Clavibacter michiganensis subsp. sepedonicus (CMS) is an important bacterial plant pathogen causing potato ring rot disease. Rapid diagnosis of CMS is crucial because of the economic losses caused by serious harvest losses. Although there are serological tests used in the rapid diagnosis of CMS, they are not widely used because of their low sensitivity. The DNA-based PCR methods, which are highly sensitive, do not have the possibility of on-site diagnosis, especially since they require serious laboratory infrastructure. In recent years, scientists have been working on alternative amplification methods to develop DNA-based point of care (POC) diagnostic methods. Accordingly, the loop-mediated isothermal amplification (LAMP) method, which was developed in the early 2000s, provides an important convenience for DNA-based tests to use in the field. Due to the unique design of primers, more amplification products could be create in a shorter time than conventional amplification methods without needing a temperature cycle, and it can be applied with the aid of a simple heater without requiring a laboratory environment. In this study, efficient LAMP method for the detection of CMS has optimized. For device-independent detection of LAMP products, colorimetric method and LFD has used.

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Introduction Tuberculosis caused by Mycobacterium tuberculosis is spread from person to person through respiratory fluids and air [1]. M. tuberculosis has a low infective dose; just below the 10 bacilli (1 to 200 bacilli) are enough for infection [2]. Tuberculosis comes right after HIV for infection-caused deaths [3]. According to the World Health Organization (WHO) reports, 1/3 of the world population today is already infected with tuberculosis. It was estimated that 10 million people developed tuberculosis and 1.6 million of these infected people died because of tuberculosis in 2017. About 1 million tuberculosis cases are seen in children. In Turkey, 12,046 new tuberculosis patients emerged in 2017 [4]. Key factors for the control of tuberculosis are a rapid diagnosis, effective treatment, and preventing transmission with scanning. The gold standard method for laboratory diagnosis of tuberculosis is culture method [5]. However, getting the results takes a few weeks long. Moreover, sensitivity of the microscopic examination following the Ziehl-Neelsen staining is low. For preventing tuberculosis spread, it is critical to diagnose the disease within 1 or 2 days and start the treatment immediately [6]. Therefore, there is a need for quick, sensitive, reliable, point-of-care, and economical methods for the laboratory diagnosis of tuberculosis. Nucleic acid amplification is one of the most effective methods for detecting infectious diseases and genetic Background/aim: Tuberculosis is still one of the most contagious diseases around the world. Key factors of tuberculosis control are rapid diagnostic, efficient treatment, and prevention of contamination by surveillance and monitoring. However, culture is the gold standard method for laboratory diagnosis of tuberculosis; the results are several weeks to obtain. In order to prevent contamination of tuberculosis, diagnosis must be made in short time and treatment should be started as soon as possible. The aim of this study is to optimize the loop-mediated isothermal amplification (LAMP) method, which provides a much faster and more sensitive result than the polymerase chain reaction (PCR) method and allows the replication of target nucleic acid sequences under isothermal conditions without the need for laboratory infrastructure. Materials and methods: Sputum samples were homogenized with 5% trypsin solution in CaCl 2 to obtain DNA. DNA was purified using QIAGEN QIAamp DNA mini kit. LAMP primers were design using Primer explorer V5 program according to IS6110 gene of Mycobacterium tuberculosis. NEB Bst 3.0 DNA polymerase kit was used for LAMP reactions. Besides, LAMP reactions were compared with TaqMan based RT-PCR method using NEB's Taq polymerase kit. Finally, for visualization of LAMP products, lateral flow dipsticks that produced by Milenia Biotec, colorimetric 2X LAMP master mix that produced by NEB and 2% w/v agarose gel electrophoresis methods were used. Results: Optimum amplification temperature for LAMP was found to be 71.4 °C. The detection limit of the method w...

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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