Detection of <i>S. pneumoniae</i> in 25 clinical isolates of oral streptococci by PCR and LAMP methods.

a<p>Classification based on API20 Strep identification testing.</p>b<p>+, optochin sensitive; −, optochin resistant.</p>c<p>+, bile soluble; −, bile insoluble.</p>d<p>Final identification from API20 strep testing, optochin sensitivity and bile solubility.</p>e<p>+, amplification occurred; −, amplification did not occur.</p

Nucleotide sequence of the <i>lytA</i> gene used to design the Sp LAMP primer.

<p>The sequences used for Sp LAMP primers are indicated by arrows (A). Structure and sequence of the primers used in the Sp LAMP reaction (B).</p

Detection limit of the LAMP and PCR assays using cerebrospinal fluid specimens spiked with <i>S. pneumoniae</i> (ATCC 6305).

a<p>Results obtained by duplicate trial: +, amplification occurred; −, amplification did not occur.</p>b<p>Results obtained by electrophoretic analysis.</p>c<p>Results determined by visual inspection.</p

Real-time sensitivity of Sp LAMP, as monitored by the measurement of turbidity.

<p>Shown from left to right in the figure are the curves of decreasing concentration (1,000,000 to 1) of bacteria. The detection limit was 10 copies (A). The relationship between the threshold time (<i>Tt</i>) of each sample and the log of the amount of initial template DNA (B).</p

Detection limits of the LAMP and PCR assays for <i>Streptococcus pneumoniae</i>.

a<p>Results obtained by triplicate trial: +, amplification occurred; −, amplification did not occur.</p>b<p>Results obtained by electrophoretic analysis.</p>c<p>Results determined by visual inspection.</p

Clinical Evaluation of a Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of <i>Neisseria meningitidis</i> in Cerebrospinal Fluid

<div><p>Background</p><p><i>Neisseria meningitidis</i> (Nm) is a leading causative agent of bacterial meningitis in humans. Traditionally, meningococcal meningitis has been diagnosed by bacterial culture. However, isolation of bacteria from patients’ cerebrospinal fluid (CSF) is time consuming and sometimes yields negative results. Recently, polymerase chain reaction (PCR)-based diagnostic methods of detecting Nm have been considered the gold standard because of their superior sensitivity and specificity compared with culture. In this study, we developed a loop-mediated isothermal amplification (LAMP) method and evaluated its ability to detect Nm in cerebrospinal fluid (CSF).</p><p>Methodology/Principal Findings</p><p>We developed a meningococcal LAMP assay (Nm LAMP) that targets the <i>ctrA</i> gene. The primer specificity was validated using 16 strains of <i>N</i>. <i>meningitidis</i> (serogroup A, B, C, D, 29-E, W-135, X, Y, and Z) and 19 non-<i>N</i>. <i>meningitidis</i> species. Within 60 min, the Nm LAMP detected down to ten copies per reaction with sensitivity 1000-fold more than that of conventional PCR. The LAMP assays were evaluated using a set of 1574 randomly selected CSF specimens from children with suspected meningitis collected between 1998 and 2002 in Vietnam, China, and Korea. The LAMP method was shown to be more sensitive than PCR methods for CSF samples (31 CSF samples were positive by LAMP vs. 25 by PCR). The detection rate of the LAMP method was substantially higher than that of the PCR method. In a comparative analysis of the PCR and LAMP assays, the clinical sensitivity, specificity, positive predictive value, and negative predictive value of the LAMP assay were 100%, 99.6%, 80.6%, and 100%, respectively.</p><p>Conclusions/Significance</p><p>Compared to PCR, LAMP detected Nm with higher analytical and clinical sensitivity. This sensitive and specific LAMP method offers significant advantages for screening patients on a population basis and for diagnosis in clinical settings.</p></div

A comparative analysis of PCR and Nm LAMP detection of <i>N</i>. <i>meningitidis</i>.

<p>A comparative analysis of PCR and Nm LAMP detection of <i>N</i>. <i>meningitidis</i>.</p

Detection limits of Nm LAMP and PCR assays targeting the <i>ctrA</i> gene and using Nm-serogroup-B-spiked CSF specimens.

<p>^a PCR results were obtained by electrophoresis. LAMP results were obtained by visual inspection of turbidity, colorimetric visual inspection dye, and by using a real-time turbidimeter.</p><p>^b +, Amplification;–, No amplification; ±, amplification occurred once.</p><p>^c Duplicate results.</p><p>Detection limits of Nm LAMP and PCR assays targeting the <i>ctrA</i> gene and using Nm-serogroup-B-spiked CSF specimens.</p

Location of the Nm LAMP primer in the capsular transport gene ctrA (1176 bp).

<p>Location of the Nm LAMP primer in the capsular transport gene ctrA (1176 bp).</p

Electrophoretic analysis of Nm LAMP products.

<p>The Nm LAMP products showed a ladder-like pattern on 2% gel electrophoresis and ethidium bromide staining. Lane M, molecular size marker (100-bp ladder; New England Biolabs, Beverly, MA); lanes 1–3, 9, 10, and 12–14: clinical samples (negative for <i>N</i>. <i>meningitidis</i>); lanes 4–8 and 11: clinical samples (positive for <i>N</i>. <i>meningitidis</i>); lane 15: negative control; lane 16: positive control</p