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Development of a visible loop mediated isothermal amplification assay for rapid detection of Bacillus anthracis
Abstract
Distressing effects on animal and human health with lethal progression, being used as bioweapon and shared
features with non-pathogenic bacteria demands sensitive, specific, safe, cost effective and rapid detection
methods for anthrax causing organisms. Conventional microbiology based diagnostics for anthrax are time
consuming and need sophisticated equipment, while molecular diagnostics require less time and labor. The Loop
mediated isothermal amplification assay (LAMP) is rapid, sensitive and specific assay and requires no specialized
equipment. In the present study, we developed a LAMP assay for rapid as well as specific detection of Bacillus
anthracis. The optimized assay produced positive results with the Sterne strain and one field isolate of B. anthracis
and, negative results with other bacteria of the same and different genera within 2 h. Sensitivity was 500 fg of
total DNA of B. anthracis, which was 100 times more sensitive than conventional PCR. The present study also
demonstrated that the simple method of total DNA extraction by repeated boiling and freezing will not adversely
affect the LAMP results. In conclusion, the optimized LAMP assay is a promising tool for the specific, sensitive,
less time-consuming diagnosis for anthrax causing bacteria and also, for detecting the virulence of suspected
B. anthracis cultures.
... DNA extracted from Formalin Fixed Paraffin Embedded (FFPE) sections was analysed in the DIMES zoonoses laboratory with the attempt of identifying pathogens causing look-alike diseases. A panPox Real-Time PCR was carried out to detect Poxviridae DNA(Luciani et al., 2021) while a Loop-Mediated Isothermal Amplification (LAMP) and a Realtime PCR were performed to detect Bacillus anthracis(Upadhyay et al., 2021;Kędrak-Jabłońska et al., 2018). The positive result to the panPox Realtime PCR was subsequently confirmed by HGC Realtime PCR(Li et al., 2010) that revealed the presence of PPV DNA. ...
Parapoxvirus (PPV) infections are considered neglected zoonoses because their incidence is often unknown or greatly underestimated despite being endemic globally. Here, we report the comprehensive diagnostic workflow that led to the identification of two cases of persistent PPV infections. The results obtained underline the importance of adopting a "One Health" approach and cross-sectoral collaboration between human and veterinary medicine for precise aetiological diagnosis and correct management of patients affected by zoonotic diseases.
The world is currently facing an adverse condition due to the pandemic of airborne pathogen SARS-CoV-2. Prevention is better than cure; thus, the rapid detection of airborne pathogens is necessary because it can reduce outbreaks and save many lives. Considering the immense role of diverse detection techniques for airborne pathogens, proper summarization of these techniques would be beneficial for humans. Hence, this review explores and summarizes emerging techniques, such as optical and electrochemical biosensors used for detecting airborne bacteria (Bacillus anthracis, Mycobacterium tuberculosis, Staphylococcus aureus, and Streptococcus pneumoniae) and viruses (Influenza A, Avian influenza, Norovirus, and SARS-CoV-2). Significantly, the first section briefly focuses on various diagnostic modalities applied toward airborne pathogen detection. Next, the fabricated optical biosensors using various transducer materials involved in colorimetric and fluorescence strategies for infectious pathogen detection are extensively discussed. The third section is well documented based on electrochemical biosensors for airborne pathogen detection by differential pulse voltammetry, cyclic voltammetry, square-wave voltammetry, amperometry, and impedance spectroscopy. The unique pros and cons of these modalities and their future perspectives are addressed in the fourth and fifth sections. Overall, this review inspected 171 research articles published in the last decade and persuaded the importance of optical and electrochemical biosensors for airborne pathogen detection.
The use of point of care diagnostics (POCD) in animal diseases has steadily increased over the years since its introduction. Its potential application to diagnose infectious diseases in remote and resource limited settings have made it an ideal diagnostic in animal disease diagnosis and surveillance. The rapid increase in incidence of emerging infectious diseases requires urgent attention where POCD could be indispensable tools for immediate detection and early warning of a potential pathogen. The advantages of being rapid, easily affordable and the ability to diagnose an infectious disease on spot has driven an intense effort to refine and build on the existing technologies to generate advanced POCD with incremental improvements in analytical performance to diagnose a broad spectrum of animal diseases. The rural communities in developing countries are invariably affected by the burden of infectious animal diseases due to limited access to diagnostics and animal health personnel. Besides, the alarming trend of emerging and transboundary diseases with pathogen spill-overs at livestock-wildlife interfaces has been identified as a threat to the domestic population and wildlife conservation. Under such circumstances, POCD coupled with non-invasive sampling techniques could be successfully deployed at field level without the use of sophisticated laboratory infrastructures. This review illustrates the current and prospective POCD for existing and emerging animal diseases, the status of non-invasive sampling strategies for animal diseases, and the tremendous potential of POCD to uplift the status of global animal health care.
Anthrax threatens human and animal health, and people's livelihoods in many rural communities in Africa and Asia. In these areas, anthrax surveillance is challenged by a lack of tools for on-site detection. Furthermore, cultural practices and infrastructure may affect sample availability and quality. Practical yet accurate diagnostic solutions are greatly needed to quantify anthrax impacts. We validated microscopic and molecular methods for the detection of Bacillus anthracis in field-collected blood smears and identified alternative samples suitable for anthrax confirmation in the absence of blood smears. We investigated livestock mortalities suspected to be caused by anthrax in northern Tanzania. Field-prepared blood smears (n = 152) were tested by microscopy using four staining techniques as well as polymerase chain reaction (PCR) followed by Bayesian latent class analysis. Median sensitivity (91%, CI 95% [84-96%]) and specificity (99%, CI 95% [96-100%]) of microscopy using azure B were comparable to those of the recommended standard, polychrome methylene blue, PMB (92%, CI 95% [84-97%] and 98%, CI 95% [95-100%], respectively), but azure B is more available and convenient. Other commonly-used stains performed poorly. Blood smears could be obtained for
There is an urgent need for convenient, sensitive, and specific methods to detect the spores of Bacillus anthracis, the causative agent of anthrax, because of the bioterrorism threat posed by this bacterium. In this study, we firstly develop a super-paramagnetic lateral-flow immunological detection system for B. anthracis spores. This system involves the use of a portable magnetic assay reader, super-paramagnetic iron oxide particles, lateral-flow strips and two different monoclonal antibodies directed against B. anthracis spores. This detection system specifically recognises as few as 400 pure B. anthracis spores in 30min. This system has a linear range of 4×10(3)-10(6)CFUml(-1) and reproducible detection limits of 200 spores mg(-1) milk powder and 130 spores mg(-1) soil for simulated samples. In addition, this approach shows no obvious cross-reaction with other related Bacillus spores, even at high concentrations, and has no significant dependence on the duration of the storage of the immunological strips. Therefore, this super-paramagnetic lateral-flow immunological detection system is a promising tool for the rapid and sensitive detection of Bacillus anthracis spores under field conditions.
The key genes required for Bacillus anthracis to cause anthrax have been acquired recently by horizontal gene transfer. To understand the genetic background for the evolution of B. anthracis virulence, we obtained high-redundancy genome sequences of 45 strains of the Bacillus cereus sensu lato (s.l.) species that were chosen for their genetic diversity within the species based on the existing multilocus sequence typing scheme. From the resulting data, we called more than 324,000 new genes representing more than 12,333 new gene families for this group. The core genome size for the B. cereus s.l. group was ∼1750 genes, with another 2150 genes found in almost every genome constituting the extended core. There was a paucity of genes specific and conserved in any clade. We found no evidence of recent large-scale gene loss in B. anthracis or for unusual accumulation of nonsynonymous DNA substitutions in the chromosome; however, several B. cereus genomes isolated from soil and not previously associated with human disease were degraded to various degrees. Although B. anthracis has undergone an ecological shift within the species, its chromosome does not appear to be exceptional on a macroscopic scale compared with close relatives.
Anthrax is a fatal disease caused by strains of Bacillus anthracis. Members of this monophyletic species are non motile and are all characterized by the presence of four prophages and a nonsense mutation in the plcR regulator gene. Here we report the complete genome sequence of a Bacillus strain isolated from a chimpanzee that had died with clinical symptoms of anthrax. Unlike classic B. anthracis, this strain was motile and lacked the four prohages and the nonsense mutation. Four replicons were identified, a chromosome and three plasmids. Comparative genome analysis revealed that the chromosome resembles those of non-B. anthracis members of the Bacillus cereus group, whereas two plasmids were identical to the anthrax virulence plasmids pXO1 and pXO2. The function of the newly discovered third plasmid with a length of 14 kbp is unknown. A detailed comparison of genomic loci encoding key features confirmed a higher similarity to B. thuringiensis serovar konkukian strain 97-27 and B. cereus E33L than to B. anthracis strains. For the first time we describe the sequence of an anthrax causing bacterium possessing both anthrax plasmids that apparently does not belong to the monophyletic group of all so far known B. anthracis strains and that differs in important diagnostic features. The data suggest that this bacterium has evolved from a B. cereus strain independently from the classic B. anthracis strains and established a B. anthracis lifestyle. Therefore we suggest to designate this isolate as "B. cereus variety (var.) anthracis".
Loop-mediated isothermal amplification (LAMP), a novel gene amplification method, enables the synthesis of larger amounts of both DNA and a visible byproduct--namely, magnesium pyrophosphate--without thermal cycling. A positive reaction is indicated by the turbidity of the reaction solution or the color change after adding an intercalating dye to the reaction solution, but the use of such dyes has certain limitations. Hydroxy naphthol blue (HNB), a metal indicator for calcium and a colorimetric reagent for alkaline earth metal ions, was used for a new colorimetric assay of the LAMP reaction. Preaddition of 120 microM HNB to the LAMP reaction solution did not inhibit amplification efficiency. A positive reaction is indicated by a color change from violet to sky blue. The LAMP reaction with HNB could also be carried out in a 96-well microplate, and the reaction could be measured at 650 nm with a microplate reader. The colorimetric LAMP method using HNB would be helpful for high-throughput DNA and RNA detection.
Virulent typical strains (Shikan, Morioka, Shizuoka) and Pasteur vaccine strains (no. 1, no. 2-H, no. 2-17JB) of Bacillus anthracis harboured two plasmid species with molecular masses of 110 MDal and 60 MDal. All of the 110 MDal plasmids isolated from the various strains showed indistinguishable patterns of digestion with restriction endonucleases. All the 60 MDal plasmids were also indistinguishable. Strain Davis, which is encapsulated but is asporogenous and avirulent, harboured only the 60 MDal plasmid while three non-encapsulated vaccine strains (34F2, Smith, Mukteswer) harboured only the 110 MDal plasmid. Four non-encapsulated variant strains obtained from the encapsulated strains Shikan, Pasteur no. 1, Pasteur no. 2-17JB and Davis had lost the 60 MDal plasmid, suggesting that encapsulation of B. anthracis may be associated with the 60 MDal plasmid.
Bacillus anthracis is the etiologic agent of anthrax, an acute fatal disease among mammals. It was thought to differ from Bacillus cereus, an opportunistic pathogen and cause of food poisoning, by the presence of plasmids pXO1 and pXO2, which encode the lethal toxin complex and the poly-gamma-d-glutamic acid capsule, respectively. This work describes a non-B. anthracis isolate that possesses the anthrax toxin genes and is capable of causing a severe inhalation anthrax-like illness. Although initial phenotypic and 16S rRNA analysis identified this isolate as B. cereus, the rapid generation and analysis of a high-coverage draft genome sequence revealed the presence of a circular plasmid, named pBCXO1, with 99.6% similarity with the B. anthracis toxin-encoding plasmid, pXO1. Although homologues of the pXO2 encoded capsule genes were not found, a polysaccharide capsule cluster is encoded on a second, previously unidentified plasmid, pBC218. A/J mice challenged with B. cereus G9241 confirmed the virulence of this strain. These findings represent an example of how genomics could rapidly assist public health experts responding not only to clearly identified select agents but also to novel agents with similar pathogenic potentials. In this study, we combined a public health approach with genome analysis to provide insight into the correlation of phenotypic characteristics and their genetic basis.
The successful use of Bacillus anthracis as a lethal biological weapon has prompted renewed research interest in the development of more effective vaccines against anthrax. The disease consists of three critical components: spore, bacillus, and toxin, elimination of any of which confers at least partial protection against anthrax. Current remedies rely on postexposure antibiotics to eliminate bacilli and pre- and postexposure vaccination to target primarily toxins. Vaccines effective against toxin have been licensed for human use, but need improvement. Vaccines against bacilli have recently been developed by us and others. Whether effective vaccines will be developed against spores is still an open question. An ideal vaccine would confer simultaneous protection against spores, bacilli, and toxins. One step towards this goal is our dually active vaccine, designed to destroy both bacilli and toxin. Existing and potential strategies towards potent and effective anthrax vaccines are discussed in this review.
The zoonotic nature and potential to be used as an agent of bioterrorism, quick onset and rapid lethal progression of the disease, ability to persist in environment for decades etc. necessitates the development of specific, sensitive, safe and reliable detection methods for the organism causing anthrax. Therefore, an attempt has been made in the present study to optimize a multiplex polymerase chain reaction (PCR) for the specific and sensitive identification of bacteria causing anthrax. Three sets of primers, one from chromosome (bxpB) and two from pXO1 plasmid (pag and cya) were designed so as to detect entire Bacillus cereus group and then specifically the bacterium causing anthrax, by single reaction. The selected primers amplified the desired products from both Sterne strain and a field isolate of Bacillus anthracis. The PCR amplification with template of other bacteria of same and different genera yielded negative results. The sensitivity was 1 pg of total DNA. The present article also delineates a very simple, reliable and less risky method for the extraction of both plasmid and chromosomal DNA from B. anthracis.
Anthrax, caused by Bacillus anthracis is primarily a zoonotic disease. Being a public health problem also in several developing countries, its early diagnosis is very important in human cases. In this study, we describe the use of an indirect enzyme-linked immunosorbent assay (ELISA) for detection of anti-lethal factor (LF) IgG in human sera samples. A panel of 203 human sera samples consisting of 50 samples from patients with confirmed cutaneous anthrax, 93 samples from healthy controls from anthrax non-endemic areas of India, 44 samples from controls from anthrax-endemic area of India, and 16 patients with a disease confirmed not to be anthrax were evaluated in an anti-LF ELISA. The combined mean anti-LF ELISA titer for the three control groups was 0.136 EU with 95% CI of 0.120 to 0.151. The observed sensitivity and specificity of the ELISA were 100% (95% CI, 92.89-100%) and 97.39% (95% CI, 93.44-99.28%) at a cut-off value of 0.375 EU as decided by ROC curve analysis. The likelihood ratio was found to be 49.98. The positive predictive value (PPV), negative predictive value (NPV), the efficiency and J index for reliability of the assay were 92.5%, 100%, 98.02% and 0.97, respectively. The false positive predictive rate and false negative predictive rate of the assay were 2.61% and 0%. The assay could be a very useful tool for early diagnosis of cutaneous anthrax cases as antibodies against LF appear much earlier than other anthrax toxins in human serum samples.
The loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method that uses only one type of enzyme. One of the characteristics of the LAMP method is its ability to synthesize extremely large amount of DNA. Accordingly, a large amount of by-product, pyrophosphate ion, is produced, yielding white precipitate of magnesium pyrophosphate in the reaction mixture. Judging the presence or absence of this white precipitate allows easy distinction of whether nucleic acid was amplified by the LAMP method. Since an increase in the turbidity of the reaction mixture according to the production of precipitate correlates with the amount of DNA synthesized, real-time monitoring of the LAMP reaction was achieved by real-time measurement of turbidity.
Bacillus anthracis is a Gram-positive, spore-forming bacterium, which causes anthrax, an often lethal disease of animals and humans. Although the disease has been well studied since the nineteenth century, it has witnessed a renewed interest during the past decade, due to its use as a bioterrorist agent in the fall of 2001 in the USA. A number of techniques aimed at rapidly detecting B. anthracis, in environmental samples as well as in point-of-care settings for humans suspected of exposure to the pathogen, are now available. These technologies range from culture-based methods to portable DNA amplification devices. Despite recent developments, specific identification of B. anthracis still remains difficult because of its phenotypic and genotypic similarities with other Bacillus species. Accordingly, many efforts are being made to improve the specificity of B. anthracis identification. This mini-review discusses the current challenges around B. anthracis identification, not only in reach-back laboratories but also in the field (in operational conditions).
We demonstrate a highly sensitive nano aptasensor for anthrax toxin through the detection of its polypeptide entity, protective antigen (PA toxin) using a PA toxin ssDNA aptamer functionalized single-walled carbon nanotubes (SWNTs) device. The aptamer was developed in-house by capillary electrophoresis systematic evolution of ligands by exponential enrichment (CE-SELEX) and had a dissociation constant (K(d)) of 112 nM. The aptasensor displayed a wide dynamic range spanning up to 800 nM with a detection limit of 1 nM. The sensitivity was 0.11 per nM, and it was reusable six times. The aptasensor was also highly selective for PA toxin with no interference from human and bovine serum albumin, demonstrating it as a potential tool for rapid and point-of-care diagnosis for anthrax.
Anthrax is the archetype zoonosis; no other infectious disease affects such a wide range of species, including humans, although most susceptible are herbivorous mammals. Although the disease appears to have been recognized for centuries, it has yet to be established scientifically how animals contract it. While primarily a disease of warmer regions, it has long been spread to cooler zones through the trade of infected animals or contaminated animal products. Today it is still endemic in many countries of Africa and Asia and non-endemic countries must remain alert to the possibility of imports from such endemic areas resulting in outbreaks in their own livestock. The epidemiology of anthrax is becoming understood better with new systems coming on stream for distinguishing different genotypes and this is covered in detail. Clinical signs and pathology in animals are described.
Loop mediated isothermal amplification (LAMP) is a powerful innovative gene amplification technique emerging as a simple rapid diagnostic tool for early detection and identification of microbial diseases. The whole procedure is very simple and rapid wherein the amplification can be completed in less than 1 h under isothermal conditions employing a set of six specially designed primers spanning eight distinct sequences of a target gene, by incubating all the reagents in a single tube. Gene amplification products can be detected by agarose gel electrophoresis as well as by real-time monitoring in an inexpensive turbidimeter. Gene copy number can also be quantified with the help of a standard curve generated from different concentrations of gene copy number plotted against time of positivity with the help of a real-time turbidimeter. Alternatively, gene amplification can be visualised by the naked eye either as turbidity or in the form of a colour change when SYBR Green I, a fluorescent dsDNA intercalating dye, is employed. LAMP does not require a thermal cycler and can be performed simply with a heating block and/or water bath. Considering the advantages of rapid amplification, simple operation and easy detection, LAMP has potential applications for clinical diagnosis as well as surveillance of infectious diseases in developing countries without requiring sophisticated equipment or skilled personnel.
Gram-positive bacilli isolated during epidemiological investigations which, on the basis of conventional tests, resemble Bacillus anthracis but which fail to produce the capsule or to induce anthrax in test animals have long been dismissed in clinical and veterinary laboratories as B. cereus or simply as unidentified Bacillus spp. and thereupon discarded as inconsequential. In this study, the application of newly available DNA probe, polymerase chain reaction and specific toxin antigen detection technology has revealed that a proportion of such strains are B. anthracis which lack the plasmid carrying the capsule gene (pXO2). While these techniques cannot, of course, be used to confirm the identities of strains resembling B. anthracis but which also lack the plasmid carrying the toxin genes (pXO1), the likelihood that these also are bona fide B. anthracis becomes more acceptable. (As yet no naturally occurring pXO1-/2+ strains have been found.) At this point, the significance of the presence of such avirulent forms of B. anthracis in specimens can only be a subject for speculation, but the possibility that they may be indicators of virulent parents somewhere in the system being examined must be considered.
Two llama calves died 3 days after inoculation with anthrax vaccine. Concurrent administration of ivermectin and other biologics may have enhanced the infectivity of the Sterne strain vaccine of Bacillus anthracis. This experience suggests that the Sterne strain of anthrax vaccine can induce fatal disease when given to young llamas and should be used only with extreme care and in face of strong "at risk" situations.
Large-molecular-weight plasmids were isolated from virulent and avirulent strains of Bacillus anthracis. Each strain contained a single plasmid species unique from the others with respect to molecular weight. Bacterial strains were cured of their resident extrachromosomal gene pools by sequential passage of cultures at 42.5 degrees C. Coincidental to the curing of plasmids was a loss of detectable lethal toxin and edema-producing activities and a dramatic decrease in lethal factor and protective antigen serological activities. The involvement of these plasmids in the production of toxin was firmly established by transformation of heat-passaged cells with plasmid DNA purified from the parent strain. The ability to produce parent strain levels of toxin was restored, and the plasmid DNA similar in molecular weight to that isolated from the parent was reisolated in all transformants examined. The exact role these plasmids play in the production of toxin remains to be elucidated. Two additional strains of B. anthracis, designated Pasteur vaccine strains, were examined for the ability to produce toxin and for the presence of plasmid DNA. Both strains were found to be nontoxigenic and contained no detectable plasmid elements. It is therefore likely that we, like Pasteur, cured B. anthracis strains of temperature-sensitive plasmids which code for toxin structural or regulatory proteins.
We have developed a novel method, termed loop-mediated isothermal amplification (LAMP), that amplifies DNA with high specificity,
efficiency and rapidity under isothermal conditions. This method employs a DNA polymerase and a set of four specially designed
primers that recognize a total of six distinct sequences on the target DNA. An inner primer containing sequences of the sense
and antisense strands of the target DNA initiates LAMP. The following strand displacement DNA synthesis primed by an outer
primer releases a single-stranded DNA. This serves as template for DNA synthesis primed by the second inner and outer primers
that hybridize to the other end of the target, which produces a stem–loop DNA structure. In subsequent LAMP cycling one inner
primer hybridizes to the loop on the product and initiates displacement DNA synthesis, yielding the original stem–loop DNA
and a new stem–loop DNA with a stem twice as long. The cycling reaction continues with accumulation of 109 copies of target in less than an hour. The final products are stem–loop DNAs with several inverted repeats of the target
and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target
in the same strand. Because LAMP recognizes the target by six distinct sequences initially and by four distinct sequences
afterwards, it is expected to amplify the target sequence with high selectivity.
Bacillus anthracis was shown to be the etiological agent of anthrax by R. Koch and L. Pasteur at the end of the nineteenth century. The concepts on which medical microbiology are based arose from their work on this bacterium. The link between plasmids and major virulence factors of B. anthracis was not discovered until the 1980s. The three toxin components are organized in two A-B type toxins, and the bacilli are covered by an antiphagocytic polyglutamic capsule. Structure-function analysis of the toxins indicated that the common B-domain binds to a ubiquitous cell receptor and forms a heptamer after proteolytic activation. One enzyme moiety is an adenylate cyclase and the other is a Zn(2+) metalloprotease, which is able to cleave MAPKKs. The capsule covers an S-layer sequentially composed of two distinct proteins. Knowledge of the toxins facilitates the design of safer veterinary vaccines. Spore-structure analysis could contribute to the improvement of human nonliving vaccines. The phylogeny of B. anthracis within the Bacillus cereus group is also reviewed.
The complete sequencing and annotation of the 181.7-kb Bacillus anthracis virulence plasmid pXO1 predicted 143 genes but could only assign putative functions to 45. Hybridization assays, PCR amplification,
and DNA sequencing were used to determine whether pXO1 open reading frame (ORF) sequences were present in other bacilli and
more distantly related bacterial genera. Eighteen Bacillus species isolates and four other bacterial species were tested for the presence of 106 pXO1 ORFs. Three ORFs were conserved
in most of the bacteria tested. Many of the pXO1 ORFs were detected in closely related Bacillus species, and some were detected only in B. anthracis isolates. Three isolates, Bacillus cereus D-17, B. cereus 43881, and Bacillus thuringiensis 33679, contained sequences that were similar to more than one-half of the pXO1 ORF sequences examined. The majority of the
DNA fragments that were amplified by PCR from these organisms had DNA sequences between 80 and 98% similar to that of pXO1.
Pulsed-field gel electrophoresis revealed large potential plasmids present in both B. cereus 43881 (341 kb) and B. thuringiensis ATCC 33679 (327 kb) that hybridized with a DNA probe composed of six pXO1 ORFs.
Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions using a set of four specially designed primers and a DNA polymerase with strand displacement activity. We have developed a method that accelerates the LAMP reaction by using additional primers, termed loop primers. Loop primers hybridize to the stem-loops, except for the loops that are hybridized by the inner primers, and prime strand displacement DNA synthesis. Although both inner and loop primers react via the loops, they do so by different mechanisms. The LAMP method presented here uses loop primers to achieve reaction times of less than half that of the original LAMP method. Since the total time of analysis including detection is less than 1h, this new method should facilitate genetic analysis, including genetic diagnosis in the clinical laboratory.
A number of genes in Bacillus anthracis encode for proteins homologous to the membrane-damaging factors known as pathogenic determinants in different bacteria. B. anthracis, however, has been traditionally considered non-hemolytic, and the recently identified hemolytic genes have been suggested to be transcriptionally silent. We found that the hemolytic genes of B. anthracis, collectively designated as anthralysins (Anls), could be induced in strict anaerobic conditions. We also demonstrate that Anl genes are expressed at the early stages of infection within macrophages by vegetating bacilli after spore germination. Cooperative and synergistic enhancement of the pore-forming and phospholipase C (PLC) activities of the Anls was found in hemolytic tests on human, but not sheep, red blood cells (RBC). These findings imply Anls as B. anthracis pathogenic determinants and highlight oxygen limitation as environmental factor controlling their expression at both early and late stages of infection.
Simultaneous analysis of three targets in three colors on any real-time polymerase chain reaction (PCR) instrument would increase the flexibility of real-time PCR. For the detection of Bacillus strains that can cause inhalation anthrax-related illness, this ability would be valuable because two plasmids confer virulence, and internal positive controls are needed to monitor the testing in cases lacking target-specific signals. Using a real-time PCR platform called MultiCode-RTx, multiple assays were developed that specifically monitor the presence of Bacillus anthracis-specific virulence plasmid-associated genes. In particular for use on LightCycler-1, two triplex RTx systems demonstrated high sensitivity with limits of detection nearing single-copy levels for both plasmids. Specificity was established using a combination of Ct values and correct amplicon melting temperatures. All reactions were further verified by detection of an internal positive control. For these two triplex RTx assays, the analytical detection limit was one to nine plasmid copy equivalents, 100% analytical specificity with a 95% confidence interval (CI) of 9%, and 100% analytical sensitivity with a CI of 2%. Although further testing using clinical or environmental samples will be required to assess diagnostic sensitivity and specificity, the RTx platform achieves similar results to those of probe-based real-time systems.
To evaluate different methods that are useful for rapid and definitive discrimination of Bacillus anthracis from other bacteria of the Bacillus cereus group in environmental samples like letters claimed to contain anthrax spores.
Characterized strains and bacteria from environmental samples were analysed by microbiological and molecular methods (PCR and restriction analysis). Environmental isolates often shared several microbiological features with B. anthracis, e.g. lack of beta-haemolysis and phospholipase C activity, and only the gamma phage assay was specific for B. anthracis. PCR assays targeting markers from the virulence plasmids exclusively detected B. anthracis, but other PCR targets were also detected in nonanthrax isolates. Additionally, the restriction pattern in an AluI restriction analysis of the SG-749 fragment is not 100% specific. The loci used for multiple-locus variable-number tandem repeat analysis of B. anthracis are also present in other members of the B. cereus group, but amplicon sizes are usually different.
Environmental samples often contain borderline isolates closely related to B. anthracis both on microbiological and genetic levels. Real-time PCR targeting plasmidal and chromosomal markers should be used for rapid and definitive exclusion of a virulent strain of B. anthracis in such samples.
This study gives an overview of the current microbiological and molecular methods used for identification of B. anthracis and shows that most assays have limits when borderline isolates present in environmental samples are analysed.