Molecular Assay Optimized for Meningitis Bacteria

By LabMedica International staff writers
Posted on 14 Feb 2011

An innovative technique enables the rapid detection of target nucleic acid sequences of Neisseria meningitidis under isothermal conditions without the need for complex instrumentation.

A loop-mediated isothermal amplification (LAMP) assay has been developed targeting a cell cycle transcription regulator gene (ctrA), for the rapid detection of capsular N. meningitidis with no cross-reactivity with other Neisseria species or with a comprehensive panel of other common human pathogens.

Image: Colored scanning electron micrograph (SEM) of Neisseria meningitidis bacteria, one of the causes of meningitis (photo courtesy the Eye of Science).

Scientists at the Royal Victoria Hospital (Belfast, UK), applied the LAMP assay to a total of 394 clinical specimens including samples from serum, ethylenediaminetetraacetic acid (EDTA) anticoagulated blood, throat swabs, cerebrospinal fluid , respiratory secretions, and fecal specimens. The majority of clinical specimens were obtained from pediatric patients who were younger than 13 years old.

An aliquot from each specimen type underwent total nucleic acid extraction, and the DNA extracts were subsequently analyzed in duplicate with a reference externally calibrated quantitative ctrA real-time polymerase chain reaction assay. This test determined meningococcal positivity status and the ctrA gene copy number present for each clinical specimen. The ctrA LAMP primers specifically amplified DNA from eight N. meningitidis-type reference strains. There was a high level of agreement between ctrA LAMP assay and real-time PCR assay with 36/394 positive for both assays and 355/394 negative for both assays.

The authors concluded that the LAMP assay was rapid and highly specific for the detection of all capsular N. meningitidis strains. The test demonstrated sensitivity for clinical specimens of 6 ctrA copies per reaction, which could be detected within 48 minutes and higher copy numbers could be detected in as little as 16 minutes.

The LAMP assay was simple to use and did not require specialized equipment, with positive specimens being readily identified by visual observation of color change or turbidity. This LAMP assay has the potential to be used for rapid detection of pathogenic meningococci in laboratories that do not have the instruments or expertise to perform real-time PCR. The study was published in February 2011 in the journal Diagnostic Microbiology and Infectious Disease.

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Royal Victoria Hospital