SAS 2009 – IEEE Sensors Applications Symposium New Orleans, LA, USA - February 17-19, 2009
Design of specific DNA primers to detect the Bacillus cereus group species Catherine Adleyb*, Khalil Arshaka, Camila Molnarb, Kamila Oliwab, Vijayalakshmi Velusamya, a
b
Electronic and Computer Engineering Department, University of Limerick, Limerick, Ireland Microbiology Laboratory, Department of Chemical and Environmental Sciences, University of Limerick, Limerick, Ireland *
[email protected]
Abstract— One of the most prevalent pathogens that cause foodborne outbreaks is the Bacillus cereus (B. cereus) group species (spp.) generally found in different types of food. Recently many researchers are focusing towards the progress of rapid methods to detect foodborne pathogens. Every year many innovative methodologies for bacterial detection are being developed to improve sensitivity and speed of detection. DNA biosensor is one of the proposed solutions for the detection of foodborne pathogens which can be used for food quality assurance. This paper reports that the DNA sequences named BCFomp1/BCRomp1 can be used for the specific detection of the B. cereus group spp. Analysis of these primers using standard PCR analysis showed that the minimum level of detection was 103 CFU/ml. The lowest number of bacterial cell per reaction tube that can be amplified was 5 CFU and the minimum quantity of DNA that can be amplified was found to be 1pg. Keywords-The B. cereus group spp.; foodborne pathogen detection; DNA; primers;PCR; biosensor.
I. INTRODUCTION Foodborne diseases are a worldwide growing health problem involving a wide spectrum of illnesses caused by microbial, viral, parasitic or chemical contamination of food. Food diarrhoeal diseases can lead to serious illnesses and in some cases, leads to death. Some diseases are caused by toxins from the “disease-causing” microbe, others by the human body’s reactions to the microbe itself. Apart from Clostridium botulinum, Campylobacter jejuni, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Spp., Shigella Spp. the most frequently isolated bacterial foodborne pathogens are the Bacillus cereus group species [1] which include: B. cereus, B. mycoides, B. pseudomycoides, B. weihenstephanensis, B. thuringiensis, B. anthracis [2, 3]. Growth of B. cereus, results in production of several highly active toxins. Therefore consumption of food containing >106 bacteria/gm may results in emetic and diarrhoeal syndromes. The most common source of this bacterium is found in liquid food products, milk powder, mixed food products and is of particular concern in the baby formula industry [4]. However they can also be found in other foods such as turkey, beef, rice and noodles. The emetic toxin type is also known to grow well in mashed potatoes, rice dishes and vegetable sprouts [5]. It has been reported in EU legislation on microbiological criteria for foodstuffs, that “foodstuffs should not contain
micro-organisms or their toxins or metabolites in quantities that present an unacceptable risk for human health”, as laid down in Regulation (EC) No 2073/2005 [6]. Recently, the World Health Assembly (WHA) established a global surveillance system for public health emergencies of international concern by adopting the International Health Regulations (IHR) on 23 May 2005 which came into force on 15 June 2007 [7]. All these current legislations on food and health provide incentives for research in the area of food pathogen detection. Therefore, importance is given to develop a DNA biosensor for the rapid detection of bacterial pathogens in food. We designed the DNA primers which is specific to the B. cereus group spp. Materials and methods used for the design of the primers are demonstrated, and the results discussed. II. MATERIALS AND METHODS
A. Conventional method of identification of B. cereus spp.
detection
and
Selective microbial culture plating methods for B. cereus group spp. are based on: bacterial growth in presence of the polymyxin B antibiotic, precipitation of hydrolysed lecithin and the inability to utilize mannitol (PEMBA-B. cereus selective agar medium, OXOID) [8, 9]. This microbial group is able to degrade starch molecules (hydrolyze of starch agar) [10] or to hemolyse red blood cells (Columbia blood agar) and grow in the presence of penicillin G antibiotic [11]. The B. cereus spp. colonies grown on a PEMBA agar (OXOID) and starch agar are shown in Fig.1.
(a)
(b)
Fig. 1. Bacillus cereus colonies grown on a (a) PEMBA agar (OXOID); (b) Starch agar
Fig.1(a) depicts the B. cereus colonies grown on a PEMBA agar (OXOID). The bacterial colonies appear turquoise blue due to their inability to ferment mannitol (carbon source in the
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media) which in turn increases the pH of the agar [8]. Fig.1(b) shows the Bacillus cereus colonies grown on Starch agar. The B.cereus group spp. are able to produce extracellular enzyme which degrades starch molecules. In Fig.1 (b), there is a clear zone around the culture on the left which indicates the ability to hydrolyse starch. The strain on the right (B. cereus BCF4) is used as a negative control and is blue [10]. The BioMerieux API 50CH is a standardized identification system, which uses biochemical tests for the study of the metabolism of these microorganisms. The API system consists of a plastic strip of individual, miniaturized test tubes containing different reagents used to determine the metabolic capabilities. During the incubation period, colour changes are observed within the tubes, what reveals the fermentation caused by the anaerobic production of acid and detected by the pH indicator in the chosen medium.
B. PCR based method of detection and identification of B. cereus spp. Cross-species polymerase chain reaction (PCR) primers were designed and conserved regions were identified from the alignment as potential cross-species primer-designing sites. The selected sequences were aligned using the multiple sequence alignment program ClustalW [12]. Alignments carried out with sequences in public databases revealed no matches other than those for sequences of the gene encoding the B. cereus group motB. BLAST (Basic Local Alignment Search Tool – a bioinformatics program that uses an algorithm for comparing primary biological sequence information), was used to check the specificity of the motB primer set. In addition, the targeted sequences of four B. cereus strains (GenBank accession numbers NC003909; NC004722; AAEK00000000; NC006274), one of B. thuringiensis (GenBank accession numbers: NC005957), and three of B. anthracis (GenBank accession numbers: NC007530; NC005945; NC003997) were aligned in order to verify the group specificity, which is shown in Fig.2.
Fig.2. Cross-species primer design in the conserved region for motB gene of B. cereus, B. thuringiensis and B. anthracis M 105 104 103 102 101 N
of
multiple
sequences
alignment
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III. RESULTS AND DISCUSSION Based on bioinformatic analysis of the B. cereus group spp. NCBI data bases, highly conserved gene within this group was found which was chosen for the specific detection of the B. cereus group spp. The gene motB which encodes for the outer membrane protein has been selected and crossspecies PCR primers (BCFomp1/BCRomp1) were designed to be used as a potential target for PCR bacterial identification. Using those primers, 76 strains of B. cereus group species were tested (except B. anthracis strains). All screened B. cereus group strains showed positive results. The size of PCR product was 575 bp as expected. BCFomp1 and BCRomp1 primers are group specific and do not react with DNA from other Bacillus and non-Bacillus species. To confirm the negative results, the primers were also tested with 13 Bacillus and non -Bacillus species. Analysis of these primers using standard PCR analysis showed that the minimum level of detection was 103 CFU/ml (Figure 3). The lowest number of bacterial cell per reaction tube that could be amplified was then 5 CFU. The minimum quantity of DNA that was amplified was 1pg.
1
600bp 500bp
2
3
4
5
575bp
Figure 3: Sensitivity of PCR detection of B. cereus with primers BCFomp1/BCRomp1. Lane 1- molecular marker (Bioline); Lane 2- negative control; Lane 3- non-Bacillus strain (Listeria monocytogenes LMF1); Lane4- Bacillus cereus NCTC 7464; Lane 5- Bacillus weihenstephanensis DSM 11821.
V. DNA BIOSENSOR Biosensors create new opportunities in the food industry sector by forming new standards for microbial monitoring. Biosensors provide the opportunity for rapid, in situ tests that can replace lengthy laboratory assays. They can be designed to monitor and quantitate a number of biological and biochemical effects as a real-time detection system [13]. Generally, DNA biosensors are constructed by the immobilization of the oligonucleotide sequence (probe) onto a transducer that is able to convert the biological event into measurable signal [14]. Sometimes the probe can be free in a solution but in all cases the principle of specific
hybridization, between single stranded DNA (ssDNA) known as the probe and “target” sequence which must be detected, plays a key role in DNA-based biosensors [15]. The converted signal is a response to hybridization of the probe and target sequence can be optical, electrochemical or pizoelectrical [16, 17]. Every year many innovative methodologies for bacterial detection are being developed to improve sensitivity and speed in detection. In particular, focus was aimed at the PCR methodologies, including multiplex and real-time PCR analysis as an answer to the traditional culture based methods, which required 5-7 days for microbial detection. Similarly, the solid phase microarray format emerged as the preferred method for high throughput, highly-parallel hybridization testing for DNA and RNA samples. Currently available microarray technology suffers from certain limitations that prohibit the exploitation of the full range of life science applications. The outcomes are not promising; the majority of the methods developed require enrichments in culture media before the analysis can begin. Problems associated with the established fluorescence-based optical detection technique include the high equipment costs and the need to use sophisticated numerical algorithms to interpret the data [18]. A DNA biosensor is one of the proposed solutions for the detection of foodborne pathogens. Over the past decade research efforts have advanced the development of DNA biosensors but improvements are still needed before DNA biosensors become real and reliable choice. In our future work, attention will be focused on developing a biological sensor to detect and monitor the B. cereus group spp. which is commonly found in milk products and the bioterrorist agent Bacillus anthracis. Individual sensors will be based on the unique DNA signature of each member of the group. Based on sensor identification of these pathogens, it will be possible to detect them directly from the food source. A biosensor based on the primers BCFomp1/BCRomp1 will be used to identify a unique signature (characteristics specific for the B. cereus group spp.) with more accuracy. It will allow for the identification of this group only among all the different species which may be present in milk products. Other objectives include designing specific primers which will be used to distinguish between the different species within the B. cereus group spp. VI. CONCLUSION Our work has shown that DNA sequences named BCFomp1/BCRomp1 can be used for the specific detection of the B. cereus group spp. Low detection limit, high sensitivity and rapid speed of detection still remains challenging. Our future work will be focused to develop a portable/handheld sensor technology that can detect the B. cereus group of spp, thereby facilitating accurate assessments of possible risks from food constituents or contaminants in consumer diets and the bioterrorism organism B. anthracis. Improving the sensitivity, selectivity, speed, simplicity and reducing the cost of food monitoring assays are important goals. It will therefore address the concerns of the European Food Safety Authority (EFSA) and the US FDA. It will enable food producers to more readily meet and adhere to food hygiene legislations through self monitoring by providing a novel platform for faster detection of food-borne pathogens.
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ACKNOWLEDGEMENTS This research work is funded by Science Foundation Ireland (SFI) Research Frontiers Programme, ID no: 07RPF-ENEF500. Camila Molnar was funded by Serosep Ltd. Limerick, Ireland. REFERENCES [1] [2]
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