Abstract
Although culture-based identification of bacteria is the gold-standard for the diagnosis of infectious diseases, it is time consuming. Recent advances in molecular diagnostics and microfluidic technologies have opened up new avenues for rapid detection of bacteria. Here, we describe a centrifugal-microfluidic chip for the detection of bacteria by integrating the cell lysis, clarification, and loop-mediated amplification (LAMP). The major advantages of this chip are as follows. Firstly, bacteria lysis was innovatively achieved by rotating a pair of magnets to generate bead-beating while the chip was kept stationary during lysis, which simplified the chip design because no additional valve was needed. Secondly, the on-chip assay time was short (within 70 min), which was competitive in emergency situations. Thirdly, results of the analysis can be interpreted by using a fluorescence detector or by the naked-eye, making it versatile in many areas, especially the resource-limited areas. The on-chip limits of detection of six types of bacteria were valued by gel electrophoresis, showing the similar results compared to the bench-top LAMP protocol. This chip can be used for rapid, sensitive, accurate and automated detection of bacteria, offering a promising alternative for simplifying the molecular diagnostics of infectious diseases.
Highlights
Bacterial infections pose a major threat to global health
Some pioneering work has already been performed towards achieving this goal, for example, Czilwik et al.[14] have shown that the steps of chemical lysis, DNA extraction, and nested polymerase chain reaction (PCR) can be integrated successfully into a single centrifugal microfluidic disk
Efficient lysis of bacteria is a pre-requisite for the success of all nucleic acids (NA) amplification procedures
Summary
Bacterial infections pose a major threat to global health. Each year, diseases like meningitis, pneumonia, and sepsis caused by bacteria continue to be the cause of numerous deaths globally[1,2,3]. Numerous commercial kits for detection of microbes based on PCR or LAMP-assisted amplification of microbial NA (DNA or RNA) have entered the markets in recent years These methods have enabled a dramatic reduction in the time required for the identification of the pathogen[7], they involve cumbersome protocols for sample preparation as well as NA amplification and detection. The instrumentation used for performing the analysis is expensive and requires well-trained personnel for performing the various steps of the analysis[11] Another serious concern about employing highly specific amplification methods like LAMP and nested PCR is the possibility of a false positive result arising out of extraneous NA contamination[12, 13]. The apparatus is relatively more expensive when compared to other methods available for detection of pathogens
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