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. 2017 Dec;7(1):48.
doi: 10.1186/s13568-017-0349-6. Epub 2017 Feb 23.

Loop-mediated isothermal amplification of Neisseria gonorrhoeae porA pseudogene: a rapid and reliable method to detect gonorrhea

Mei-Ling Liu  1 , Yong Xia  1 , Xing-Zhong Wu  2 , Jian-Qiong Huang  3 , Xu-Guang Guo  4
Affiliations

Loop-mediated isothermal amplification of Neisseria gonorrhoeae porA pseudogene: a rapid and reliable method to detect gonorrhea

Mei-Ling Liu et al. AMB Express. 2017 Dec.

Abstract

Background and objective: Gonorrhea is a sexually transmitted disease caused by the bacterium Neisseria gonorrhoeae. Rapid detection is crucial for effective prevention and treatment. This study developed and tested a low-cost effective method for detecting N. gonorrhoeae, especially in developing countries.

Methods: DNA from a N. gonorrhoeae standard strain, as well as from 26 genital secretion samples of gonorrhea patients, were isolated and used for loop-mediated isothermal amplification (LAMP) assay, which was conducted using either an automatic real-time PCR analyzer or a water bath. The amplified porA pseudogene sequence was compared with the NCBI database and the LAMP results were compared with that of the traditional culture method for its sensitivity and specificity.

Results: LAMP was able to detect Neisseria DNA at a concentration as low as 1 pg/µL (1 × 103 CFU/mL cells). The LAMP assay results obtained using an automatic real-time PCR analyzer was similar to that of the water bath. Relative to traditional culture, the sensitivity and specificity of the LAMP assay were 94.7 and 85.7%, respectively.

Conclusion: LAMP was sensitive and reliable for detecting the porA gene of N. gonorrhoeae. It could be used as a rapid, low cost, and effective method for detecting N. gonorrhoeae.

Keywords: Gonorrhea; LAMP; Neisseria gonorrhoeae detection; porA gene.

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Figures

Fig. 1
Fig. 1
LAMP detection of the ATCC 49926 porA gene using an automatic PCR cycler
Fig. 2
Fig. 2
Specificity of LAMP for porA detection. The LAMP product (fluorescence) was detected from DNA of the Neisseria gonorrhoeae standard strain, but not in any of the 23 non-Neisseria bacteria
Fig. 3
Fig. 3
Sensitivity test for LAMP using different concentrations of DNA templates from Neisseria gonorrhoeae ATCC 49926
Fig. 4
Fig. 4
LAMP assay using 20 different DNA templates isolated from the same Neisseria gonorrhoeae standard strain (ATCC 49926), but from different colonies, at a concentration of 1 pg/µL
Fig. 5
Fig. 5
A representative example of LAMP using the clinical sample and the positive control (DNA from ATCC 49926)
Fig. 6
Fig. 6
LAMP assay using a water bath and direct observation of the LAMP products via fluorescence dye
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