Reverse Line Blot Macroarray for Simultaneous Detection and Characterization of Four Biological Warfare Agents

BackgroundBrucella, a Gram-negative bacterium, is classified as a potential bioterrorism agent mainly due to the low dose needed to cause infection and the ability to transmit the bacteria via aerosols. Goats/sheep, cattle, pigs, dogs, sheep and rodents are infected by B. melitensis, B. abortus, B. suis, B. canis, B. ovis and B. neotomae, respectively, the six classical Brucella species. Most human cases are caused by B. melitensis and B. abortus. Our aim was to specifically detect Brucellae with ‘smooth’ lipopolysaccharide (LPS) using a highly sensitive monoclonal antibody (mAb) based immunological assay.MethodsTo complement molecular detection systems for potential bioterror agents, as required by international biodefense regulations, sets of mAbs were generated by B cell hybridoma technology and used to develop immunological assays. The combination of mAbs most suitable for an antigen capture assay format was identified and an immunoassay using the Luminex xMAP technology was developed.ResultsMAbs specific for the LPS O-antigen of Brucella spp. were generated by immunising mice with inactivated B. melitensis or B. abortus cells. Most mAbs recognised both B. melitensis and B. abortus and antigen binding was not impeded by inactivation of the bacterial cells by γ irradiation, formalin or heat treatment, a step required to analyse the samples immunologically under biosafety level two conditions. The Luminex assay recognised all tested Brucella species with ‘smooth’ LPS with detection limits of 2 × 102 to 8 × 104 cells per mL, depending on the species tested. Milk samples spiked with Brucella spp. cells were identified successfully using the Luminex assay. In addition, the bead-based immunoassay was integrated into a multiplex format, allowing for simultaneous, rapid and specific detection of Brucella spp., Bacillus anthracis, Francisella tularensis and Yersinia pestis within a single sample.ConclusionOverall, the robust Luminex assay should allow detection of Brucella spp. in both natural outbreak and bio-threat situations.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0534-1) contains supplementary material, which is available to authorized users.

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“…Currently available molecular identification assays for Brucella spp . offer comparable or even lower detection limits [ 32 , 33 , 38 ].…”

Section: Discussionmentioning

confidence: 99%

Development of a bead-based Luminex assay using lipopolysaccharide specific monoclonal antibodies to detect biological threats from Brucella species

et al. 2015

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“…The multiplex PCR assay can successfully identify Y. pestis with high sensitivity (99). Vanlalhmuaka et al (100) developed a multiplex PCR-based reverse line blot macroarray for simultaneous detection and characterization of four pathogens, including B. anthracis, Y. pestis, B. melitensis and B. pseudomallei. This assay is able to detect 8x10 2 cfu/ml for Y. pestis.…”

Section: Multiplex Pcrmentioning

confidence: 99%

Applications of polymerase chain reaction‑based methods for the diagnosis of plague (Review)

Zhang

Wang

et al. 2022

Exp Ther Med

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Plague is an acute bacterial infection caused by Yersinia pestis. The three major clinical forms of plague are bubonic, pneumonic and septicemic, which have high case-mortality rates. Therefore, rapid and reliable diagnostic tools are crucial. Currently, bacteriological means and traditional serological assays are used for detecting infection with Y. pestis. However, such methods have their limitations. Polymerase chain reaction (PCR) is one of the most useful tools for rapid diagnosis of plague. The present review introduced the main PCR techniques and their applications for detecting and confirmation of Y. pestis. The advantages and disadvantages of the different PCR methods were also summarized.

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“…Meningitis is usually detected by biochemical tests, microscopy, latex agglutination, PCR, microarray and biosensors [2][3][4][5]. Recently, blot microarray and SPR biosensor were reported for detection of biological warfare agents [6,7]. All these methods are time consuming, expensive and cumbersome.…”

mentioning

confidence: 99%

Quick Diagnosis of Human Brain Meningitis Using Omp85 Gene Amplicon as a Genetic Marker

Dash

Sharma²,

Khare³

et al. 2013

Indian J Microbiol

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The usual diagnosis of life-threatening human brain bacterial meningitis are expensive, time consuming or non-confirmatory. A quick PCR based diagnosis of meningitis in cerebrospinal fluids (CSF) using specific primers of virulent Omp85 gene of Neisseria meningitidis can detect as low as 1.0 ng of genomic DNA (G-DNA) in 80 min for confirmation of bacterial meningitis caused by N. meningitidis infection. The 257 bp amplicon of Omp85 gene does not show homology with other suspected pathogens in CSF and can be used as a specific genetic marker for diagnosis of the disease.Keywords Genetic marker Á Meningitis Á N. meningitidis Á Omp85 gene Meningitis causes inflammation of the meninges (outer membrane covering) in the brain and spinal cord of the patients [1]. Meningitis is usually detected by biochemical tests, microscopy, latex agglutination, PCR, microarray and biosensors [2][3][4][5]. Recently, blot microarray and SPR biosensor were reported for detection of biological warfare agents [6,7]. All these methods are time consuming, expensive and cumbersome. Boving et al. [8] reported an eight-plex PCR for simultaneous detection of Neisseria meningitidis, S. pneumoniae, E. coli, S. aureus, L. monocytogenes, S. agalactiae, herpes simplex virus and varicella-zoster virus which was time consuming. Omp85 (outer membrane protein) gene which is highly conserved in all the strains of N. meningitidis and assists for effective insertion of lipids and integral proteins into the outer membrane [9]. Omp85 gene can be used to develop genetic marker due to its virulence.The patient 0.5 ml CSF samples (from National Centre for Disease Control) was centrifuged at 16,0009g for 1 min to remove the CSF and the pellet was used in 25 ll of PCR mix containing 19 PCR buffer (1. For quantification of bacterial G-DNA, the pellet obtained from CSF containing bacteria was suspended in 0.5 ml of TE (10 mM Tris and 1 mM EDTA) buffer, pH 8.0 and heated at 95°C for 5 min for cell lysis. The heated solution was further centrifuged at 16,0009g for 1 min to remove cell debris and the supernatant containing G-DNA was quantified by Nanodrop spectrophotometer. The sensitivity of the PCR method was performed using different concentrations of N. meningitidis G-DNA (0.5-5.0 ng) in

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Reverse Line Blot Macroarray for Simultaneous Detection and Characterization of Four Biological Warfare Agents

Cited by 5 publications

References 16 publications

Development of a bead-based Luminex assay using lipopolysaccharide specific monoclonal antibodies to detect biological threats from Brucella species

Development of a bead-based Luminex assay using lipopolysaccharide specific monoclonal antibodies to detect biological threats from Brucella species

Applications of polymerase chain reaction‑based methods for the diagnosis of plague (Review)

Quick Diagnosis of Human Brain Meningitis Using Omp85 Gene Amplicon as a Genetic Marker

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