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

Abstract

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...

Real-time fluorometric and end-point colorimetric isothermal assays for detection of equine pathogens C. psittaci and equine herpes virus 1: validation, comparison and application at the point of care

“…Interestingly, the commercial colorimetric mix also gave false positive results for EHV-1 when using the primer set described by Nemoto et al [ 19 ], whereas EHV-1 primers designed in this study did not cause any false positive test results. Other studies using this colorimetric method also observed false positive test results, which may have arisen due to primer dimerization, non-specific amplification due to primer design, change of pH or other factors [ 13 , 25 , 26 ]. Moreover, we demonstrated that the LAMP reagents were thermostable at a storage temperature of 4 °C for at least six weeks .…”

Portable and Battery-Operated Isothermal Amplification Device Validation for Onsite Analysis of M. tuberculosis “DNA Hunter”

“…The primers were optimized according to the protocol described in the previous study. 23 The LAMP primers consisted of two outer primers (F3 and B3) and two inner primers [FIP (F1c + F2) and BIP (B1c + B2)], and two loop primers [FLP: (forward loop primer) and BLP (backward loop primer)]. 21 For the preparation of samples, the standard strain of M. tuberculosis H37Rv was used.…”

“…The limit of detection (LOD) of the M. tuberculosis LAMP test was determined as 10 2 CFU/mL in the previous study. 23 In this study to test whether the DNA hunter device performed the LAMP reaction correctly, five different concentrations (10 1 -10 5 CFU/mL) were studied above and below the LOD limit. A bacteria-free sputum sample was used for negative control.…”

“…In this study, a colorimetric LAMP master mix kit was used according to the optimized protocol. 23 LAMP assay was performed by a thermal cycler (Bio-Rad, Hamburg, Germany) at the same time as carrying out the DNA Hunter device. LAMP reaction operated under the same conditions (65°C for 30 minutes).…”

Tri-primer-enhanced strand exchange amplification combined with rapid lateral flow fluorescence immunoassay to detect SARS-CoV-2

“…[23][24][25] The coordinated use of Bst 3.0 DNA polymerase and WarmStart™ RTx greatly improves the consistency and specificity of one-step isothermal amplification. 26,27 By optimizing the reaction system and conditions, the amplification of two target genes can be performed at the same reaction temperature using one device. In order to improve the sensitivity of LFIA to fluorescence sensing, functional nanoparticles are used as signal amplifiers.…”