sample” may be obtained from a cell or plurality of cells, such as microbial cells and bacterial cells, wherein the structural integrity of a substantial portion of the cells present is disrupted. To this end, the cell wall, if present, may be destroyed for releasing the contents within the cell. To release the contents of disrupted cells, such as bacterial cells, the material is treated with certain agents to disintegrate, make porous, dissolve, degrade, and/or denature the cell walls of the microbial cells. In addition to the above, the cellular membranes may be destroyed.
  • the material may be treated with enzymes or with chemicals to dissolve, degrade or denature the cellular walls and cellular membranes of the cells or such organisms.
  • lysis lysis
  • the lysis (“lytic”) process will also disrupt the structural integrity of any bacterial contaminant (within the sample), thereby liberating nucleic acids of a contaminant.
  • chaotropic agents such as a guanidinium salt and/or anionic, cationic, zwitterionic, or non-ionic detergent when nucleic acids are set free in the process.
  • proteases which rapidly degrade enzymes with nucleolytic activity and other unwanted proteins.
  • the particulate matter can be separated from the lysate to result in a cleared lysate which may be done by filtering or centrifugation, for example.
  • the term “lysate” encompasses a cleared lysate.
  • “Purification of nucleic acids” from a processed sample can be done using a wide variety of methods which are standard techniques. These may include, for example, precipitation of nucleic acids from an aqueous solution using an alcohol (e.g., ethanol or isopropanol, for example), and isolation of the precipitate. Other methods may include adsorption of nucleic acids onto a solid phase (for example, with an oxidic surface such as silica), separating the solid phase, and eluting the nucleic acids from the solid phase.
  • an alcohol e.g., ethanol or isopropanol, for example
  • Other methods may include adsorption of nucleic acids onto a solid phase (for example, with an oxidic surface such as silica), separating the solid phase, and eluting the nucleic acids from the solid phase.
  • PCR polymerase chain reaction
  • Primers DNA oligonucleotides
  • Primers DNA oligonucleotides
  • a “reaction mixture” in this regard comprises a DNA polymerase, dNTP, ions, buffer and all other compounds necessary to affect extension of the primers hybridized (annealed) to the target DNA sequence.
  • PCR occasionally produces irregular amplicons or artifacts (i.e. non-specific amplification of fragments differing in size from the desired target DNA fragment).
  • irregular amplicons or artifacts i.e. non-specific amplification of fragments differing in size from the desired target DNA fragment.
  • PCR performed using a primer pair of SEQ ID NOs: 1 and 2 (according to the PCR reaction conditions set forth in Eldering, J. A., et al., Biologicals 32 (2004) 183-193, which discloses a system for bacterial testing of Chinese hamster ovary cells, the disclosure of which is hereby incorporated by reference in its entirety) occasionally produces non-specific amplification products. This is especially so when the DNA template is prepared from sample material from cultures of cells derived from other animal species or humans is used as template.
  • the formation of non-specific amplification products and artifacts can be significantly suppressed when the PCR (utilizing pairs according to SEQ ID NOs: 1 and 2 and the reaction conditions set forth in Eldering, et al.) is performed in the presence of CHO cell DNA.
  • this desired technical effect is most beneficial when the CHO cell DNA is free of contamination with bacterial DNA which can be amplified by the primer pair.
  • a method for performing PCR includes processing and/or purifying nucleic acid from a sample (such as a eukaryotic sample suspected as having a bacterial contamination). Preparing a PCR reaction mixture including primers specific for a first target, such as a contaminant nucleic acid sequence (from a prokaryotic source, for example) within the sample, and adding in nucleic acid from a second source, for example a second eukaryotic sample. The method further includes performing PCR and detecting the specific amplification product. As disclosed herein, the instant method surprisingly and unexpectedly reduces and/or eliminates non-specific amplification products.
  • an improved method for performing PCR is provided.
  • a specific amplification product with a size in the range of about 100 bp to about 1500 bp is formed by DNA polymerase-catalyzed extension of a pair of oligonucleotide primers during PCR.
  • the primers each hybridize to a target sequence comprising the genomic complement of the 16S-rRNA of one or more prokaryotic organism(s) and the pair of oligonucleotide primers is capable of forming (in PCR) a specific amplification product from a first template (under predetermined reaction conditions and compositions).
  • the first template comprises genomic DNA of a eukaryotic organism and genomic DNA of one or more prokaryotic organism.
  • the pair of primers hybridizes to target sequences within the genomic complement of rRNA of the one or more prokaryotic organisms and does not form an amplification product (under the same conditions) from genomic DNA of the eukaryotic organism of the first template. Furthermore, the pair of primers does not form an amplification product (under the same conditions) from genomic DNA from an additional eukaryotic organism (such as CHO cells).
  • the improved method comprises the steps of: (a) providing a pair of oligonucleotide primers; (b) providing genomic DNA of a first eukaryotic organism; (c) providing a third template comprising genomic DNA of a second eukaryotic organism which is suspected to contain genomic DNA of one or more prokaryotic organism(s); (d) mixing in the reaction mixture the primer pair of (a), the third template of (c), and a measured amount of the genomic DNA of the first eukaryotic organism of (b), and performing PCR under the conditions; wherein the formation of a non-specific PCR amplification product is suppressed.
  • the reaction conditions of the PCR reflect parameters including temperature regime, incubation times, number of PCR cycles and other physical parameters as known in the art of PCR.
  • the reaction mixture comprises all components of the final mixture with which the PCR process is conducted, including all compounds needed for primer elongation.
  • the reaction mixture also includes the pair of primers, each at its respective predetermined concentration.
  • reaction parameters and mixtures may vary based on variables such as the PCR platform being used, the volume of the reaction mixture, etc.
  • the present disclosure, and the methods disclosed herein, are not limited to any one amplification platform and are applicable with all platforms for amplifying and/or detecting the presence (and/or quantifying) DNA or RNA. Further, and for the sake of clarity, the reaction mixture in this sense does not comprise the template DNA which is added separately (following its processing and purification).
  • a template DNA may comprise genomic DNA of a first organism (such as a eukaryotic organism suspected of having bacterial contamination) and then additionally, DNA from another additional organism in a measured amount (e.g., based on the amount of DNA from the first organism).
  • the additional organism may be CHO cells.
  • CHO cell DNA is added to the PCR reaction mixture in a measured amount (i.e. at a predetermined concentration which may be based on the amount of target and/or volume of the reaction mixture, for example).
  • the pair of oligonucleotide primers hybridizes to a plurality of prokaryotic species, and between 0 to 3 mismatches may occur in the hybridization of the primers with their target region of the 16S-rRNA gene of such prokaryotic species. According to some embodiments, a mismatch (if present) would not be at the terminal nucleotide providing the 3′-OH group of the respective oligonucleotide.
  • the one or more prokaryotic organism(s) may be a species selected from the group consisting of a Mollicutes species, a Bacillus species, a Clostridium species, a Corynebacterium species, a Micrococcus species, a Staphylococcus species, and a Streptococcus species.
  • the species may be a Mycoplasma species such as M. hyorhinis, M. arginini, M. pneumoniae, M. fermentans, M. orale, and M. pirium .
  • the species may be an Acholeplasma species, Acholeplasma laidlawii , or a Spiroplasma species such as Spiroplasma mirium.
  • the additional eukaryotic organism is a CHO cell or a culture comprising a plurality of CHO cells.
  • the pair of primers comprises either one of, or both of, SEQ ID NO:1 and SEQ ID NO:2.
  • the conditions and the reaction mixture comprise those as specified in the MYCOTOOL assay by Roche Diagnostics GmbH, Mannheim (Germany) described in the manuals of the MYCOTOOL test kits (catalog numbers, 05200709001; 05184592001; 05184240001, for example).
  • Another embodiment of the present disclosure provides an improved method for determining the presence or absence of a bacterial contaminant in a liquid sample, said method comprising the steps of: (a) processing the sample and purifying the nucleic acids from the processed sample; followed by (b) forming a composition for a PCR-based amplification reaction, the composition including a first primer according to SEQ ID NO:1, a second primer according to SEQ ID NO:2, and the purified nucleic acids of step (a) or a measured fraction thereof as a template; followed by (c) performing a polymerase chain reaction (PCR) with the composition of step (b), whereby a target sequence comprised in a prokaryotic 16S-rRNA gene, if present in the template, is amplified; followed by (d) detecting the presence or absence of an amplified target sequence, whereby the presence of said amplified target sequence indicates the presence of the bacterial contaminant in the sample, and the absence of said amplified target sequence indicates the absence of the bacterial contaminant in
  • the predetermined amount of DNA per ml of the liquid sample is the DNA content from about 5 ⁇ 10 6 CHO cells.
  • the liquid sample is selected from the group consisting of cell culture medium with cultured cells, cell-free culture supernatant, and amniotic fluid.
  • the amniotic fluid is from embryonated eggs.
  • the bacterial contaminant is a genus selected from the group consisting of Acholeplasma, Bacillus, Clostridium, Corynebacterium, Micrococcus, Mycoplasma, Spiroplasma, Staphylococcus, and Streptococcus .
  • the bacterial contaminant is a Mycoplasma species selected from the group consisting of M. hyorhinis, M. arginini, M. pneumoniae, M. fermentans, M. orale , and M. pirium , or the bacterial contaminant is Acholeplasma laidlawii , or the bacterial contaminant is Spiroplasma mirium.
  • a composition comprising amniotic fluid from embryonated eggs and DNA from CHO cells is provided.
  • a lysis reagent selected from a chaotropic agent and a protease is also provided.
  • compositions comprising (i) purified nucleic acids from a sample selected from the group consisting of amniotic fluid, a suspension of eukaryotic cells, and a supernatant from a suspension of eukaryotic cells, and (ii) DNA from CHO cells which is free of prokaryotic DNA.
  • the composition further comprises a first primer according to SEQ ID NO:1, a second primer according to SEQ ID NO:2, nucleotide triphosphates, and a thermostable DNA polymerase.
  • the composition may also comprise an intercalating dye in some embodiments.
  • Some embodiments of the instant disclosure include a use of the disclosed composition according for amplifying DNA of a prokaryotic contaminant from DNA isolated from a sample selected from the group consisting of amniotic fluid, a suspension of eukaryotic cells, and a supernatant from a suspension of eukaryotic cell.
  • kits comprising in separate containers (i) a lysis reagent, (ii) purified DNA from CHO cells at a predefined concentration, said DNA being free of prokaryotic DNA, and (iii) a first primer according to SEQ ID NO:1 and a second primer according to SEQ ID NO:2.
  • Still other embodiments of the instant disclosure comprise an improved method for performing an improved polymerase chain reaction (PCR), wherein a specific amplification product with a size in the range of about 100 bp to about 1500 bp is formed by DNA polymerase-catalyzed extension of a pair of oligonucleotide primers.
  • the primers each hybridize to a target sequence comprised in the genomic complement of the 16S-rRNA of one or more prokaryotic organism(s) and are capable of forming in PCR a specific amplification product from a first template.
  • the PCR is conducted under predetermined conditions in a reaction mixture with a predetermined composition, and the first template comprises genomic DNA of a first eukaryotic organism and genomic DNA of the one or more prokaryotic organism(s). Also, during PCR the pair of oligonucleotide primers does not form an amplification product from a second template under the same conditions in the reaction mixture. The genomic DNA of the one or more prokaryotic organism(s) is in the second template, but genomic DNA of the first eukaryotic organism is.
  • the improved method comprises the steps of:
  • the MYCOTOOL PCR Mycoplasma Detection Kit is an in vitro nucleic acid amplification test optimized for the detection of bacteria belonging to the Mollicutes. These include Mycoplasma hyorhinis, M. arginini, M. pneumoniae, M. fermentans, M. orale, M. pirium, M. salivarum, M. hominis, M. synoviae, Spiroplasma mirium, S. citri , and Acholeplasma laidlawii.
  • the MycoTool Kit comprises two subkits: Subkit 1 (“Detection Prep Kit”; Roche Applied Science Catalog No. 05184592001) and Subkit 2 (“Detection Amplification Kit”; Roche Applied Science Catalog No. 05184240001).
  • the kit was used exactly according to the instructions of the manufacturer.
  • a liquid sample selected from (i) cell culture supernatant, (ii) a suspension of cultured cells and (iii) amniotic fluid was lysed by adding an aqueous buffer containing a guanidinium salt and proteinase K, mixing the buffer with the sample, and incubating the mixture to effect lysis.
  • CHO cell DNA was added to the lysate and mixed.
  • the CHO cell DNA was free of any contaminating prokaryotic DNA as tested separately.
  • the nucleic acids were precipitated from the mixture by adding alcohol.
  • the precipitate was recovered by centrifugation, washed with 70% ethanol and dried. The dried pellet was dissolved in the prescribed buffer and subjected to PCR analysis.
  • the MYCOTOOL kit also includes a primer pair for the GAPDH housekeeping gene.
  • uracil-N-glycosylase enzyme component of the kit.
  • PCR was performed exactly according to the instructions by the manufacturer. PCR products were electrophoresed on polyacrylamide gels under standard conditions. Bands were visualized with the RESOLIGHT compound and UV illumination, band detection was at 520 nm.
  • the primers of the MYCOTOOL kit used for detection of Mollicutes are those of SEQ ID NO:1 and SEQ ID NO:2.
  • Control primers specific for GAPDH are also provided (SEQ ID NO:3 and SEQ ID NO:4).
  • Each MYCOTOOL kit further comprises a control plasmid containing Mycoplasma DNA, however producing a PCR fragment with a discernably different size compared to the PCR fragments amplified from isolated bacterial DNA (positive control).
  • a control plasmid containing Mycoplasma DNA, however producing a PCR fragment with a discernably different size compared to the PCR fragments amplified from isolated bacterial DNA (positive control).
  • DNA prepared from Acholeplasma laidlawii or Mycoplasma orale was spiked to either sample material prior to lysis, or to isolated DNA prepared from sample material (if not indicated otherwise).
  • DNA was prepared from reference cultures (standard conditions) of Acholeplasma laidlawii (ATCC 27556) and Mycoplasma orale (ATCC 23714).
  • cfu colony forming units
  • Mollicutes DNA as indicated above was used in the spiking experiments described below.
  • the size of a typical specific PCR fragment amplified from Mollicutes DNA (specific amplification product) was in the range of about 430-470 bp.
  • a culture of MDCK cells free of any prokaryotic organisms and having a cell titer of 0.79 ⁇ 10 6 cells/ml was used.
  • Acholeplasma laidlawii DNA was added to the sample at a concentration of 3 colony forming units (cfu) per 1 ml sample.
  • Nucleic acids were isolated from the spiked sample material as specified in the instruction manual of the MYCOTOOL kit (see also Example 1). Two different nucleic acid preparations were made, the first without addition of CHO cell DNA, the second with the addition of CHO cell DNA (50 mg per 1 ml sample) to the sample material.
  • nucleic acids were prepared with or without CHO cell DNA.
  • FIGS. 1 and 2 show the gels with the bands indicating the PCR products which were obtained.
  • FIG. 1 depicts the results obtained without CHO cell DNA added
  • FIG. 2 sows the PCR products obtained with CHO cell DNA added.
  • FIG. 1 Lane Comment/Dilution 1-5 GAPDH control PCR; DNA isolated from MDCK cells; no CHO cell DNA; spiked with A. laidlawii DNA 1 undiluted 2 10 ⁇ 1 3 10 ⁇ 2 4 10 ⁇ 3 5 10 ⁇ 4 6 Size marker (50 bp steps) 7-11 GAPDH control PCR; DNA isolated from MDCK cells, no CHO cell DNA, not spiked 7 10 ⁇ 1 8 10 ⁇ 2 9 10 ⁇ 3 10 10 ⁇ 4 11 Size marker (50 bp steps)
  • FIG. 2 Lane Comment/Dilution 1-5 GAPDH control PCR; DNA isolated from MDCK cells, CHO cell DNA added, spiked with A. laidlawii DNA 1 undiluted 2 10 ⁇ 1 3 10 ⁇ 2 4 10 ⁇ 3 5 10 ⁇ 4 6 Size marker (50 bp steps) 7-11 GAPDH control PCR; DNA isolated from MDCK cells, CHO cell DNA added, not spiked 7 10 ⁇ 1 8 10 ⁇ 2 9 10 ⁇ 3 10 10 ⁇ 4 11 Size marker (50 bp steps)
  • ASC free of any prokaryotic organisms were sedimented by centrifugation.
  • the cleared supernatant (cell culture medium) was used for DNA isolation as specified in the instruction manual of the MYCOTOOL kit (see also Example 1).
  • Mycoplasma orale DNA was added to the sample at a concentration of 3 colony forming units (cfu) per 1 ml sample. The sample was then divided into two equal volumes. To one aliquot CHO cell DNA was added at a concentration of 100 mg per 1 ml of supernatant. Total DNA was isolated from both aliquots separately.
  • FIGS. 3 and 4 show the gels with the amplification products obtained by PCR and after electrophoresing the DNA fragments.
  • FIG. 3 Lane Comment/Dilution 1-8 Primers as in SEQ ID NO: 1 and SEQ ID NO: 2; DNA isolated from ASC, spiked with M. orale DNA, no CHO cell DNA 1-8 Primers as in SEQ ID NO: 1 and SEQ ID NO: 2; PCR with 8 independently drawn aliquots 6 The size of the band is in line with the size range observed for a specific target DNA amplification product
  • FIG. 4 Lane Comment/Dilution 1-8 Primers as in SEQ ID NO: 1 and SEQ ID NO: 2; DNA isolated from ASC, spiked with M. orale DNA, with CHO cell DNA added 1-8 Primers as in SEQ ID NO: 1 and SEQ ID NO: 2; PCR with 8 independently drawn aliquots 9-12 Primers as in SEQ ID NO: 1 and SEQ ID NO: 2; PCR with buffer (“no template” control) 13 Size marker (50 bp steps)
  • FIGS. 5 and 6 show the gels with the amplification products obtained by PCR and after electrophoresing the DNA fragments.
  • FIG. 5 Lane Comment/Dilution 1-8 Primers as in SEQ ID NO: 1 and SEQ ID NO: 2; Tris buffer spiked with A. laidlawii DNA, no CHO cell DNA 1-4 10 cfu A. laidlawii DNA 5-8 1 cfu A. laidlawii DNA 9-12 Primers as in SEQ ID NO: 1 and SEQ ID NO: 2; Tris buffer, not spiked, no CHO cell DNA 13, 14 Primers as in SEQ ID NO: 1 and SEQ ID NO: 2; Tris buffer, about 10 copies of positive control plasmid (part of MYCOTOOL kit) per PCR reaction mixture, no CHO cell DNA 15 Size marker (50 bp steps)
  • FIG. 6 Lane Comment/Dilution 1-8 Primers as in SEQ ID NO: 1 and SEQ ID NO: 2; Tris buffer spiked with A. laidlawii DNA, with CHO cell DNA added 1-4 10 cfu A. laidlawii DNA 5-8 1 cfu A. laidlawii DNA 9-12 Primers as in SEQ ID NO: 1 and SEQ ID NO: 2; Tris buffer, not spiked, with CHO cell DNA added 13, 14 Primers as in SEQ ID NO: 1 and SEQ ID NO: 2; Tris buffer, about 10 copies of positive control plasmid (part of MYCOTOOL kit) per PCR reaction mixture, with CHO cell DNA added 15 Size marker (50 bp steps)
  • the lanes with the positive control plasmid illustrate the discernible size difference compared with the specific amplification products of A. laidlawii target sequence.
  • Suspensions of Vero cells with a cell titer in the range of 10 5 to 10 6 cells per ml from a culture which was free of any prokaryotic organisms were spiked with A. laidlawii or M. orale DNA at a concentration of 3 or 10 cfu per 1 ml of cell suspension.
  • To the culture spiked with 3 cfu/ml CHO cell DNA was added at a concentration of 10, 30, 50, 70, 85, 100, and 150 ⁇ g per 1 ml of cell suspension.
  • FIGS. 7 and 8 show the gels with the amplification products obtained by PCR and after electrophoresing the DNA fragments.
  • the samples shown in FIG. 8 contained CHO cell DNA at a concentration of 150 ⁇ g per 1 ml of cell suspension. Comparable results were obtained when using the concentrations of 10, 30, 50, 70, 85, and 100 ⁇ g CHO cell DNA per 1 ml of cell suspension.
  • FIG. 7 Lane Comment/Dilution 1-4 Primers as in SEQ ID NO: 1 and SEQ ID NO: 2; Vero cell suspension spiked with A. laidlawii DNA, no CHO cell DNA 1-4 10 cfu A. laidlawii DNA 5-8 Primers as in SEQ ID NO: 1 and SEQ ID NO: 2; Vero cell suspension, not spiked, no CHO cell DNA 9, 10 Primers as in SEQ ID NO: 1 and SEQ ID NO: 2; Tris buffer, about 10 copies of positive control plasmid (part of MYCOTOOL kit) per PCR reaction mixture, no CHO cell DNA 11, 12 Primers as in SEQ ID NO: 1 and SEQ ID NO: 2; PCR with buffer (“no template” control) 13 Size marker (50 bp steps)
  • FIG. 8 Lane Comment/Dilution 1-4 Primers as in SEQ ID NO: 1 and SEQ ID NO: 2; Vero cell suspension spiked with A. laidlawii DNA, with CHO cell DNA added 1-4 3 cfu A. laidlawii DNA 5-8 Primers as in SEQ ID NO: 1 and SEQ ID NO: 2; Vero cell suspension, not spiked, with CHO cell DNA added 9, 10 Primers as in SEQ ID NO: 1 and SEQ ID NO: 2; Tris buffer, about 10 copies of positive control plasmid (part of MYCOTOOL kit) per PCR reaction mixture, with CHO cell DNA added 11, 12 Primers as in SEQ ID NO: 1 and SEQ ID NO: 2; PCR with buffer (“no template” control) 13 Size marker (50 bp steps)
  • Allantois fluid free of mycoplasma contamination was harvested from embryonated chicken eggs (9 to 11 days) which were inoculated with a viral vaccine strain.
  • a liquid culture of A. laidlawii with a titer of 2.45 ⁇ 10 3 cfu was diluted with TE buffer 1:1,000. An aliquot of this dilution was diluted 1:10 with allantois fluid. A volume of 12.2 ⁇ l of this 1:10,000 inoculum was added per 1 ml of allantois fluid to result in an equivalent titer of 3 cfu per 1 ml of inoculated allantois fluid. The inoculated allantois fluid was subjected to DNA isolation without further incubation, i.e. the bacteria were not allowed to grow in the allantois fluid.
  • Example 7 The experiment was conducted as described in Example 7 with the exception that no CHO cell DNA was added prior to DNA purification. Instead, CHO cell DNA was added to the reaction mixture prior to starting PCR.
  • the concentration of CHO cell DNA in the reaction mixture was 0, 2.5, 5, and 10 ⁇ g per 50 ⁇ l (volume of the PCR reaction mixture). This corresponds to 0 ⁇ g/ ⁇ l, 0.05 ⁇ l/ ⁇ l, 0.1 ⁇ g/ ⁇ l, and 0.2 ⁇ g/ ⁇ l in the final reaction mixture, i.e. the reaction mixture just prior to starting the PCR reaction.
  • FIGS. 9-12 show the results (the arrow indicates the region of the get in which non-specifically amplified PCR products migrate).
  • FIG. 9 Lane Comment/Dilution 1-4 Primers as in SEQ ID NO: 1 and SEQ ID NO: 2; allantois fluid “inoculated” with A. laidlawii , no CHO cell DNA 1-4 3 cfu A. laidlawii DNA 5-8 Primers as in SEQ ID NO: 1 and SEQ ID NO: 2; allantois fluid, not inoculated, no CHO cell DNA 9, 10 Primers as in SEQ ID NO: 1 and SEQ ID NO: 2; Tris buffer, about 10 copies of positive control plasmid (part of MYCOTOOL kit) per PCR reaction mixture, no CHO cell DNA 11 Size marker (50 bp steps)
  • FIG. 10 Lane Comment/Dilution 1-4 Primers as in SEQ ID NO: 1 and SEQ ID NO: 2; allantois fluid “inoculated” with A. laidlawii , 2.5 ⁇ g/50 ⁇ l CHO cell DNA 1-4 3 cfu A. laidlawii DNA 5-8 Primers as in SEQ ID NO: 1 and SEQ ID NO: 2; allantois fluid, not inoculated, 2.5 ⁇ g/50 ⁇ l CHO cell DNA 9, 10 Primers as in SEQ ID NO: 1 and SEQ ID NO: 2; Tris buffer, about 10 copies of positive control plasmid (part of MYCOTOOL kit) per PCR reaction mixture, no CHO cell DNA 11 Size marker (50 bp steps)
  • FIG. 11 Lane Comment/Dilution 1-4 Primers as in SEQ ID NO: 1 and SEQ ID NO: 2; allantois fluid “inoculated” with A. laidlawii , 5 ⁇ g/50 ⁇ l CHO cell DNA 1-4 3 cfu A. laidlawii DNA 5-8 Primers as in SEQ ID NO: 1 and SEQ ID NO: 2; allantois fluid, not inoculated, 5 ⁇ g/50 ⁇ l CHO cell DNA 9, 10 Primers as in SEQ ID NO: 1 and SEQ ID NO: 2; PCR with buffer (“no template” control) 11 Size marker (50 bp steps)
  • FIG. 12 Lane Comment/Dilution 1-4 Primers as in SEQ ID NO: 1 and SEQ ID NO: 2; allantois fluid “inoculated” with A. laidlawii , 10 ⁇ g/50 ⁇ l CHO cell DNA 1-4 3 cfu A. laidlawii DNA 5-8 Primers as in SEQ ID NO: 1 and SEQ ID NO: 2; allantois fluid, not inoculated, 10 ⁇ g/50 ⁇ l CHO cell DNA 9, 10 Primers as in SEQ ID NO: 1 and SEQ ID NO: 2; PCR with buffer (“no template” control) 11 Size marker (50 bp steps)
  • Example 5 The experiment was conducted as described in Example 5 with the exception that the concentration of A. laidlawii DNA was 3 cfu per 1 ml of buffer and instead of CHO cell DNA calf thymus was added at a concentration of 200 ⁇ g per 1 ml of buffer prior to DNA purification.
  • FIG. 13 shows the results.
  • FIG. 13 Lane Comment/Dilution 1-8 Primers as in SEQ ID NO: 1 and SEQ ID NO: 2; Tris buffer spiked with A. laidlawii DNA, 1-4 3 cfu A. laidlawii DNA, no calf thymus DNA 5-8 1 cfu A.
  • the PCR fragments generated in the presence of calf thymus DNA show a certain variation in size, unlike the fragments produced in the presence of CHO cell DNA, e.g. those shown in FIG. 6 (in particular).
  • calf thymus DNA does not appears to be suited to suppress the formation of PCR artifacts.
  • CHO cell DNA's (rather than calf thymus DNA or other similarly suited eukaryotic genomic DNAs) ability to provide the desired effect is due to the fact that the MYCOTOOL PCR is based on the primers of SEQ ID NO:1 and SEQ ID NO:2 and was initially adapted to CHO cell cultures and culture supernatants.
  • the genomic DNA of the CHO cells provided a “background” which for the primers appears to beneficial in that the “background” apparently suppresses the formation of artifacts.
  • Genomic DNA from other species is different in composition and apparently not able to suppress formation of non-specific artifacts (or less efficient in doing so).
  • Example 7 The experiment was conducted as described in Example 7 with the exception that instead of CHO cell DNA, calf thymus was added prior to DNA purification.
  • FIG. 14 shows the results.
  • FIG. 14 Lane Comment/Dilution 1-8 Primers as in SEQ ID NO: 1 and SEQ ID NO: 2; allantois fluid “inoculated” with A. laidlawii , with calf thymus DNA added 1-4 3 cfu A.
  • Example 9 Basically, the conclusion is similar as in Example 9. Again, non-specific amplification products were produced, in contrast to PCR in the presence of CHO cell DNA
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