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    Optimization of lyophilized LAMP and RT-PCR reaction mixes for detection of tuberculosis
    (Sciendo, 2020) Ağel, Esra; Sağcan, Hasan
    Undoubtedly, one of the most infectious diseases in the world is tuberculosis. Key factor for tuberculosis control is to prevent possible contagion with rapid diagnosis and effective treatment. The culture method, which it takes several weeks to obtain results, is the gold standard method for laboratory diagnosis of tuberculosis. In order to prevent possible contagion of tuberculosis, diagnosis must be made in short time and treatment should be started as soon as possible. Normally, clinical samples are studied in advanced laboratories designed for this purpose. However, especially after the screening in rural areas, the transmission of the samples to the centers has many negative effects on the clinical material. Therefore, the latest trend molecular techniques in microbiological diagnosis are developing into point of care systems that can be applied in the field without laboratory infrastructure. The major challenge for molecular-based point-of-care tests is the need to store polymerase enzymes and some of the ingredients used in the cold chain. The aim of this study is to increase the resistance of the amplification reaction mixtures by lyophilizing the tuberculosis diagnosis. Lyophilization was performed on Loop-mediated isothermal amplification (LAMP) and Real-time PCR mixtures. For the lyophilization of LAMP and RT-PCR mixtures, two different experimental setups were tried from the literature except for the developed content. Chemicals such as stachyose, trehalose, glycerol and PEG 8000 are widely using as cryoprotectants. As a result, the developed content (0.5% PEG 8000, 2.0 % Stachyose) was determined the best cryoprotectant mixture. Accordingly, amplification mixtures can be produced with the developed lyophilization method and point of care kits can be developed.
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    Rapid molecular diagnosis of group a streptococcus with a novel loop mediated isothermal amplification method
    (Verlag Klinisches Labor GmbH, 2022) Toptan, Hande; Ağel, Esra; Sağcan, Hasan; Ertunç, Yiğit M.; Elmas, Bahri; Köroğlu, Mehmet; Şengil, Ahmet Zeki; Altındiş, Mustafa
    Background: Group A Streptococcus (GAS) is the most common bacterial cause of acute tonsillopharyngitis. In this study, it was aimed to evaluate the performance of a novel Loop Mediated Isothermal Amplification (LAMP) method in the rapid diagnosis of GAS in samples taken from children with a pre-diagnosis of acute bacterial tonsillopharyngitis by comparing it with culture and rapid antigen test (RAT) methods. Methods: A total of 100 throat swab samples taken from children at the pediatrics outpatient clinic with suspected tonsillopharyngitis were included in the study. Throat swab samples were analyzed by RAT, throat culture, and LAMP method. GAS suspected colonies were identified with MALDI-TOF MS system. The isothermal amplification reaction for LAMP was conducted by a novel LAMP instrument. Results: According to the results of throat cultures; 53 of them were positive and 47 were negative in terms of GAS. Six (11.32%) of the culture positive samples were found to be negative by the RAT (sensitivity; 88.68%, specificity 100%). While the antigen test was positive, no culture negative sample was detected. One of the culture positive samples was found negative by LAMP. In two samples, while throat culture was negative, it was observed that LAMP was positive (sensitivity; 98.11%, specificity; 95.74%). In one of these samples, the bacteria grown in the culture were identified as Streptococcus dysgalactiae by mass spectrophotometry. Conclusions: In this study, it was determined that the LAMP method used in the diagnosis of throat infections caused by GAS has high sensitivity and specificity. We believe that the instrument is easy to use, low cost, portable, and adaptable to point of care tests. There are very few studies in the literature regarding the use of the instrument in this field, and it should be evaluated in terms of its usability in daily practice with new studies.