Dataset Information

Optimization of the Isothermal Amplification Method for Mycobacterium tuberculosis Detection and Visualization for Fieldwork.

ABSTRACT: 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 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 CaCl2 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 was 102 CFU/ml and sensitivity was determined 100% compared to five different Mycobacterium species. CONCLUSION:The current study indicated that the LAMP-LFD and colorimetric LAMP protocol optimized with sputum samples can be reliable used as a rapid, sensitive and specific assay in the diagnosis of tuberculosis in the field.

SUBMITTER: Agel HE

PROVIDER: S-EPMC7379476 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Optimization of isothermal amplification method for Mycobacteriumtuberculosisdetection and visualization method for fieldwork

Ağel Hatice Esra HE Sağcan Hasan H Ceyhan Ismail I Durmaz Riza R

Turkish journal of medical sciences 20200623 4

<h4>Background/aim</h4>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 ...[more]

PMID: 32151124

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Project description:MicroRNAs (miRNAs) play important roles in a wide range of biological processes, and their aberrant expressions are associated with various diseases. The levels of miRNAs can be useful biomarkers for cellular events or disease diagnosis; thus, sensitive and selective detection of microRNAs is of great significance in understanding biological functions of miRNAs, early-phase diagnosis of cancers, and discovery of new targets for drugs. However, traditional approaches for the detection of miRNAs are usually laborious and time-consuming, with a low sensitivity. Here, we develop a simple, rapid, ultrasensitive colorimetric assay based on the combination of isothermal Exponential Amplification Reaction (EXPAR) and AuNP-labeled DNA probes for the detection of miRNAs (taking let-7a as a model analyte). In this assay, the presence of let-7a is converted to the reporter Y through EXPAR under isothermal conditions. The subsequent sandwich hybridization of the reporter Y with the AuNP-labeled DNA probes generates a red-to-purple color change. In other words, if the reporter Y is complementary to the AuNP-labeled DNA probes, the DNA-functionalized AuNPs will be aggregated, resulting in the change of solution color from red to purple/blue, while when the AuNP-labeled DNA probes are mismatched to the reporter Y, the solution remains red. This assay represents a simple, time-saving technique, and its results can be visually detected with the naked eye due to the colorimetric change. The method provides superior sensitivity, with a detection limit of 4.176 aM over a wide range from 1 nM to 1 aM under optimal conditions. The method also shows high selectivity for discriminating even single-nucleotide differences between let-7 miRNA family members. Notably, it is comparable to the most sensitive method reported to date, thus providing a promising alternative to standard approaches for the direct detection of let-7a miRNA. Importantly, through combination with specific templates, different miRNAs can be converted to the same reporter Y, which can hybridize with the same set of AuNP-labeled DNA probes to form sandwich hybrids. The color change of the solution can be observed in the presence of the target miRNA. This technique has potential as a routine method for assessing the levels of miRNAs, not only for let-7, but also for various miRNAs in the early phase of cancers. In addition, it can be a useful tool in biomedical research and clinical diagnosis, as well as diagnosis or surveillance programs in field conditions.

Dataset Information

Optimization of the Isothermal Amplification Method for Mycobacterium tuberculosis Detection and Visualization for Fieldwork.

Publications

Optimization of isothermal amplification method for Mycobacteriumtuberculosisdetection and visualization method for fieldwork

Similar Datasets

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