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Indian Journal of Medical Microbiology, Vol. 29, No. 4, October-December, 2011, pp. 372-378 Original Article Detection of anthrax toxin genetic sequences by the solid phase oligo-probes KC Addanki1, M Sheraz1, K Knight1, K Williams1, DG Pace2, O Bagasra1 1 South Carolina Center for Biotechnology, Claflin University, Orangeburg, SC 29115, USA Date of Submission: 06-Jul-2011 Code Number: mb11091 PMID: 22120797 Abstract Purpose: There is an urgent need to detect a rapid field-based test to detect anthrax. We have developed a rapid, highly sensitive DNA-based method to detect the anthrax toxin lethal factor gene located in pXO1, which is necessary for the pathogenicity of Bacillus anthracis. Keywords: Biological warfare, biological weapon, class A pathogens, microRNA, RNAi, terrorism, toxin Introduction Threats of bioterrorism have prompted renewed research interest in the development of rapid and sensitive assays for the detection of Bacillus anthracis, which is of particular concern for bioterrorism, particularly in light of post 9/11 anthrax attack that caused several casualties. [1],[2],[3],[4] Because of the severity of the illness, the ease of respiratory infection, and the extreme resistance of the spores to unfavourable environmental conditions, B. anthracis is considered a potential biological warfare agent, [1],[2],[3],[4] and in recent years, the need for novel, reliable and rapid diagnostic means have increased. [5],[6],[7],[8],[9],[10],[11] B. anthracis is the causative agent of anthrax. It is a gram-positive spore-forming bacterium that rarely causes a fatal disease in ordinary situations. [1],[2],[3],[4],[5] The three toxin polypeptides encoded by the B. anthracis pXO1 plasmid combine in binary combinations to form two toxins, lethal toxin (LT) and edema toxin (ET), which are responsible for the lethality and symptoms associated with anthrax. [1],[2],[3],[4],[5] Although the pXO2 plasmid-encoded capsule is an important virulence factor for the establishment of disease, the symptoms associated with anthrax are the result of the two-toxin production, following exposure to the spores. Thus, antibiotics that clear bacteria from infected hosts cannot protect against the toxic effects of LT and ET after they have been produced. [1],[2],[3],[4],[5],[6],[7] However, in this case passive immunization is administered. [5] Currently, the major concern with B. anthracis is its potential use as a bioweapon because the weaponized form of the spores can be quickly inhaled upon release. [1],[2],[3],[4],[5],[6],[7] Upon inhalation, B. anthracis spores enter the alveolar spaces, taken up by alveolar macrophages, and germinate into vegetative bacilli which eventually invade the bloodstream where they multiply on a massive scale and secrete both LT and ET. The genes that encode the two exotoxin components are located on the native virulence plasmid pXO1. Genes that encode proteins with functions involved in the synthesis of the second major B. anthracis virulence determinant (an immunologically inert polyglutamyl capsule that protects bacteria from phagocytosis) are located on a second native virulence plasmid, pXO2. [1],[3],[6] The current recommendation for prophylaxis of persons exposed to aerosolized anthrax is treatment with antibiotics (ciprofloxin) for 8 weeks, in the absence of vaccine, or 4 weeks and until three doses of vaccine have been given. [1],[2],[3],[4],[5] The amount of antibiotics required for post-exposure prophylaxis of large populations could be enormous and could easily overwhelm emergency response teams and extend beyond the capacity of those responsible for consequence management. [1],[2],[3],[4],[5] In addition, the development of side effects, the promotion of resistant strains and the encouragement of other socio-economic externalities can be significant. The major aim of our research was to develop a highly sensitive and specific method of anthrax detection which would be able to identify toxin. We believe that in the case of a national emergency there is a strong possibility that a weaponized anthrax could be a chimera or bioengineered agent where the antibody-based assay may not be useful or give correct results. [6],[7],[8],[9],[10],[11] Therefore, we devised a DNA-based method that targets the detection of highly conserved portions of the LT that would be required for pathogenicity, which portions would be present even in a genetically engineered agent or atypical agents. [4],[12] Therefore, we selected a highly conserved DNA sequence in the LT: Lethal factor endopeptidase sequences located in pXO1, which must be present in order for the anthrax to be an effective bioweapon. [1],[2],[3],[4],[5] Here, we describe this new DNA solid phase approach to detect very low amounts of the anthrax LT. Materials and Methods Probes, oligonucleotides and reagents The DNA-BIND 96 well Plates were purchased from Corning Inc. All the oligonucleotides were purchased from Sigma Genosis (St. Loius, MO, 63103USA). All the oligonucleotides were custom manufactured according to our specifications and purified by gel electrophoresis. All the chemical reagents were purchased from Sigma-Aldrich, Inc., St. Louis, MO, 63103, USA. All the chemicals were molecular biology grade. Preparation of capture probe In a 1.5 mL centrifuge tube, amine modified oligonucleotide (i.e., Capture probe) was added into an oligo-binding buffer (50 mM Na 2 PO 4 , pH 8.5, 1 mM EDTA) in a ratio of 1:100, respectively, or 100 pMoles/well. Preparation of linker In a 1.5 mL centrifuge tube, amine modified oligonucleotide (i.e., Linker) was added into a hybridization solution (5X saline-sodium citrate (SSC), 1.0% casein, 0.1% N-lauroylsarcoine, 0.02% sodium dodecyl sulfate (SDS)) in a ratio of 1:100, respectively, or 100 pMoles/well. Preparation of biotinylated probe In a 1.5 mL centrifuge tube, amine modified oligonucleotide (i.e., Biotinylated probe) was added into a hybridization solution at a ratio of 1:100, respectively, or 100 pMoles/well. Colorimetric detection A volume of 100 mL per well of Capture probe was added to the DNA-BIND plate and incubated for 1 h at 37°C. Following the incubation, the wells were washed three times with phosphate buffer saline (PBS). A volume of 200 mL per well of blocking solution (3% Bovine serum albumin (BSA) in oligo binding buffer) were added and incubated for 30 min at 37°C, then decanted. Then, 200 mL/well of hybridization solution containing both the linker and biotinylated probes in a 1:1 ratio were added. The plate was heated on a Thermal Cycler using the following protocol: Step 1 = 80°C for 30 s, Step 2 = Ramp 1.0°C/s to 45.0°C, Step 3 = 45.0°C Hold. The wells were then washed with preheated (45°C) wash solution (2X SSC, 0.1% SDS) twice and soaked for 5 min on the third wash. A volume of 100 mL per well of Streptavidin-peroxidase diluted 1:1000 in an assay diluent was added and incubated for 30 min at 37°C. The wells were then washed three times with PBS, and 100 mL/well of a substrate was added. The optical density (OD) reading was read every 10 min for 1 h. The schematic and sequences of the capture, Linker and detection oligonucleotides are shown in [Figure - 1]. The wells were organized as follows: Empty well, capture probe only well, linker only well, Biotinylated probe only well, and capture probe plus linker well all served as negative controls while the capture probe, linker and biotinylated probe well served as the testing well. An additional well was utilized to determine whether doubling the linker probe would render a significant positive outcome compared to the initial positive control well. This was initiated to determine whether increasing the linker added to the positive control well would elevate the chance of colorimetric indicator detection. The detail sequences of oligonucleotides are shown in [Table - 1]. Results The colorimetric detection was performed using a 96-well plate coated with the DNA annealing and hybridization chemistry necessary for directly retaining the capture probe and indirectly retaining the linker and biotinylated probe. The testing process employed using multiple wells to test and verify that colour was obtained only in the event that all of the oligonucleotide constructs annealed in a well as illustrated in [Figure - 1]. Samples were run in duplicates and repeated three times. Also, the variation of the pH of the blocking solution was devised to determine whether the efficiency of this solution would vary when compared to known data on subsequent experimentations. The noted steps for colorimetric detection were adhered to when testing the various samples. The probe combination of CP1, Linker16, BP6; CP2, Linker25, BP5; and CP3, Linker34, BP4 were used in order to project the best oligonucleotide construct for detecting the toxin DNA sequence found in anthrax bacterium. There was a faint blue colour observed in the negative control wells that is a common observation in this kind of colorimetric assay, whereas a strong blue colour was observed in the test wells. The development of colour was allowed to form over a period of 60 min with reading taken at an interval of 10 min. The initial positive colour indication was observed at 10 min; the wells consisting of the negative controls still indicated a non-determinant to faint colour verification of the colorimetric detection assays. As shown in [Figure - 2], when we utilized capture probe, linker probe, biotinylated probe or linker + biotinylated probes, lower signals were detected as compared to the complete detection cascade that was necessary to identify the anthrax toxin LT DNA fragment. This was most evident in the results obtained for the construct of CP2, Linker25, BP5, indicated by the colour blue in [Figure - 2] which confirms the colorimetric results. The enzyme-linked immunosorbent assay (ELISA) confirmed elevation of the colorimetric results for the negative controls was observed both electronically and visually over the 60 min trial period. Analysis of the colorimetric assay for all utilized wells was performed using the solid-phase ELISA detection system for absorbance wavelengths of 405 nm, 450 nm, 492 nm, 540 nm and 620 nm. The results show that the negative controls did not give a strong signal on the ELISA-based detection system at 492 nm and after 60 min. When determining the best wavelength in which to analyze the wells, we consulted the known visible colour spectrum in conjunction with the intended colorimetric indicator requested from the manufacturer. Since the known colour indicator for our assay was designed to be a variant of blue, we determined that the absorbance of this indicator would best be detected at 492 nm. Adhering to the same protocol illustrated in the ′Materials and Methods′ section, an additional comparison of the probes was performed but with a change in the pH of the blocking solution. The results for this deviation from the standard protocol are provided in [Figure - 3]. In [Figure - 3], there is a clear difference in the results obtained for each of the oligonucleotide constructs tested among one another and compared to those of [Figure - 2]. Once again, the construct of CP2, Linker25 and BP5 was clearly the best indicator of the presence of the LT toxin gene fragment presence. Another side effect of the decrease in pH of the blocking solution revealed a decrease in the indicated absorbance of the negative controls across all of the oligonucleotide constructs. However, the positive controls still displayed a favourable indication of complete oligonucleotide construct annealing when coupled with the colorimetric indicator. The change in pH of the blocking solution appears to have a downgrading effect on the absorbance values for the negative and positive controls in [Figure - 3]. There is a 0.1 to 0.2 decrease in absorbency for the positive control well consisting of any oligonucleotide construct for CP#, Linker#, and BP#. Although the decrease in absorbance values for [Figure - 3] is observable, the oligonucleotide construct of CP2, Linker25, and BP5 demonstrates its potential for being the best indicator of the presence of toxins for anthrax due to its substantial indication of absorbance. The complete system for colorimetric detection (i.e., capture probe, linker, and Biotinylated probe well) shows significant increase (P>0.001) in the identification of the genetic sequence from the successful combining of reactant components compared to the negative controls for both experiments. Discussion Currently, the most common method to detect anthrax is the sandwich-based ELISA that utilizes monoclonal, polyclonal antibodies or their combinations. [10] In recent years, several excellent antibody ELISA-based methods have been developed that are highly sensitive and specific for anthrax. For example, Tang et al. [10] have described a europium nanoparticle-based immunoassay for the sensitive detection of anthrax protective antigen (PA). The assay reportedly exhibited a linear dose-dependent pattern within the detection range of 0.01 to 100 ng/mL, and was approximately 100-fold more sensitive than the ELISA more commonly used for the PA detection. [11] The major disadvantages of antibody-based immunodetection methods are twofold: (1) they target protective antigen that is not necessary for the toxicity of anthrax and can be deleted from a bioengineered anthrax, and (2) with the advent of biotechnology and genetic engineering, it is unlikely that a bioweapon deployed in a civilian population would bear any resemblance to the natural agent. It would most likely be a chimera agent carrying a pseudo envelope of a common agent, and the genetic material of a highly pathogenic agent. In this scenario, the antibody-based ELISA assay may not be useful, or it may detect the wrong agent. Therefore, we believe that the results presented in this research article may have a much wider application in bioweapon detection than may appear from a cursory examination of the article′s content. We have adopted a versatile DNA-based solid phase detection system that also utilizes a sandwich assay system that can identify the precise genetic sequences to any agent. The main advantage of our method is that it can essentially identify any natural or genetically engineered biological agent. As mentioned above, we believe that in the case of bioterrorism it is highly unlikely that a naturally occurring type of anthrax would be utilized. The weaponized versions of anthrax are already known to be genetically engineered, and the bacterial spore surface is modified so that it is not sticky to create maximum dispersion of spores in an aerosolized formation. [1],[2],[3],[4],[5] However, it is most likely that the natural toxins would be modified enough to make them more toxic. [13] In this case, the antibody-based assays may prove useless. [8],[9],[12] A toxin-based terroristic attack may cost many lives, increase public fear, and erode the trust in health care personnel. Previously, Kumar et al. [14] have described a sensitive multiplex PCR method to detect the virulence factor genes located on two plasmids, pXO1 and pXO2 of the bacterium. However, current multiplex PCR methodology is time consuming, and may not be suitable for rapid detection field utility. [14] Biagini et al. [15] have also described a highly sensitive immunoassay utilizing lateral flow immunochromatographic device that can detect as low as 3μg/mL of anti-anthrax antibodies. Carter et al. [16] have described a lateral flow nucleic acid based miniaturized detection method that uses only 10μL of fluid that can detect as low as two bacteria. Very recently, Oh et al. [17] have reported a biosensor based detection system that utilizes fluorescent europium-modified polymer nanoparticles that can detect as low as 10 pM of dipicolinic acid, the anthrax spores′ outer coat component. However, the same compound is found on almost all the Bacillus species spires and makes this rather attractive method non-specific. Baeummer et al. [18] have described a membrane-strip-based biosensor assay that combines nucleic acid sequence-based PCR assay that can detect as low as ten the anthrax bacteria. The problem is the PCR that takes several hours to complete and thus delays the detection process. Similarly, Tims et al. [19] have described a biosensor assay that can be assayed from the spiked powder of suspected envelope or samples. Here, the problem is that generally the weaponized forms of anthrax are in the air upon discovery. There are numerous other methods for detecting anthrax; however, they require both long and tedious pre-preparation or very heavy equipment (i.e. NMR) that are not suitable for field or rapid analyses. [20],[21],[22],[23],[24],[25],[26] The method described in our report can be used in the detection of any DNA or RNA. Ideally, for pathogens with particularly robust "shells," such as anthrax spores, an additional highly aggressive mechanical disruption step may be required. The end result would be a very effective matrix-disrupter apparatus that destroys all the structural elements, while leaving the DNA or RNA molecules intact and stable, thereby freeing them for recovery and separation. These agents would then be no longer infectious. In summary, here we describe a novel method of highly sensitive, solid phase anthrax toxin detection that is versatile enough to be adopted for more widespread applications. References
Copyright 2011 - Indian Journal of Medical Microbiology The following images related to this document are available:Photo images[mb11091f2.jpg] [mb11091f1.jpg] [mb11091f3.jpg] [mb11091t1.jpg] |
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