Rapid Detection of Pathogens Based on Loop-Mediated Isothermal Amplification (LAMP) for Point-of-Care Diagnosis

Abstract

Infectious diseases, to this day, remain a major health threat worldwide. Molecular diagnostics, based on nucleic acid (NA) amplification technologies, are in the forefront for the detection of pathogens in order to prevent the transmission of diseases. Polymerase chain reaction (PCR) is one of the most widely used methods for nucleic acid amplification. However, PCR requires thermocycling for the amplification process. The requirement for thermocycling causes challenges for implementing PCR in the lab-on-chip system at point-of-care (POC) settings. One of the major hurdles is the formation of bubbles during thermocycling in microchannels resulting in unreliable experimental results. These problems severely hamper the commercialization of such lab-on-chip systems. Isothermal amplification methods demonstrate a possibility of amplifying DNA under isothermal conditions, without thermocycling, which can overcome the difficulties of PCR in lab-on-chip systems at POC setting. The aim of this Ph.D. project is to develop, optimize and evaluate one of the isothermal amplification methods called loop-mediated isothermal amplification (LAMP), suitable for on-line or at-site rapid detection of pathogens toward POC diagnostics applications. This Ph.D. project includes two main parts. In the first part, conventional LAMP assays are developed and optimized for singleplex detection of pathogens (Chapter 2-5). In the second part, LAMP is developed in solid phase amplification for enhancing multiplex detection capacity (Chapter 6). In Chapter 2, a LAMP assay was developed, optimized and used for specific, sensitive, simple and cost-effective detection of Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli) in the poultry production chain. By combining this LAMP reaction with a visual detection approach using a DNA intercalating dye, LAMP amplified product was observed directly by naked eyes under a small, low-cost portable commercial blue LED trans-illuminator. Under optimized conditions, a detection limit of 50 CFU/mL was achieved when testing for C. jejuni and C. coli in spiked chicken feces within 60–70 min from receiving the samples to the final results (no enrichment step needed). The optimized LAMP assay was comparable to RT-PCR with 98.4% relative accuracy, 97.9% specificity , and 100% sensitivity. The study showed the potential of LAMP assay for rapid detection of C. coli and C. jejuni at poultry production to prevent transmission of foodborne disease. The visual detection method using a DNA intercalating dye is simple and does not require an optical detection part, but the requirement of adding a DNA intercalating dye after completing the reaction may cause carry-over contamination of LAMP products. To overcome this problem, in Chapter 3, a Cod-Uracil-DNA-Glycosylase real-time reverse transcriptase LAMP assay (Cod-UNG-rRT-LAMP) was developed. SARS-CoV-2 virus was chosen as the target in this assay. Using the Cod-UNG-rRTLAMP assay, a detection limit of 2 copies/µL (8 copies/reaction) of the SARS-CoV-2 virus was achieved within 45 min of amplification, and simultaneously 10-20 pg of contaminants were eliminated per reaction. Moreover, the investigation of different detection methods for POC diagnostics in CodUNG-rRT-LAMP assay demonstrated the feasibility of using the Cod-UNG-rRT-LAMP assay for applications toward POC diagnosis of SARS-CoV-2 and on-site testing of other pathogens. Besides the visual detection method, wherein the LAMP reaction was confirmed at the end of the reaction, another approach that can monitor LAMP reaction in real-time was developed using DNA intercalating dyes and is presented in Chapter 4. To select a suitable DNA intercalating dye for monitoring a real-time LAMP assay, an investigation was performed with twenty-three DNA dyes having different properties on real-time LAMP. Based on the LAMP inhibition effects, the dyes were classified into four different groups such as dyes with non-inhibition effect, medium inhibition effect, high inhibition effect, and very high inhibition effect. The performance of these dyes was compared under similar LAMP reaction conditions, under different LAMP reaction conditions, and also in different detection systems. Of the twenty-three dyes tested, SYTO 9, SYTO 82, SYTO 16, SYTO 13, and Miami Yellow showed the best performance due to no inhibitory effect, possibility to achieve a low limit of detection, and high signal-to-noise ratio in the real-time LAMP reactions. This classification of the dyes will simplify the selection of fluorescence dyes in future aimed at developing real-time LAMP assays for POC setting. With the aim of developing technologies for POC diagnostics, a gelification strategy was investigated, in Chapter 5, in order to store all the reagents in a ready-to-use format. Storage of reagents through gelification strategy is easy and simple, and enables easy handling, testing, and transportation. The ready-to-use reaction tube with gelified reagents was stable at room temperature for 7 weeks and at 2- 8 °C up to 3 years. Besides studies of LAMP for singleplex detection of pathogens (Chapters 2-5), the multiplexing capacity of LAMP was investigated in this Ph.D. Since the conventional LAMP reaction exposed the challenges of developing multiplex detection of pathogens at POC setting, another approach, solid phase LAMP (SP-LAMP), was developed to study the multiplexing capacity of LAMP for detection of multiple pathogens in a single reaction (Chapter 6). SP-LAMP was developed successfully for singleplex detection of different pathogens (Campylobacter spp., Salmonella spp., C. coli, C. jejuni, avian influenza virus, and pan avian for internal control). The investigation of SP-LAMP reactions on heterodimers of different primer sets for multiplex detection on liquid phase was investigated. However, the multiplex detection of SP-LAMP on microchips remains challenges.  In summary, different methods based on LAMP assay have been developed and optimized for the rapid detection of pathogens toward POC diagnostics applications in this thesis. The simple visual detection was suitable for rapid detection of C. coli and C. jejuni in the poultry production line. In addition, the Cod-UNG-rRT-LAMP was used for the elimination of carry-over contamination of LAMP products, not only in SARS-COV-2 but also in other targets. Moreover, the classification of dyes based on inhibition effects in LAMP would simplify the selection of dyes for monitoring real-time LAMP assay at the POC setting. Furthermore, the gelification of LAMP reagents in a ready-to-use format facilitated the use, storage and transportation. Finally, SP-LAMP was developed successfully and used for singleplex detection of different pathogens. However, multiplex SP-LAMP was still a challenge. The multiplexing capacity of SP-LAMP itself is not yet completely developed and, therefore, is one of the major research in the field. The initial promising results of SP-LAMP opens the way towards possible success for multiplexing of the SP-LAMP in the near future.