Rapid detection of multiple pathogens by the combined loop-mediated isothermal amplification technology and microfluidic chip technology.

Abstract

A rapid detection system for multiple pathogens that combines loop-mediated isothermal amplification (LAMP) technology and microfluidic chip technology was established. Primers were designed for the specific conservative genes of the multiple pathogens. A rapid detection method for multiple pathogens based on LAMP technology was established using the primer screening and optimization of reaction conditions. The microfluidic chip was designed. manufactured, and combined with the LAMP method for detection pathogenic bacteria as detected by a chip detector. After this, the detection limit and anti-interference ability of the chip were evaluated, and the accuracy of chip was verified by clinical samples. A rapid microfluidic detection system for Staphylococcus aureus, Escherichia coli, Pneumoniae klebsiella, Shigella, Methicillin-resistant Staphylococcus Aureus (MRSA) and Candida albicans was established. The detection limits of the 6 strains above were 6.95, 44.6, 3.89, 15.33, 16.45, and 463 pg/µL, respectively; there was no cross-reaction with the other 15 strains; analysis of ROC curve showed the best cut-off values for the 6 strains are 38.5, 21.25, 31.5, 36.5, 22.5 and 33.75 respectively and the area under the curve for the 6 strains was 0.91, 0.91, 0.83, 0.97, 0.96, and 0.9, respectively, as analyzed by receiver operating characteristic curve (ROC). A total of 278 clinical samples collected including blood, urine, sputum and drainage fluid were analyzed, and the total coincidence rates were 0.91, 0.83, 0.75, 0.99, 0.92 and 0.76, respectively. The establishment of microfluidic detection chips for multiple pathogen types can be used for the rapid detection of bacteria and may be particularly useful in environments with the limited equipment and personnel, such as infection sites, grassroots hospitals, disaster area rescue, etc.