The Detection of Tuberculosis by Loop-Mediated Isothermal Amplification (LAMP) Combined with a Lateral Flow Dipstick [chapter]

Thongchai Kaewphinit, Somchai Santiwatanakul, Kosum Chansiri
Handbook of Research on Diverse Applications of Nanotechnology in Biomedicine, Chemistry, and Engineering  
Diphtheria outbreaks occurred in endemic areas and imported and indigenous cases are reported in UE/EEA. Because of the high infectiveness and severity of the disease, early and accurate diagnosis of each suspected case is essential for the treatment and management of the case and close contacts. The aim of the study was to establish simple and rapid testing methods based on Loop-Mediated Isothermal Amplification (LAMP) assay for the detection of Corynebacterium diphtheriae and differentiation
more » ... nd differentiation between toxigenic and non-toxigenic strains. Methods Corynebacterium diphtheriae and Corynebacterium ulcerans isolates from the National Institute of Public Health-National Institute of Hygiene collection were used for the development of LAMP assay for the diagnosis of diphtheria and nontoxigenic C. diphtheriae infections. Various colorimetric methods for visualization of results were investigated. Sensitivity and specificity of the assay were examined using a collection of DNA samples from various gram-positive and gramnegative bacteria. Results The LAMP assay for tox and dtxR genes was developed. The sensitivity and specificity of the assay were calculated as 100%. The detection limit was estimated as 1.42 pg/µl concentration of DNA template when the reaction was conducted for 60 min. However, the detection limit was lowered 10 times for every 10 minutes of reduction in the time of incubation during the reaction. Positive results were successfully detected colorimetrically using hydroxynaphthol blue, calcein, QuantiFluor, and lateral flow Milenia HybriDetect dipsticks. Conclusion The assay developed in the study might be applied for point-of-care testing of diphtheria and other C. diphtheriae infections as well as for other infections caused by diphtheria-toxin producing Corynebacterium species. It is highly sensitive, specific, inexpensive, easy to use, and suitable for low-resource settings. Background Diphtheria is an acute, highly infectious and potentially lethal infectious disease of humans. The disease is caused by diphtheria toxin-producing strains of Corynebacterium diphtheriae, Corynebacterium ulcerans, and Corynebacterium pseudotuberculosis. The infection can be Streptococcus salivarius, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus epidermidis, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus, Corynebacterium pseudodiphtheriticum, Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae. DNA extraction was performed for 24 h bacterial cultures on the medium appropriate for the bacterial species by using DNeazy Blood and Tissue Kit (Qiagen, Germany) according to the manufacturer's instructions for Gram-positive and Gram-negative bacteria, respectively. Bacteria identification Corynebacterium strains were identified and biotyped based on the colony morphology on tellurite agar plates and ApiCoryne tests (Biomerieux, France). Toxigenicity was tested using Elek test. Additionally, the presence of tox gene was verify by PCR, according to WHO Manual for Laboratory Diagnosis of Diphtheria [13] . Other non-Corynebacterium strains mentioned above were identified using conventional microbiology Neutral red (Sigma-Aldrich, USA) and phenol red (Sigma-Aldrich, USA) were dissolved in deionized water or 1 M NaOH, respectively, at 50 mM to prepare a stock solution and diluted to 2.5 mM. For the optimization of the concentration of the indicators in the reaction solution, the following final concentrations were tested: 0.2, 0.15, 0.1, 0.05, and 0.01 mM. HNB was dissolved in deionized water at 50mM to prepare a stock solution. Then, the solution was diluted and tested in the LAMP reaction at the following final concentrations: 1, 0.5, 0.32, 0.25, 0.16, 0.125, 0.08, and 0.04 mM. The calcein stock solution consisted of 0.5 mM calcein (Novazym, Poland) and 10 mM MnCl 2 (Sigma-Aldrich, USA) in deionized water. To select an optimal concentration, the following volumes of the stock solution were added to the reaction solution: 0.25, 0.5, 1, 1.5, and 2 µl. The amount of QuantiFluor (Promega, Germany) in the post-reaction solution was optimized by the addition of the following volumes of the dye: 2, 1, and 0.5 µl. Determination of specificity, sensitivity, detection limit, and minimal reaction time Specificity and sensitivity of the LAMP were investigated using abovementioned bacterial species that can be present in respiratory tracts. The sensitivity was calculated as follows: A/(A + C) × 100%, and the specificity was calculated as follows: D/(B + D) × 100%, where A is the number of true positive Not Applicable
doi:10.4018/978-1-4666-6363-3.ch013 fatcat:ndfsgcfofre6bgnffid4osqbya