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Clinical evaluation of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Neisseria meningitidis in cerebrospinal fluid.

Lee D, Kim EJ, Kilgore PE, Kim SA, Takahashi H, Ohnishi M, Anh DD, Dong BQ, Kim JS, Tomono J, Miyamoto S, Notomi T, Kim DW, Seki M - PLoS ONE (2015)

Bottom Line: 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.This sensitive and specific LAMP method offers significant advantages for screening patients on a population basis and for diagnosis in clinical settings.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy, College of Pharmacy, Hanyang University, Ansan, Republic of Korea; Institute of Pharmacological Research, Hanyang University, Ansan, Republic of Korea.

ABSTRACT

Background: Neisseria meningitidis (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).

Methodology/principal findings: We developed a meningococcal LAMP assay (Nm LAMP) that targets the ctrA gene. The primer specificity was validated using 16 strains of N. meningitidis (serogroup A, B, C, D, 29-E, W-135, X, Y, and Z) and 19 non-N. meningitidis 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.

Conclusions/significance: 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.

No MeSH data available.


Related in: MedlinePlus

Real-time monitoring of Nm LAMP reaction mixture turbidity.N. meningitidis serogroup B reference DNA; dilution series from 1,000,000 copies to 1 copy. The molecular weight of the genomic DNA is 2.2 Mbp. The molecular weight of one copy is 2.5 fg. The LAMP assay detected 10 copies of N. meningitidis serogroup B within 60 min.
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pone.0122922.g002: Real-time monitoring of Nm LAMP reaction mixture turbidity.N. meningitidis serogroup B reference DNA; dilution series from 1,000,000 copies to 1 copy. The molecular weight of the genomic DNA is 2.2 Mbp. The molecular weight of one copy is 2.5 fg. The LAMP assay detected 10 copies of N. meningitidis serogroup B within 60 min.

Mentions: We developed a rapid and sensitive LAMP reaction for detecting N. meningitidis that reduced the occurrence of nonspecific reactions. Using the primer set we developed, the LAMP assay successfully amplified the 194-bp target sequence of the ctrA gene within 45 min (Fig 2). No nonspecific amplification reactions were observed within 90 min even under stringent conditions (Table 2). The amplified product was visible on an agarose gel (Fig 3) and showed a ladder-like pattern that was characteristic of the LAMP reaction, indicating the production of stem-loop DNAs with inverted repeats of the target sequence [10].


Clinical evaluation of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Neisseria meningitidis in cerebrospinal fluid.

Lee D, Kim EJ, Kilgore PE, Kim SA, Takahashi H, Ohnishi M, Anh DD, Dong BQ, Kim JS, Tomono J, Miyamoto S, Notomi T, Kim DW, Seki M - PLoS ONE (2015)

Real-time monitoring of Nm LAMP reaction mixture turbidity.N. meningitidis serogroup B reference DNA; dilution series from 1,000,000 copies to 1 copy. The molecular weight of the genomic DNA is 2.2 Mbp. The molecular weight of one copy is 2.5 fg. The LAMP assay detected 10 copies of N. meningitidis serogroup B within 60 min.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390149&req=5

pone.0122922.g002: Real-time monitoring of Nm LAMP reaction mixture turbidity.N. meningitidis serogroup B reference DNA; dilution series from 1,000,000 copies to 1 copy. The molecular weight of the genomic DNA is 2.2 Mbp. The molecular weight of one copy is 2.5 fg. The LAMP assay detected 10 copies of N. meningitidis serogroup B within 60 min.
Mentions: We developed a rapid and sensitive LAMP reaction for detecting N. meningitidis that reduced the occurrence of nonspecific reactions. Using the primer set we developed, the LAMP assay successfully amplified the 194-bp target sequence of the ctrA gene within 45 min (Fig 2). No nonspecific amplification reactions were observed within 90 min even under stringent conditions (Table 2). The amplified product was visible on an agarose gel (Fig 3) and showed a ladder-like pattern that was characteristic of the LAMP reaction, indicating the production of stem-loop DNAs with inverted repeats of the target sequence [10].

Bottom Line: 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.This sensitive and specific LAMP method offers significant advantages for screening patients on a population basis and for diagnosis in clinical settings.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy, College of Pharmacy, Hanyang University, Ansan, Republic of Korea; Institute of Pharmacological Research, Hanyang University, Ansan, Republic of Korea.

ABSTRACT

Background: Neisseria meningitidis (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).

Methodology/principal findings: We developed a meningococcal LAMP assay (Nm LAMP) that targets the ctrA gene. The primer specificity was validated using 16 strains of N. meningitidis (serogroup A, B, C, D, 29-E, W-135, X, Y, and Z) and 19 non-N. meningitidis 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.

Conclusions/significance: 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.

No MeSH data available.


Related in: MedlinePlus