A Real-Time PCR Assay Targeting 16S rDNA for Direct Quantification

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Food Targeting: A real-time PCR assay targeting 16S rDNA for direct quantification of Alicyclobacillus spp. spores after aptamer-based enrichment Tim Hünniger, Christine Felbinger, Hauke Wessels, Sophia Mast, Antonia Hoffmann, Anna Schefer, Erwin Märtlbauer, Angelika Paschke-Kratzin, and Markus Fischer J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b00874 • Publication Date (Web): 16 Apr 2015 Downloaded from http://pubs.acs.org on April 22, 2015

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Journal of Agricultural and Food Chemistry

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Food Targeting: A real-time PCR assay targeting 16S rDNA for direct

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quantification of Alicyclobacillus spp. spores after aptamer-based enrichment

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Tim Hünniger1, Christine Felbinger1, Hauke Wessels1, Sophia Mast2, Antonia Hoffmann1,

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Anna Schefer1, Erwin Märtlbauer2, Angelika Paschke-Kratzin1, and Markus Fischer1*

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1

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Grindelallee 117, 20146 Hamburg, Germany,

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2

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HAMBURG SCHOOL OF FOOD SCIENCE; Institute of Food Chemistry, University of Hamburg,

Lehrstuhl für Hygiene und Technologie der Milch, Tierärtzliche Fakultät, Ludwig-

Maximilians-Universität, Schönleutnerstraße 8/219, 85764 Oberschleißheim, Germany

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*Corresponding

author:

Tel.:

+49-40-428384357;

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[email protected]

Fax:

+49-40-428384342;

E-Mail:

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1 ACS Paragon Plus Environment


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ABSTRACT

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Spore forming Alicyclobacillus spp. are able to form metabolites which induce even in small

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amounts an antiseptical or medicinal off-flavor in fruit juices. Microbial contaminations

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could occur by endospores which overcame the pasteurization process. The current

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detection method for Alicyclobacillus spp. can take up to one week due to microbiological

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enrichment.

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In a previous study, DNA aptamers were selected and characterized for an aptamer-driven

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rapid enrichment of Alicyclobacillus spp. spores from orange juice by magnetic separation.

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In the present work, a direct quantification assay for Alicyclobacillus spp. spores was

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developed in order to complete the two-step approach of enrichment and detection. After

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mechanical treatment of the spores, the isolated DNA was quantified in a real-time PCR

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assay targeting 16S rDNA. The assay was evaluated by the performance requirements of the

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European Network of GMO Laboratories. Hence, the presented method is applicable for

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direct spore detection from orange juice in connection with an enrichment step.

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KEYWORD

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real-time PCR, Alicyclobacillus, spores, detection, 16S rDNA, off-flavour, orange juice

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INTRODUCTION

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Due to metabolic products which induce an antiseptical or medicinal off-flavor, the

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detection of Alicyclobacillus spp. is of high importance for fruit beverage industries.1-3

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Although only vegetative cells of this ubiquitous and non-pathogenic microorganisms are

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forming the metabolic products guaiacol (2-methoxyphenol), 2,6-dibromophenol, and 2,6-

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dichlorophenol among others, pasteurization does not eliminate the problem thoroughly.4-7

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Alicyclobacillus spp contaminations remain due to the formation of endospores leading to a

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significant economic problem for juice producers.8-10

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Membrane filtration or similar techniques for enrichment and subsequent detection are

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not applicable for non-clarified juices, so the International Federation of Fruit Juice

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Producers (IFU), the Australian Fruit Juice Association (AJA), and the Japan Fruit Juice

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Association (JFJA) recommend microbiological cultivation.5, 11-13 Due to the low division rates

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of Alicyclobacillus spp. this procedure can take up to one week.

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available kits (e.g. Vermicon AG, Germany) could accelerate the detection time by utilization

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of DNA probes targeting specific genes of Alicyclobacillus spp.. However, incubation times of

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several days are still required for cultivation.

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5, 14

Currently commercial

Alternatives are both antibody- and aptamer-based enrichment techniques, which make

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use of the high specificity and affinity of these acceptors.15,

16

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antibodies is limited by directive 2010/63/EU due to the use of animals during production,

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the aptamer-technology is fully independent of animals.17, 18 Aptamers are single-stranded

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oligonucleotides which are able to form distinct three-dimensional structures and interact

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highly specific with various targets.19-21 The selection and characterization of aptamers with

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an affinity to Alicyclobacillus spp. spores, which could be applicable for the mentioned

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enrichment techniques, has been recently published.22

While the application of



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To complete the two-step method of enrichment and detection, a reliable quantification

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assay is necessary. In the course of this strategy a quantitative SYBR Green real-time PCR

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assay seems to be appropriate.23-25 The vast majority of published real-time PCR methods for

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the detection of Alicyclobacillus spp. are focusing on the detection of vegetative cells, so that

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a time-consuming microbiological enrichment is still necessary.26,

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techniques and utilization of commercially available kits are equally time-consuming.28 In the

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presented work a direct spore quantification method after mechanical treatment without

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microbiological enrichment has been developed.

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Spore preparation

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Because the pathway responsible for off-flavor formation in Alicylobacillus spp. is still

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unknown, the nuclear 16S rDNA gene was chosen as target.29, 30 This gene is also used for

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phylogenetic studies and enables quantification in a low concentration range due to its high

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copy number.31 For validation of the developed real-time PCR assay the performance

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requirements of the European Network of GMO Laboratories (ENGL) were used to gain a

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maximum of reliability.32 The assay was evaluated in terms of correlation coefficient, slope

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of regression line, amplification efficiency, dynamic range, relative repeatability standard

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deviation, limit of detection (LOD), and limit of quantification (LOQ).

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MATERIALS AND METHODS

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Spore Lysis. The spores used in the presented work (Table 1) were from our in-house

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collection and also provided by Dr. B. Schütze, LADR GmbH (Geesthacht, Germany). In order

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to obtain the 16S rDNA, spores were diluted in ddH2O to a total volume of 400 µL, about

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250 mg of glass beads (Ø=0,5mm, Sigma Aldrich Chemie GmbH Munich, Germany) were

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added, and subsequently grinded twice in a TissueLyser (Qiagen GmbH, Hilden, Germany) for

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5 min at 30 Hz. The resulting fragmented spore mixtures were centrifuged for 5 min at

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12000 g to precipitate the glass beads and the spore fragments. The supernatant was used

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directly as template for real-time PCR.

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real-time PCR Design. To design primers, which are suitable for a wide variety of

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Alicyclobacillus spp. strains, the 16S rDNA sequences of various Alicyclobacillus spp. were

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collected from NCBI (National Center for Biotechnology Information, Bethesda, USA).

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Suitable primers (Table 2) were designed using the PrimerQuest® Tool (Integrated DNA

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Technologies Inc., Leuven, Belgium).

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The real-time PCR was performed in 96 well PCR plates (Sarstedt Inc. & Co, Nümbrecht,

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Germany) sealed with sealing tape (Sarstedt Inc. & Co, Nümbrecht, Germany) in a total

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volume of 20 µL. Each PCR reaction mix consisted of the following components: 1x

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DreamTaq buffer (10x, Fisher Scientific-Germany GmbH, Schwerte, Germany), 0,8 mM

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dNTPs (10 mM, Bioline GmbH, Luckenwalde, Germany), 0.25 U DreamTaq-Polymerase

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(0.5 U/µL, Fisher Scientific-Germany GmbH, Schwerte, Germany), 0,2 µM of each primer (16S

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rDNA Fw and 16S rDNA Rv, Integrated DNA Technologies Inc., Leuven, Belgium), 0,3125x

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SYBR Green® I nucleic acid gel stain (10 000x, Fa. Invitrogen GmbH, Karlsruhe, Germany),

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various concentrated spore lysates, and ddH2O filled up to 20 µL. The PCR amplification was

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carried out in an iCycler iQTM5 (BioRad Laboratories Inc., Hercules, USA). The real-time PCR 5 ACS Paragon Plus Environment


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starts with a 5 min denaturation step at 95°C, followed by 35 cycles of denaturation at 95°C

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for 30 s, annealing at 58°C for 30 s, elongation at 72°C for 30 s, and detection at 84°C after

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30 s. The last step is a final elongation at 72°C for 5 min. The fluorescence was excited at

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485 nm, resulting emission measured at 530 nm, and the threshold cycle was calculated

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using the Optical System Software (V 2.0, BioRad Laboratories Inc., Hercules, USA). Real-time

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PCR reaction curves with CT values ˃ 32 were defined as no amplification and CT values ≤ 32

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as positive results together with the expected amplicons. The calibration lines were obtained

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by plotting CT values against the formed logarithm of diluted and prepared spore

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suspensions.

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RESULTS AND DISCUSSION

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Spore Lysis. For the DNA extraction from Alicyclobacillus spp. spores, several spore lysis

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methods were tested, e.g. (i) chemical lysis by using surfactants, acids, and alkali, (ii)

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temperature treatment by using liquid nitrogen, heat, and autoclaving, (iii) various DNA

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extraction kits, (iv) mechanical spore treatment by using glass beads or sonic waves, and (v)

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different combinations of above mentioned techniques.28,

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TissueLyser with glass beads (0.5 mm diameter) and the subsequent centrifugation provides

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a very simple method for a rapid extraction of DNA from spores. Due to the fact that the

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spore lysis was performed in ddH2O, the resulting supernatant is directly useable for the

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real-time PCR assay to quantify Alicyclobacillus spp. spores.22

33-38

The application of a

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real-time PCR Design. The 16S rDNA primers used in the presented work were designed

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regarding A. acidoterrestris DSM 2498 (accession no. AB059675.1, Figure 1). This sequence

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was previously aligned with several A. acidoterrestris strains (e.g. DSM 3922, DSM 12489,

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ATCC 49025, NBRC 106291, NBRC 106287, and NBRC 106295) and other Alicyclobacillus spp.,

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for example A. acidiphilus (DSM 14558), and A. herbarius (DSM 13609). Sequences were 6 ACS Paragon Plus Environment

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obtained from NCBI (National Center for Biotechnology Information, Bethesda, USA) and

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aligned using GENtle (V 1.9.4, University of Köln). The 16S rDNA primers were designed by

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PrimerQuest® Tool (Integrated DNA Technologies Inc., Leuven, Belgium) and possess melting

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temperatures of 61.5 and 62.1 °C (Table 2). After optimization of the PCR assay 58.0 °C

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seems to be the optimal annealing temperature resulting in an amplicon with a length of 143

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bp (Figure 1). To avoid detection of primer dimers and PCR artifacts, fluorescence monitoring

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was performed at 84 °C.39 By this temperature the detection of byproducts is suppressed,

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resulting in an overall increased sensitivity of the PCR assay (Figure 2).40

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The DNA amplification was monitored by using the intercalating fluorescence dye SYBR

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Green® I. These real-time PCR assays have various advantages such as high dynamic range

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for quantification and superior reproducibility.24,

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used as template, SYBR Green® I enables sensitive and precise quantification results (Figure

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3 and Table 3).43 Moreover, it is also useful for high throughput screening in incoming

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control of fruit juice industry due to robustness, practicability, high standardization

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potential, and running expenses.32, 44

41, 42

Even though crude spore DNA was

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real-time PCR Evaluation. The performance requirements of the European Network of

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GMO (genetically modified organisms) Laboratories (ENGL) were used to evaluate the real-

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time PCR for quantification of Alicyclobacillus spp. spores in terms of correlation coefficient,

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slope of regression line, amplification efficiency, dynamic range, relative repeatability

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standard deviation, limit of detection, and limit of quantification.32, 45

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For the real-time PCR evaluation spore concentrations from 8.45∙102 to 8.45∙106 cfu/mL

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of a strain mix (A. acidoterrestris DSM 2498, A. acidoterrestris wild type, 1/1, v/v) were used.

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To increase the dynamic range of spore quantification, the lysed spore suspension of

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8.45∙102 cfu/mL was further diluted in 10-fold serial steps to a theoretically spore 7 ACS Paragon Plus Environment


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concentration of 0.845 cfu/mL. Mentioned dilutions of the applicable DNA templates were

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possible due to the high copy number of the 16S rDNA.29-31

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During the extensive validation of the real-time PCR assay a dynamic range of

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Alicyclobacillus spp. spores concentrations from 8.45 to 8.45∙105 cfu/mL was established

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(Figure 3 and Table 3). The evaluation of higher spore concentration than 8.45∙105 cfu/mL is

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not necessary because it can be assumed that these concentration ranges never occur in

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orange juice samples or could be bypassed by dilution.

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A correlation coefficient (R2) of 0.983 was determined by linear regression analysis (Figure

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3B and Table 3) which fulfills the performance requirements of the ENGL (according to ENGL

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the correlation coefficient should be ≥ 0.98). Hence, this evaluation parameter confirms a

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linear correlation and therefore justifies the usage of a linear fit.32 In addition, the slope of

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the regression line (m) of -3.42 (Figure 3 and Table 3) fulfills the performance requirements

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of the ENGL (-3.1 ≥ m ≥ -3.6) and is therefore considered as appropriate. Moreover, the

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amplification efficiency (E) was calculated by use of the regression line slope (formula: E=10-

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1/m

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which is also within the requirements of ENGL (Figure 3 and Table 3).

-1). For the developed real-time PCR assay an efficiency of approx. 96 % was achieved,

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The relative repeatability standard deviation (RSDr) of the assay results indicates the

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reproducibility of the developed real-time PCR assay and should be ≤ 25 % over the whole

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dynamic range of the developed method according to ENGL.23 Moreover, the relative

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repeatability should be determined by a sufficient number of test results (at least 15) as

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indicated in ISO 5725-3.32, 46 During the whole assay development the relative repeatability

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standard deviation was in the range of 3.0 - 15.4 %. This is in accordance with the ENGL-

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criteria and was obtained by considerably more than 15 approaches per amplification point

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(Figure 3 and Table 3). 8 ACS Paragon Plus Environment

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Due to the above mentioned aspect of unknown target concentrations after aptamer-

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based spore enrichment, one of the ENGL criteria (limit of detection, LOD, should be less

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than 1/20th of target concentration) is not determinable. But according to ENGL the LOD can

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also be determined by ensuring that ≥ 95 % of false negative results are detectable. Under

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the assumption that CT values ˃ 32 are blank samples , this evaluation parameter could be

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fulfilled.15 Therefore, the limit of detection of the present real-time PCR assay is below the

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dynamic range of quantification. A further equimolar dilution of spores would lead to a

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concentration ˂ 1 cfu/mL which is not relevant for the applicaYon. However, to indicate a

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theoretical limit of detection the LOD was calculated by using a CT of 32. Thus, the

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theoretical detection limit value is 0.28 cfu/mL.

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According to ENGL, the limit of quantification (LOQ) should be less than 1/10th of the

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target concentration which is also not determinable due to mentioned aspect of unknown

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target concentrations after spore enrichment. However, this spore concentration can be

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determined in consideration of the mentioned and defined performance requirements of

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the ENGL. Moreover, a further LOQ criterion (≤ 25 % RSDr) is achieved for the estimated

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spore concentration of 8.45 cfu/mL. Nevertheless, the statistical probability to receive

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precise results in a lower range of spore concentration is significantly decreased and leads to

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CT values beyond the RSDr range of ˃ 25 %.

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In addition, the selectivity of the developed real-time PCR was assessed by an end-point

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PCR experiment and analysis by agarose gel electrophoresis (AGE). Due to the final

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application in conjunction with an aptamer-based spore enrichment for a rapid and direct

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proof and quantification of Alicyclobacillus spp. spores in orange juice, the selectivity

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towards several for the fruit juice industry relevant spores is especially important. In this

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context 13 spores of various Alicyclobacillus spp. DSM strains, 34 Alicyclobacillus spp. wild 9 ACS Paragon Plus Environment


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types, five Bacillus cereus, one B. weihenstepanensis, two B. thuringiensis, one

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B. licheniformis, one B. subtilis, one B. coagulans, one B. polymyxa, one B. circulans, one

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B. sphaericus, and two Clostridium perfringens strains were analyzed (Table 1). All 47

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Alicyclobacillus spp. spore strains yielded the calculated 143 bp amplicon whereas neither

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Bacillus spp. nor C. perfringens spore strains were amplified.

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In general, the developed method is suitable for spore quantification subsequent to an

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affinity enrichment procedure for instance from orange juice. Due to the intended spore

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elution with water after the enrichment process, the determination of spore suspensions in

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water is required. Moreover, this simple approach enables the variation of spore lysis

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volumes during the spore elution after the enrichment step. In summary, the aim of the

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whole technique includes (i) the aptamer-based enrichment step by magnetic spore

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separation with (ii) consecutive determination of Alicyclobacillus spp. spore concentration by

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real-time PCR.

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As described by Hünniger et al., the selected aptamers possess a substantially high affinity to

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Alicyclobacillus spp. spores and a rather low affinity to Bacillus spp. or Clostridium spp.

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spores (Figure S3, Supporting Information).22 In the end this given selectivity is substantially

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increased by the presented PCR which allows the distinction between Alicyclobacillus spp.,

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Bacillus spp. and Clostridium spp.. However, if further non Alicyclobacillus spp. strains will be

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identified in orange juices in the future their sequences have to be further characterized in

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order to assess the selectivity of the test system developed in this study.47

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On the whole, the combination of the prior spore collecting step by immobilized aptamers in

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combination with the very selective real-time PCR assay developed in this study appears as

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the scientific basis for the development of a rapid and reliable incoming control in the


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beverage plant. In an upcoming study the whole system will be assembled, evaluated and

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assessed using real and spiked orange juice samples of different origins.

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SUPPORTING INFORMATION

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FUNDING

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This research project was supported by the German Ministry of Economics and Technology

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(via AiF) and the FEI (Forschungskreis der Ernährungsindustrie e. V., Bonn, Germany). Project

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AiF 17245 N.

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ACKNOWLEDGEMENT

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The authors gratefully acknowledge Dr. Burkhard Schütze (LADR GmbH, Geesthacht,

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Germany) for the provision of various Alicyclobacillus spp. strains (spores and vegetative

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cells) and the Verband der deutschen Fruchtsaft-Industrie e.V. (Bonn, Germany) for support.

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LIST OF FIGURE CAPTIONS Figure 1: 16S rDNA gene sequence of Alicyclobacillus acidoterrestris (DSM 2498; accession no. AB059675.1, position 821 - 1000 with the designed primers (16S rDNA Fw and 16S rDNA Rv) marked by arrows. Figure 2: Melting temperature analysis of two different prepared spore concentrations (8.45 cfu/mL and 8.45∙105 cfu/mL.). Figure 3: (A) Example of observed reaction curves during real-time PCR validation with different spore concentrations (cfu/mL). Spore concentrations from left to right: 8.45∙105 cfu/mL, 8.45∙104 cfu/mL, 8.45∙103 cfu/mL, 8.45∙102 cfu/mL, 8.45∙101 cfu/mL, and 8.45 cfu/mL. (B) Resulting calibration line during real-time PCR validation with E=96 % (m=-3,42) and R2=0,983. Spore concentrations from left to right: 8.45 cfu/mL, 8.45∙101 cfu/mL, 8.45∙102 cfu/mL, 8.45∙103 cfu/mL, 8.45∙104 cfu/mL, and 8.45∙105 cfu/mL.



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FIGURES AND TABLES

Figure 1


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Figure 2



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Figure 3


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Table 1: Alicyclobacillus spp., Bacillus spp., and Clostridium spp. spores used for selectivity determination of real-time PCR.

Alicylobacillus spores

Bacillus spores

A. acidoterrestris (DSM 2498)

5x B. cereus (wild types)


B. weihenstepanensis (wild type)


B. thuringiensis (ATCC 10792)


B. thuringiensis (DSM 6029)

A. cycloheptanicus (DSM 4005)

B. licheniformis (DSM 13)


B. subtilis (DSM 2109)


B. coagulans (ATCC 7050)

A. acidocaldarius (DSM 446)

B. circulans (ATCC 9966)

A. herbarius (DSM 13609)

B. sphaericus (ATCC 245)

A. acidiphilus (DSM 14558)

Paenibacillus polymyxa (ATCC 10401)

A. pomorum (DSM 14955) A. sacchari (DSM 17974) A. fastidiosus (DSM 17978)

Clostridium spores 2x C. perfringens (wild types)

34x undefined wild types



Page 24 of 26

Table 2: 16S rDNA primers and corresponding PCR product of the real-time PCR assay.

Name

Sequence (5‘-3‘)

16S rDNA Fw

CGAAGGAAACCCAATAAGCAC

16S rDNA Rv

GGATGTCAAGCCCTGGTAAG

Length (bp)

Tm (°C)

% GC

839-859

21

61.5

47.6

962-981

20

62.1

55.0

Pos.

PCR product (bp)

143


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Table 3: Summary of evaluation parameters of developed real-time PCR validation according to the European Network of GMO Laboratories. evaluation parameters according to ENGL

evaluation parameters of developed real-time PCR

-

8.45 - 8.45∙105 cfu/mL

slope of regression line

-3.1 ≥ m ≥ -3.6

-3.42

amplification efficiency

110 - 90 %

96 %

correlation coefficient

≥ 0.98

0.983

relative repeatability standard deviation

≤ 25 %

3.0 - 15.4 %

limit of quantification (LOQ)

-

8.45 cfu/mL

limit of detection (LOD) (calculated by regression line)

-

0.28 cfu/mL

dynamic range



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TOC graphic


A Real-Time PCR Assay Targeting 16S rDNA for Direct Quantification

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