| Background:In clinical laboratory test,biological warfare,food industry and environmental monitoring,rapid detection and accurate identification of pathogens are very important.The traditional microbiological methods for bacterial identification include selective culture,biochemical and serological identification.These conventional approaches almost rely on the isolation and selection of viable bacterial cells using a specific agar medium.For example,culture is the most commonly used identification method in the laboratory.Using a variety of media,samples from infected individuals were seeded on the plate,allowing the microorganisms to grow on them.Identification of bacteria based on microbial phenotype and growth medium.However,these culture experiments for bacterial analysis need days or weeks to provide accurate results.In fact,over the past few decades,many rapid methods have been developed to reduce the detection time.So far,the research on the rapid detection method include enzyme linked immunosorbent assay(ELISA),lateral flow immunoassay and polymerase chain reaction(PCR),the detection time can be reduced to 10-24 h and 4-6 h,the detection limit can reach 102-106 cfu/ml.However,these tests require the use of higher cost instruments and professional operators to limit their wide application,especially in developing countries and regions.They are not the best choice for Point-of-Care Test(POCT)and the popularization of the grass-roots unit.Therefore,the development of a rapid,simple and highly specific detection technology for pathogenic microorganisms detection has important clinical significance.Nucleic acid isothermal amplification is characterized by amplification from beginning to end at a constant temperature.Compared with common PCR,it reduces the cost of temperature control equipment and shortens the amplification reaction steps and time,an ordinary thermostatic apparatus can meet.Therefore,in the diagnosis of POCT,nucleic acid isothermal amplification technology has more practical value than the common PCR technology.In nucleic acid isothermal amplification,Loop mediated isothermal amplification(LAMP)is a more stable techniques.By using a kind of Bst DNA polymerase and specific primers,the target sequence can be amplified by isothermal(~65℃)in a short period of time(30-60min),with high sensitivity and specificity.In addition,it only needs a thermostatic apparatus instead of an expensive thermal cycle,which significantly reduces the detection cost.Therefore,the aim of this study is to develop a simple and rapid method for the detection nucleic acid using loop mediated isothermal amplification(LAMP).Microfluidic chip technology is also known as the "lab-on-a-chip",first proposed by Manz A and Widmer in 1990,which is a integration sample preparation,biochemical reaction,test results analysis in a few square centimeters of chip technology.The features of Microfluidic chip technology include high efficiency,low energy consumption(including time,sample and reagent),miniaturization and high-throughput analysis of experimental.Through a combination of sensor or sample pretreatment module,the Microfluidic chip technology provides a rapid diagnosis for POCT.Paper material,has a rich source,cheap,sustainable,stable,easy processing,easy transportation and easy modification,good biocompatibility and other characteristics,combined with modern printing technology can be easily prepared and can be used for the microfluidic device with isolation,analysis and detection.However,the use of microfluidic chip is limited in current market,mainly used to detect the immune protein and nucleic acid detection in the use of less.It may be due to the change of the temperature of PCR method,so there are some difficulties in the operation of the chip.In this study,we explored the LAMP on the microfluidic chip and the detection of the amplified product,which can improve the use value of the microfluidic chip for POCT.Objective:The present study aims to fully integrate the characteristics and advantages of LAMP technology and paper-based microfluidic chip technology,the design and construction of LAMP-paper microfluidic chip,to establish a rapid and simple detection method based on LAMP technology for the early diagnosis and early treatment of pathogens on the spot or field.Methods:1.Establishment of a rapid and simple detection method for pathogenic microorganisms and drug resistance genes by a visual LAMP method.1)Collection of Pseudomonas aeruginosa clinical isolates,Antimicrobial susceptibilities test of imipenem resistance were detected by the agar dilution method.2)Rapid extraction of DNA by a simple boiling method.3)Design of LAMP primers for OprL and Opr D2 gene of Pseudomonas aeruginosa.4)Establishment of a visual LAMP reaction system.5)Specificity and sensitivity of the visual LAMP assay.6)Detection of clinical specimens.7)The resistance of OprD2 negative and Opr D2 positive strains to imipenem was statistically analyzed.2.Design and fabrication of a microfluidic chip for isothermal amplification of nucleic acids.1)Detection of pathogenic microorganism by LAMP on bioactive paper.2)Design of a microfluidic chip for isothermal amplification of nucleic acids.3)Simple fabrication of paper microfluidic chips using Whatman#1 filter paper.4)Microstructure observation of a paper microfluidic chip by field emission scanning electron microscopy.3.Establishment and optimization of a streptavidin-biotin LAMP system.1)Establishment of a streptavidin-biotin LAMP system: according to the covalent coupling effect of biotin and streptavidin,the biotinylated LAMP product was immobilized on the surface of the paper chips labeled with gold nanoparticles-streptavidin.2)The optimal temperature,time,the concentration of d NTPs,BstDNA fragment polymerase,MgSO4,Betaine and the internal and external primers were optimized by fluorescence quantitative PCR.3)The sensitivity and specificity of streptavidin-biotin LAMP reaction were analyzed by fluorescence quantitative PCR.4)Clinical isolates detection.Results:1.successfully simplified the extraction of pathogenic microorganisms DNA operation,shorten the experimental time.A rapid,sensitive and simple method for detection of Opr L and Opr D2 genes in imipenem resistant Pseudomonas aeruginosa was successfully established by a visual LAMP.Its specificity was good.Sensitivity(17.41μg/L)was 10 times higher than that of general PCR method.the traditional method(bacterial culture,VITEK 2 system,and agar dilution method)were used to test the collected clinical isolates while simultaneously using 1.0 μL of DNA for visual LAMP and conventional PCR methods.For Opr L gene,the sensitivity and specificity of the visual LAMP was 100%,and the general PCR was 94.9% and 87%,respectively.For OprD2 gene,the sensitivity and specificity of the visual LAMP was 75% and 73.7%,and the general PCR was 94.9% and 87%,respectively.Statistical analysis showed that there was significant difference in imipenem resistance between Opr D2-negative strains and –positive strains(P < 0.05).2.Pseudomonas aeruginosa was used as an example,and the nucleic acid DNA was successfully amplified on paper by LAMP.3.Design and fabrication of a paper microfluidic chip for isothermal amplification of nucleic acids.Apply for and authorize a national utility model patent(Paper microfluidic for isothermal amplification of nucleic acids,ZL 201420583698.X),a national invention patent(Method for isothermal amplification of nucleic acid using paper microfluidic,201410528335).4.The streptavidin-biotin LAMP reaction system was successfully constructed,which laid a solid foundation for the next step of fixation and signal amplification detection in the microfluidic chip.And the LAMP reaction conditions were optimized: get the best reaction temperature was 65℃,reaction time was 40 mins,the optimum concentration of MgSO4 was 6 m M,Bst DNA polymerase concentration was 0.32 U/μL and d NTPs mixture concentration was 1.2 m M,b-FIP/BIP inter primer concentration was 1.6 μM,F3/B3 outer primer concentration was 0.4 μM,the Betaine concentration was 0.6 m M.Pseudomonas aeruginosa as an example,streptavidin-avidin LAMP analysis method using fluorescence quantitative PCR detection based on its good specificity,sensitivity(0.402 μg/L)100 times higher than the visual LAMP,1000 times higher than conventional PCR.The clinical isolates of Pseudomonas aeruginosa were detected by this method.The detection rate was similar to that of visual LAMP(14/14,100%),while the conventional PCR(13/14,93%).Conclusions:1.Visual LAMP method for rapid detection of pathogenic microorganisms and their resistance genes has the characteristics of short detection time,simple process,high sensitivity and specificity,and the results can be seen by the naked eye.In this study,Opr L and OprD2 gene of Pseudomonas aeruginosa was taken as an example to establish a visual LAMP detection technology which result is simple and convenient.Statistical analysis showed that Opr D2 negative strains had higher risk of imipenem resistance than Opr D2 positive strains.Early identification of the Opr L and OprD2 genes will help us better select antibiotic treatment options for IRPA infection,which will reduce costs and time.This method is more suitable for POCT and primary medical institutions.2.Microfluidic chip technology,also known as a "lab-on-chip",is a technology that integrates sample preparation,biological or chemical reactions,the results of detection and analysis on a few square centimeter chips.It has the characteristics of high efficiency and low consumption(including time,sample and reagent),miniaturization and quantitative analysis,and provides a rapid diagnosis method for field detection and timely detection by combining the sensor or sample pretreatment module.The paper has many advantages as the biological sensing material.Its use makes the biosensor more compact,convenient and easy to carry,and low detection cost.In this study,the integration of LAMP technology and paper-based microfluidic chip technology,successfully designed and fabricated a paper microfluidic chip based on isothermal nucleic acid amplification for the next step in the field early qualitative or quantitative detection of pathogenic microorganism infection. |
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