Duplex recombinase polymerase amplification assays incorporating competitive internal controls for bacterial meningitis detection. 2018

Owen Higgins, and Eoin Clancy, and Matthew S Forrest, and Olaf Piepenburg, and Martin Cormican, and Teck Wee Boo, and Nicola O'Sullivan, and Claire McGuinness, and Deirdre Cafferty, and Robert Cunney, and Terry J Smith
Molecular Diagnostics Research Group, School of Natural Sciences and National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland. Electronic address: owen.higgins@nuigalway.ie.

Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification technology that provides rapid and robust infectious disease pathogen detection, ideal for point-of-care (POC) diagnostics in disease-prevalent low-resource countries. We have developed and evaluated three duplex RPA assays incorporating competitive internal controls for the detection of leading bacterial meningitis pathogens. Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae singleplex RPA assays were initially developed and evaluated, demonstrating 100% specificity with limits of detection of 4.1, 8.5 and 3.9 genome copies per reaction, respectively. Each assay was further developed into internally controlled duplex RPA assays via the incorporation of internal amplification control templates. Clinical performance of each internally controlled duplex RPA assay was evaluated by testing 64 archived PCR-positive clinical samples. Compared to real-time PCR, all duplex RPA assays demonstrated 100% diagnostic specificity, with diagnostic sensitivities of 100%, 86.3% and 100% for the S. pneumoniae, N. meningitidis and H. influenzae assays, respectively. This study details the first report of internally controlled duplex RPA assays for the detection of bacterial meningitis pathogens: S. pneumoniae, N. meningitidis and H. influenzae. We have successfully demonstrated the clinical diagnostic utility of each duplex RPA assay, introducing effective diagnostic technology for POC bacterial meningitis identification in disease-prevalent developing countries.

UI MeSH Term Description Entries
D009345 Neisseria meningitidis A species of gram-negative, aerobic BACTERIA. It is a commensal and pathogen only of humans, and can be carried asymptomatically in the NASOPHARYNX. When found in cerebrospinal fluid it is the causative agent of cerebrospinal meningitis (MENINGITIS, MENINGOCOCCAL). It is also found in venereal discharges and blood. There are at least 13 serogroups based on antigenic differences in the capsular polysaccharides; the ones causing most meningitis infections being A, B, C, Y, and W-135. Each serogroup can be further classified by serotype, serosubtype, and immunotype. Diplokokkus intracellularis meningitidis,Meningococcus,Micrococcus intracellularis,Micrococcus meningitidis,Micrococcus meningitidis cerebrospinalis,Neisseria weichselbaumii
D004269 DNA, Bacterial Deoxyribonucleic acid that makes up the genetic material of bacteria. Bacterial DNA
D006193 Haemophilus influenzae A species of HAEMOPHILUS found on the mucous membranes of humans and a variety of animals. The species is further divided into biotypes I through VIII. Bacterium influenzae,Coccobacillus pfeifferi,Haemophilus meningitidis,Hemophilus influenzae,Influenza-bacillus,Mycobacterium influenzae
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D013296 Streptococcus pneumoniae A gram-positive organism found in the upper respiratory tract, inflammatory exudates, and various body fluids of normal and/or diseased humans and, rarely, domestic animals. Diplococcus pneumoniae,Pneumococcus
D016133 Polymerase Chain Reaction In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships. Anchored PCR,Inverse PCR,Nested PCR,PCR,Anchored Polymerase Chain Reaction,Inverse Polymerase Chain Reaction,Nested Polymerase Chain Reaction,PCR, Anchored,PCR, Inverse,PCR, Nested,Polymerase Chain Reactions,Reaction, Polymerase Chain,Reactions, Polymerase Chain
D016920 Meningitis, Bacterial Bacterial infections of the leptomeninges and subarachnoid space, frequently involving the cerebral cortex, cranial nerves, cerebral blood vessels, spinal cord, and nerve roots. Bacterial Meningitis,Bacterial Meningitides,Meningitides, Bacterial
D045522 Recombinases A broad category of enzymes that are involved in the process of GENETIC RECOMBINATION. Recombinase,Resolvase,Resolvases
D019095 Point-of-Care Systems Laboratory and other services provided to patients at the bedside. These include diagnostic and laboratory testing using automated information entry. Bedside Computing,Point of Care Technology,Bedside Technology,Point-of-Care,Bedside Technologies,Computing, Bedside,Point of Care,Point of Care Systems,Point-of-Care System,Systems, Point-of-Care,Technologies, Bedside,Technology, Bedside
D021141 Nucleic Acid Amplification Techniques Laboratory techniques that involve the in-vitro synthesis of many copies of DNA or RNA from one original template. DNA Amplification Technic,DNA Amplification Technique,DNA Amplification Techniques,Nucleic Acid Amplification Technic,Nucleic Acid Amplification Technique,RNA Amplification Technic,RNA Amplification Technique,RNA Amplification Techniques,Amplification Technics, Nucleic Acid,Amplification Techniques, Nucleic Acid,DNA Amplification Technics,Nucleic Acid Amplification Technics,Nucleic Acid Amplification Test,Nucleic Acid Amplification Tests,RNA Amplification Technics,Technics, Nucleic Acid Amplification,Techniques, Nucleic Acid Amplification,Amplification Technic, DNA,Amplification Technic, RNA,Amplification Technics, DNA,Amplification Technics, RNA,Amplification Technique, DNA,Amplification Technique, RNA,Amplification Techniques, DNA,Amplification Techniques, RNA,Technic, DNA Amplification,Technic, RNA Amplification,Technics, DNA Amplification,Technics, RNA Amplification,Technique, DNA Amplification,Technique, RNA Amplification,Techniques, DNA Amplification,Techniques, RNA Amplification

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