Abstract
Molecular diagnostics is one of the most important tools currently in use for clinical pathogen detection due to its high sensitivity, specificity, and low consume of sample and reagent is keyword to low cost molecular diagnostics. In this paper, a sensitive DNA isothermal amplification method for fast clinical infectious diseases diagnostics at aM concentrations of DNA was developed using a polycarbonate (PC) microfluidic chip. A portable confocal optical fluorescence detector was specifically developed for the microfluidic chip that was capable of highly sensitive real-time detection of amplified products for sequence-specific molecular identification near the optical diffraction limit with low background. The molecular diagnostics of Listeria monocytogenes with nucleic acid extracted from stool samples was performed at a minimum DNA template concentration of 3.65[Formula: see text]aM, and a detection limit of less than five copies of genomic DNA. Contrast to the general polymerase chain reaction (PCR) at eppendorf (EP) tube, the detection time in our developed method was reduced from 1.5[Formula: see text]h to 45[Formula: see text]min for multi-target parallel detection, the consume of sample and reagent was dropped from 25[Formula: see text][Formula: see text]L to 1.45[Formula: see text][Formula: see text]L. This novel microfluidic chip system and method can be used to develop a micro total analysis system as a clinically relevant pathogen molecular diagnostics method via the amplification of targets, with potential applications in biotechnology, medicine, and clinical molecular diagnostics.
Highlights
Infectious diseases account for more than half of child deaths worldwide, and one in nine child deaths are caused by diarrhea, making diarrhea the second leading cause of death among children under the age ofve.[1]
There are isothermal amplication methods have been developed to overcome the drawbacks of polymerase chain reaction (PCR), which mainly consist of loop-mediated isothermal amplication (LAMP),[7] nucleic acid sequence-based amplication (NASBA),[8] strand displacement amplication (SDA),[9] and rolling circle amplication (RCA).[10]
The sensitivity of the isothermal amplication assay on the micro°uidic chip was assessed using genomic DNAs of Listeria monocytogenes
Summary
Infectious diseases account for more than half of child deaths worldwide, and one in nine child deaths are caused by diarrhea, making diarrhea the second leading cause of death among children under the age ofve.[1] A fundamental requirement for successful disease control is to diagnose pathogens to guide the timely and appropriate treatment and development of e®ective interventions such as vaccines.[2] Clinically, point-of-care diagnostics for the identication of diarrhea pathogens is mainly based on bacterial culture, ELISAs, and molecular techniques.[3] The conventional bacterial culture method is still used as the gold standard for pathogen identication, but the process usually takes several days to complete.[4] ELISA is rapid and precise, but its application is limited by the availability of pure antigens and subsequent production of specic antibodies.[5] Nucleic acid amplication-based methods are preferred because they enable the accurate identication of target nucleic acid sequences with high sensitivity and specicity, and nucleic acid amplication methods are less time consuming and less vulnerable to cross-contamination. LAMP has attracted considerable interest because it can be implemented with a single enzyme (strand displacing polymerase) using simple electrodes or heating elements.[11]
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