Detection of Neisseria meningitidis in Cerebrospinal Fluid Using a Multiplex PCR and the Luminex Detection Technology
Rapid clinical and laboratory diagnoses are the foundation for a successful management of serious infections with Neisseria meningitidis. A species-specific multiplex polymerase chain reaction (PCR) coupled with fluidic microarrays using microbeads (the L
- PDF / 185,543 Bytes
- 17 Pages / 504.57 x 720 pts Page_size
- 54 Downloads / 221 Views
l meningitis is a serious infection affecting the central nervous system (CNS), with high morbidity and mortality. Neisseria meningitidis is one of the major causes of meningitis. Meningococcal meningitis may rapidly progress and result in permanent damage of the CNS. N. meningitidis is also known to cause epidemic outbreaks in crowded surroundings such as schools, refugee camps, and military recruit training centers. Rapid clinical and laboratory diagnoses
Myron Christodoulides (ed.), Neisseria meningitidis: Advanced Methods and Protocols, Methods in Molecular Biology, vol. 799, DOI 10.1007/978-1-61779-346-2_3, © Springer Science+Business Media, LLC 2012
37
38
J.K. Møller
are therefore mandatory for the successful management of meningococcal meningitis patients, as well as for interventions preventing the spread of meningococcal disease. The traditional laboratory diagnostic investigations comprise Gram stain and culture of cerebrospinal fluid (CSF), a blood culture, a naso-pharyngeal culture and if relevant, a sample taken from a petechial hemorrhage in the skin. However, culture for the identification of bacterial pathogens takes 24 h or more. Furthermore, the practice of starting antimicrobial therapy before clinical sample collection decreases the ability to confirm the pathogenic microorganisms of bacterial meningitis by culture (1). Nonculture methods have therefore been recognized as an important tool for optimal laboratory diagnosis of meningococcal infections. Various Nucleic Acid Amplification Tests (NAATs) from the early 1990s were employed in detection of N. meningitidis in CSF (2). Later on, detection of nucleic acids of the 16S rRNA gene by polymerase chain reaction (PCR) was introduced for the diagnosis of meningococcal disease (3). Recently, real-time PCR assays have included other targets, e.g., the porA gene encoding the outer membrane PorA porin (4), the conserved regulatory gene, crgA (5), and the ctrA gene (6) involved in the transport of the capsular polysaccharide in meningococci (7). A species-specific multiplex PCR coupled with fluidic microarrays using microbeads (Luminex xMAP™ Technology) for the detection of bacterial pathogens most frequently found in patient’s CSF has recently been described (8). The eightplex PCR described includes primers targeting the ctrA gene in N. meningitidis. Compared to real-time PCR, the advantage of the Luminex microsphere-based suspension array platform is that up to 100 different reactions in a single reaction vessel may be analyzed and reported at a time. The Luminex system based on MicroPlex™ Microspheres (formerly named xMAP Multianalyte Carboxylated Microspheres) is based on a unique combination of existing technologies (flow cytometry, microspheres, lasers, digital signal processing, and traditional chemistry) that use distinctly color-coded bead sets, each of which can be conjugated with a different specific analyte, e.g., a protein or nucleic acid reactant (9). A reporter molecule, specific for the analyte, is used to quantify the interaction. A sample
Data Loading...
