| Since the reform and opening-up,China’s aquaculture industry has developed rapidly and China has become the country with the world’s largest aquaculture yield.Under high-density and intensive aquaculturing conditions,various diseases have also occurred frequently and spread widely,which has become a major factor plaguing the aquaculture industry.E.tarda,V.harveyi and V Splendidus are three common pathogens in marine aquaculture in northern China and have caused huge economic loss to the global aquaculture industry.It is of great significance for pathogen monitoring and effective prevention to establish rapid and accurate detection technology to achieve early diagnosis of pathogenic bacteria and immediate detection in basic level operators.Loop-mediated isothermal amplification(LAMP)technology uses DNA polymerase with highly active property of strand displacement to perform amplification under constant temperature conditions(60-65℃)for 30-60 minutes,which is less demanding to template purity and temperature control.This method has the advantages of simplicity,rapidity,sensitivity,specificity,low cost,etc.,and has been widely applied to rapid detection of pathogens on site.Microfluidics is a kind of science and technology characterized by manipulation of fluids in micrometer scale,which is also known as the lab-on-a-chip.This microfluidic technology provides a possible solution to the main problems related to pathogen detection,such as portability,low cost,automation,etc.Aiming at the major issues of real-time on-site detection of pathogenic bacteria,this work uses the nucleic acid analysis microfluidic system to detect the three bacteria of E.tarda,V.harveyi and V.Splendidus.The research includes microfluidic-based nucleic acid extraction of pathogenic bacteria,construction of LAMP detection system for pathogenic bacteria,and rapid detection of multi-sample and multi-target.The main research work and related results are as follows:(1)A novel method was provided for nucleic acid extraction,which was based on microfluidic technology integrating rotating three-dimensional magnetic field and horizontally moving magnetic field.We developed the multi-channel/multi-chamber microchips and a supporting magnetic control microdevice for nucleic acid extraction.The functionality was verified and the performance was investigated preliminarily.(2)A new set of LAMP primers for V Splendidus was designed and screened,and the temperature for the LAMP reaction was optimized.Basic performance was tested for the LAMP detction of V.Splendidus by using the microfluidic real-time LAMP system.It was initially constructed for the LAMP detection method of V.Splendidus on microfluidic system.(3)The main performances,including specificity,sensitivity and reproducibility,were investigated for the LAMP detection of E.tarda and V.harveyi on the microfluidic system.An on-chip LAMP method was constructed for multi-sample and multi-target detection.The proposed microfluidic-based nucleic acid extraction process can be automated,fast,efficient and easy to integrate;the operation of on-chip LAMP detection is simple and reliable(primers can be pre-dehydrated in reaction wells on the LAMP chip;only one step of adding samples and reagents is required;the chip is sealed and fixed with a dedicated plastic shell,which can effectively avoid contamination),and the detection instrument is low-cost and portable(size 353 mm × 340 mm×178 mm,weight about 7 kg),enabling field application and promotion.The results of this study will provide effective technical support for on-site rapid detection of marine aquaculture pathogens as well as for effective prevention and control of related diseases. |
PDF Full Text Request