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Robust Multiplex Quantitative PCR Assay for Universal Detection of Microorganisms in Fuel Osman Radwan, Thusitha S. Gunasekera, and Oscar N. Ruiz Energy Fuels, Just Accepted Manuscript • DOI: 10.1021/acs.energyfuels.8b02292 • Publication Date (Web): 31 Aug 2018 Downloaded from http://pubs.acs.org on September 10, 2018
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Robust Multiplex Quantitative PCR Assay for Universal Detection of Microorganisms in
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Fuel
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Osman Radwan†, Thusitha S. Gunasekera†, Oscar N. Ruiz*‡
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†
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USA
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‡
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Wright-Patterson AFB, Ohio, USA
Environmental Microbiology Group, University of Dayton Research Institute, Dayton, Ohio,
Fuels and Energy Branch, Aerospace Systems Directorate, Air Force Research Laboratory,
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*
Corresponding author (e-mail address:
[email protected])
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ABSTRACT
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Rapid detection of microbial contamination in conventional and alternative fuels is hampered by
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the lack of sensitive and cost-effective assays to detect total and specific microorganisms in
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fuels. Here, we report a simple and highly sensitive TaqMan qPCR assay for universal detection
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and quantification of fungi, bacteria, and archaea in fuel in a single multiplexed reaction.
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Universal primers and probes targeting conserved regions of the 16S and 18S rRNA genes were
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designed and validated for specific amplification of total fungi, bacteria, and archaea in fuel. The
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assay is able to detect as low as 10 pg of fungal and bacterial DNA. The combination of a simple
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liquid-liquid extraction to recover cells from fuel with a freeze-and-heat method to release DNA
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for direct qPCR amplification eliminates the need for DNA extractions from contaminated
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samples, thus making the assay much faster, inexpensive, and less laborious. The universal
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microbial multiplex qPCR assay demonstrated a high capacity to detect and quantify a wide
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range of microbial contaminants. The assay was validated to accurately detect and quantify the
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intended microorganisms in the presence of high levels of non-target DNA and in fuel from field
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samples. This robust microbial qPCR assay can be applied to microbial detection in
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environmental and industrial settings to facilitate risk assessment and mitigation of microbial
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contamination.
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KEYWORDS: Quantitative Polymerase Chain Reaction, qPCR, TaqMan, Multiplex, DNA,
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Bacteria, Fungi, Archaea, Rapid Detection, Fuel, Biofuel, Risk Assessment
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Energy & Fuels
INTRODUCTION Quantitative polymerase chain reaction (qPCR) or real-time PCR is one of the most
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sensitive, rapid, and specific tools for high-throughput analysis of DNA samples (1, 2). By the
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second half of the 1990s, the first qPCR procedure had been developed (3). The real-time PCR
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method had the ability to monitor the amplicon accumulation in each PCR cycle by measuring
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fluorescence emission (4). One common method of perfoµrming qPCR is by using a fluorescent
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dye that intercalates with double-stranded DNA to allow detection and quantification of an
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amplicon. In order to use a fluorescent dye for quantification, the qPCR assay has to be
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developed on the concept of selective amplification; where the forward and reverse primers
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provide the specificity for the target DNA (5). The dye SYBR® Green (SYBR is a registered
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trademark of Life Technologies, Carlsbad, CA) is a robust fluorescent dye for qPCR applications
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(4). A melting curve analysis is performed with each SYBR Green qPCR run to ensure that a
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single melt peak corresponding to the target amplicon melting temperature is produced. Having
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to run a melt curve analysis increases the compulsory time to perform qPCR analysis. A major
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drawback of SYBR Green-based qPCR is its inability to detect multiple DNA targets in a single
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test reaction; this reduces throughput and increases the use of sample, cost, and the time required
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for results.
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Alternatively, qPCR assays can be developed using the concept of selective detection (6-
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9). In this type of assay, a single-stranded DNA probe containing a covalently linked fluorophore
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molecule provides both target specificity and detection by fluorescence. The specific probe can
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be combined with a set of selective primers to further improve specificity. Accordingly,
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amplicon specific detection is the method of choice for accurate quantification of microbial
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contamination. The TaqMan chemistry is the most widely used fluorescent-probe chemistry
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today (10). A TaqMan probe is a target-specific oligonucleotide with attached 5’ fluorophore and
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3’quencher molecules designed to anneal to the target DNA at a melting temperature (Tm)
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approximately 10 ºC higher than the amplification primers’ Tm. The higher annealing
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temperature of the probe ensures the probe has bound to every molecule of single-stranded target
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DNA before amplification can proceed. Once DNA elongation proceeds, the 5’ to 3’ exonuclease
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activity of the Taq DNA polymerase or similar thermostable polymerases degrades the probe
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releasing the fluorophore and preventing fluorescence resonance energy transfer (FRET)
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between the close proximity 5’ fluorophore and 3’ quencher, producing the fluorescence signal
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(7, 10, 11). Although TaqMan qPCR has been widely used for detection of specific
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microorganisms in medical (12-15) and agricultural applications (11), a universal multiplex
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qPCR assay capable of detecting the bacteria, fungi, and archaea domains in complex
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environmental samples including fuel has not been developed.
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In the field of fuel microbiology, PCR has been used to characterize microbial
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contamination in fuel tanks and assess bacterial community dynamics (16, 17, 18). The use of
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PCR overcame important drawbacks of culture-dependent method including a propensity to
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underestimates the viable population density in fuel, and long wait times for results. Fuel system
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biodeterioration is an important problem affecting both commercial and military systems
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containing conventional and alternative fuels (16-31). Consequences of biodeterioration in the
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fuel system include reduced fuel stability and quality, tank corrosion, coating degradation,
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deactivation of fuel-water separators, injector fouling and filter plugging. More environmentally
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friendly biofuels, such as biodiesel, can show a higher propensity for biocontamination due to the
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increased biodegradability of fatty acid methyl esters (32-34). The use of SYBR Green-based
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qPCR assays for detection of total bacteria and fungi in fuel samples has been demonstrated
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previously (19-21, 35). Recently, a SYBR Green-based qPCR assay for specific detection of
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Hormoconis resinae was published (36). To address the drawbacks of SYBR Green-based qPCR
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assays and facilitate the implementation of qPCR in testing for microbial contamination in fuels
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and other complex matrices, here we describe the development, characterization, and validation
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of a universal TaqMan qPCR assay that when combined with a simple freeze-heat step to recover
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microbial DNA provides a simple method to rapidly detect and quantify total bacteria, fungi, and
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archaea in a single test reaction. Because environmental samples contain a mixture of unknown
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microorganisms, it is critical to employ a qPCR assay that is able to detect and quantify all the
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microorganisms in a sample. Previous research efforts have focused on designing universal
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primers targeting the amplification of the 16S and 18S rRNA genes from bacteria (12, 37-39),
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archaea (40, 41) and fungi (42) in separate test reactions. In the current work, we designed a new
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universal microbial qPCR assay for multiplexed quantification of bacteria, fungi and archaea in
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fuel. The use of a universal multiplexed qPCR assay without the need for DNA purification is
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validated; dramatically reducing the time required to process and test a field sample. The
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multiplex qPCR assay demonstrated here may be used in other environmental and industrial
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settings to detect and quantify microbiological contamination.
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EXPERIMENTAL SECTION
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Microbial Materials and DNA Sequences for Assay Development
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Twenty-three fungal isolates and twenty-three bacterial strains isolated from fuel-
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containing environments and capable of hydrocarbon degradation were used in this study (Supp.,
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Table S1). Additionally, Hormoconis resinae (ATCC 22711), Yarrowia lipolytica (ATCC
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20496) and Pseudomonas aeruginosa (ATCC 33988) obtained from the American Type Culture
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Collection (Manassas, VA, USA) were used. Bacterial and fungal species were grown in Luria-
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Bertani (LB) medium and Potato Dextrose Broth (PDB) medium, respectively, at 28 °C with
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agitation for up to 72 hours. DNA from the microbial cultures was obtained by using the
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UltraClean Microbial DNA isolation kit (Cat No. 12224-250, QIAGEN, MD, USA) and the
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genomic DNA used in PCR. The quantity and purity of the DNA were confirmed by Nanodrop
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using the 260/280 nm function. DNA integrity was confirmed by running the DNA samples in a
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2100 Bioanalyzer chip-based microcapillary electrophoresis system (Agilent Technologies,
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Santa Clara, CA, USA). A 423 base-pair (bp) region of the fungal 18S rRNA (42) and 521 bp
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region of the bacterial16S rRNA (37) was PCR amplified using bacteria-specific and fungi-
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specific sequencing primer sets. PCR products were purified by agarose electrophoresis,
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sequenced (GenScript Inc., Piscataway, NJ, USA), and the DNA sequences analyzed by BLAST
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(Basic Local Alignment Search Tool, National Center for Biotechnology Information, U.S.
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National Library of Medicine, Bethesda. http://blast.ncbi.nlm.nih.gov/Blast.cgi) to identify the
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genus, and when possible, the species of each microorganism.
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Primers and Probes Design
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Thirty-one 18S rRNA fungal sequences were aligned using Clustral-X (43). A 168 bp
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region with high homology between different fungal sequences was selected to design fungal
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primers for the universal qPCR assay. A specific primer set that amplifies a 132 bp amplicon was
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finally selected (Table 1 and Supp., Figure S1). Similarly, 22 archaeal 16S rRNA sequences were
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aligned to verify the specificity of a publicly available universal qPCR primer set for archaea
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(40, 41) that amplifies a 137 bp region (Table 1). A publicly available universal 16S rRNA
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primer set (12) that produces a 147 bp amplicon was used as the basis for the universal qPCR
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assay for bacteria (Table 1). Three different fluorogenic probes for the bacterial, fungal and
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archaeal universal qPCR assays (Table 1) were designed and ordered from Integrated DNA
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Technologies, Inc. (Coralville, IA, USA). The Texas (Red) fluorophore with an emission peak of
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615 was used in combination with the Quencher Iowa Black RQ in the fungal probe while FAM
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(blue) with an emission peak of 517 and Quencher Iowa Black FQ were used for the bacterial
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probe, and for archaea double quenched probe HEX (green) fluorophore with an emission peak
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of 556 and two quenchers one internal ZEN™ quencher (ZEN is a trademark of Integrated DNA
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Technologies, Coralville, IA) and 3’ quencher Iowa Black FQ was used. Polyacrylamide gel
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electrophoresis (PAGE) purified synthetic oligonucleotides spanning the 132, 147 and 137 bp of
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the bacterial, fungal, and archaeal universal qPCR amplicon region, respectively, were
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synthesized (Integrated DNA Technologies Inc., Coralville, IA, USA), serially diluted from
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1x109 to 1x104 gene copies/2µL and used as absolute standards.
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Establishing a TaqMan qPCR Assay To examine the specificity of designed primers, qPCR assays were performed using
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SYBR Green with melting curve analysis. For the SYBR Green qPCR, 2 µL of DNA or
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synthetic oligonucleotide standard was utilized in a 12.5 µl PCR reaction containing 6.25 µl of
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Taq™ universal SYBR® Green Supermix (Cat No. 172-5122, Bio-Rad, CA, USA) and 0.2
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µmol/L of each primer (Supp., Table S2). Quantitative PCR was carried out using a two-step
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amplification procedure with an initial denaturation step at 95 °C for 4 min followed by 40
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cycles of annealing/elongation for 30 s at 58 °C; fluorescence was measured after each cycle.
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After the qPCR amplification run, a melt curve analysis was performed by heating the samples
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from 68 to 95 °C in 0.2 ºC increments.
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The TaqMan qPCR assay was conducted in a final volume of 20 µL using 10 µL of
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iTaqTM Universal Probes Supermix (Cat No. 172-5132, Bio-Rad, CA, USA), 0.2 µmol/L of each
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primer, 0.2 µmol/L of each probe, and 2 µL of either DNA template, synthetic oligonucleotide
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standard or 1:10 dilution of cells and spores recovered from cultures and fuel samples, and heat-
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shock treated using the freeze-and-heat method before qPCR. The simple “freeze-and-heat”
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method comprised freezing samples for 5 min in the -80 °C freezer and then heating the sample
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to 95 °C for 5 min to lyse cells and spores to release DNA. PCR was performed in Bio-Rad CFX
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real-time PCR instrument using the previously explained two-step amplification procedure.
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Three independent biological and technical replicates were included in each qPCR analysis.
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Pearson correlation (r), from R (ggpubr Package), was used to calculate the linear correlation
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between singleplex and multiplex qPCR.
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Synthetic Standards for qPCR The sequences and characteristics of the PAGE purified synthetic oligonucleotide
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standards that used in the current study are presented in Table 1. The oligos were chemically
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synthesized by Integrated DNA Technology using their Ultramer synthesis. Once the synthetic
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oligonucleotide that span the amplicon region of the universal qPCR assays were received, the
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oligo copies were calculated from the nmoles amount. To achieve this, the amount in nmoles
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provided by the vendor is converted to moles and then multiplied by the Avogadro’s constant
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(6.022×1023 molecules−1) to get the number of molecules of the oligo; at this point, the term
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molecules can be substituted by gene copies. The synthetic oligonucleotides are then serially
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diluted using ten-fold dilutions from 1x109 to 1x102 copies/2 µL.
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Characterization of the TaqMan qPCR Assay using Fuel-Derived Microbial Materials The initial validation experiments were carried out using filter sterilized Jet A fuel spiked
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with a known number of microorganisms. A 0.22µm filter was used to sterilize fuel. Similarly,
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contaminated fuel samples collected from different locations across the USA were used for
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validation of the TaqMan qPCR assay. Hormoconis resinae (ATCC 77211) and other fungi were
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grown in Potato Dextrose Agar (PDA) for two weeks. Fungal spores were collected in 10 mL of
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sterilized distilled water and then used as spiking material. The collected fungal spores were
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washed twice with Bushnell Haas (BH) and spore concentration was estimated using a
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hemocytometer (Hausser Scientific, Horsham, PA, USA). For bacterial inoculum preparation,
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Pseudomonas aeruginosa (ATCC 33988) and other bacteria were grown in Luria-Bertani (LB)
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medium overnight at 28 °C, and cells were collected by centrifugation at 11,000 RPM for 10
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min. Bacterial cells were washed twice using BH medium and the concentration of bacteria
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determined by measuring optical density (OD) at 600 nm in the spectrophotometer. The initial
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inoculum level was adjusted to 3x107 cells/mL and 1x106 spores/mL for P. aeruginosa and H.
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resinae, respectively. Twenty mL of BH medium overlaid with 4 mL of Jet A fuel in 50 mL
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polystyrene tubes (note: polystyrene is compatible with middle distillate fuels including diesel
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and jet fuel but incompatible with gasoline and ethanol-blended gasoline) were inoculated either
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with H. resinae or P. aeruginosa, and incubated at 28 °C in a shaker at 200 rpm. Samples were
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collected at 3, 6, 9 and 12 days after inoculation for qPCR and Colony Forming Unit (CFU)
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quantification. A liquid-liquid extraction was performed to recover microorganism from
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hydrophobic fuel phase to hydrophilic aqueous phase. The liquid-liquid extraction protocol
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entailed collecting 500 mL of fuel in a sterile high-density PVDF bottle and adding 10 mL of
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sterile 1X phosphate buffer (1X PBS) followed by vigorous shaking. Samples were allowed to
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stand for 5 minutes for separation of the aqueous phase and fuel phase. Finally, the PBS
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containing microorganisms were aseptically recovered with a serological pipette and transferred
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to a sterile 10 mL conical tube for qPCR as follows. Each contaminated sample was diluted 1:10
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with sterile ultrapure water and subjected to freeze-and-heat protocol to extract DNA from
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samples for direct qPCR amplification. Two µL of each contaminated sample was used as a
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template for qPCR in 20 µL final volume reaction. Serial dilutions of 1:10, 1:100 and 1:1000
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from each contaminated sample were prepared and 100 µL of each dilution was spread on either
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Tryptone Soya Agar (TSA) or PDA plates for enumeration of bacterial and fungal populations,
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respectively. Plates were incubated at 28 °C for 3 days and the number of colonies/mL was
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calculated. Three independent biological and technical replicates were included in each
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experiment. For the field testing, 23 contaminated fuel samples collected from different locations
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across the USA were tested by multiplex qPCR and culture-based methods. Pearson correlation
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(r), from R (ggpubr Package), was used to calculate the linear correlation between different
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factors.
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RESULTS AND DISCUSSION
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Establishing a TaqMan qPCR Assay
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Energy & Fuels
The development of a simple qPCR method for detection and quantitation of the total
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microbial population in fuel is a crucial step toward prevention and mitigation of
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biodeterioration. To advance qPCR as a mainstream method for rapid detection of
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biocontamination in fuel systems, multiple factors need to be addressed including: 1) the qPCR
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test should be able to detect all possible microbial contaminants using the smallest amount of
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sample; 2) the qPCR method should not require purified DNA as test input; 3) the qPCR reaction
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protocol, data analysis, and interpretation should be simple.
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The main objective of this work was to establish a robust multiplex qPCR assay that could be eventually used for on-site detection and quantify total bacteria, fungi, and archaea in fuel
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samples. Toward this goal, we developed a universal microbial qPCR assay for simultaneous
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detection of fungi, bacteria and archaea in a single test reaction. A universal qPCR primer set
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capable of detecting all types of fungi was not publicly available, thus, we started by performing
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sequence alignment of 18S rRNA gene sequences from 31 different fuel-degrading fungal
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species to find a conserved gene region for development of universal fungal qPCR primers and
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probe capable of detecting filamentous fungi and yeasts (Supp., Figure S1). The results showed
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that a 168 bp region of the fungal 18S rRNA was highly conserved. Therefore, we designed a
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forward primer, reverse primer, and a TaqMan probe targeting this region (Table 1). To ensure
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the primers and probe were fully compatible with the target DNA, degenerate nucleotides were
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incorporated at polymorphic positions within the oligonucleotides; the primers and probes were
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named FW-Fungal-Universal, RV-Fungal-Universal, and Probe-Fungal-Universal (Table 1).
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The optimal oligo (i.e. primers and probe) annealing temperature and concentration for increased
22
specificity, amplification rate and total fluorescence were experimentally determined by
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performing temperature gradient qPCR with post-amplification melt-curve analysis using SYBR
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Green dye (Suppl., Table S2).
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We used previously validated universal primers for detection of bacteria and archaea (12, 37,
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38, 39, 40, 41). For bacterial detection, we selected the 16S universal primer pair established by
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Maeda et al., (12). This primer set has been successfully used in the detection and quantification
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of fuel-degrading bacteria using SYBR Green-based qPCR analysis (30, 44-46). For universal
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detection of archaea, the primer set demonstrated by Bayer et al., (41) was selected. The
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published bacterial and the archaeal qPCR primers were designed with degenerate nucleotides to
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account for polymorphisms in different species. The bacterial and archaeal amplicon region was
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used as a reference for TaqMan probe development (Table 1). Table 1 presents the sequences of
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the bacterial and archaeal qPCR primers and probes. The specificity of the bacterial qPCR
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primers and probe was confirmed by BLAST analysis for more than one hundred bacterial
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species including the hydrocarbon-degrading bacteria Nocardioides luteus, Gordonia sihwensis,
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Acinetobacter venetianus, Marinobacter hydrocarbonoclasticus, Rhodovulum sp., Pseudomonas
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aeruginosa, P. putida, P. stutzeri, P. frederiksbergensis, Alcanivoras sp., Bacillus licheniformis,
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and Achromobacter sp. Similarly, the specificity of the archaeal qPCR primers and probe was
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confirmed by BLAST analysis for more than 22 sequences from different archaeal species
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including Methanobacterium ferruginis, Methanobacterium petrolearium, Halorubrum
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halodurans, and many uncultured archaeal species.
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Synthetic oligonucleotide standards spanning the 132 bp, 147 bp and 137 bp amplicon region
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covered by the fungal, bacterial and archaeal qPCR assays, respectively, were developed to allow
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absolute quantification of the sample DNA. This strategy facilitated the generation of precise
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standard curves for each qPCR run that allowed absolute quantification of the copy number of
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16S and 18S rRNA genes in each tested sample. Accordingly, the amplification efficiency of
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each qPCR assay was determined from the standard curves produced from serial dilutions of
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synthetic oligonucleotide standards ranging from 1x104 to 1x109 gene copies/µL (Figure 1). The
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amplification efficiency of the fungal and bacterial universal qPCR assays in singleplex reactions
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was 112.9% and 99.7%, respectively (Figure 1A, B). Similarly, the efficiency of the fungal and
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bacterial qPCR assay was determined from multiplex reactions, which showed efficiency (E) of
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97.9% and 100.8%, respectively (Figure 1C). The linear correlation of standard curves (R2) was
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exceptional with R2 ≥ 0.985 (Figure 1). The fact that multiplexing the assays did not reduce the E
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and the R2 demonstrated the high specificity and efficiency of the development assays.
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Furthermore, the current microbial multiplex qPCR assay demonstrated its suitability to detect
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and quantify all three microbial domains of life (i.e. bacteria, fungi, and archaea) efficiently by
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multiplexing (Figure 2).
15 16 17
Evaluating the specificity and sensitivity of TaqMan qPCR Assay The specificity and sensitivity of the microbial qPCR assay were confirmed by using
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different concentrations (10 pg to 100 ng) of the target DNA, fungal (Byssochlamys sp. isolate
19
BYSS01; 47) or bacterial (P. aeruginosa ATCC 33988; 48), in a high concentration (100 ng) of
20
non-target DNA background. For the bacterial qPCR assay, the non-target DNA was from
21
Byssochlamys sp. and for the fungal qPCR assay, the non-target DNA was from P. aeruginosa.
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Results showed that the microbial qPCR assay was able to detect as low as 10 pg of either fungal
23
(Figure 3A) or bacterial DNA (Figure 3B) in the presence of 100 ng of non-target DNA (Supp.,
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Table S3). Furthermore, the assay showed high specificity with negligible background
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amplification in the negative control containing 100 ng of non-target DNA, which showed after
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cycle 32 (Figure 3). Pearson correlation (r) test indicated a correlation of 1 between template
4
DNA concentrations and gene copy number/mL (Supp., Figure S2) demonstrating the high
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sensitivity of the qPCR assay for very low abundance target DNA in the presence of high
6
background non-target DNA. This high level of specificity and sensitivity is required for
7
accurate detection and quantification of microbiological contamination in complex
8
environmental matrices such as fuel.
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To evaluate the ability of the microbial qPCR assay for detection of a wide range of
10
environmental target microorganisms, the DNA of twenty-three fungal species and twenty-three
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bacterial species (Supp., Table S1) was used as a qPCR template. Results (Figure 4 and Suppl.
12
Table S4) demonstrated the ability of qPCR to detect every fungal (Figure 4A) and bacterial
13
(Figure 4B) species tested. Interestingly, the capacity of qPCR to quantify DNA was not altered
14
by multiplexing all the fungal and bacterial species together (Figure 4C; Suppl., Table S4).
15
Pearson correlation (r) was used to calculate the linear correlation between singleplex and
16
multiplex qPCR. The results showed a strong correlation (r=0.99 and P-value
