A Rapid Detection Method For Multi-bacteria Pathogens In Chinese Medicinal Decocting Pieces Based On Microfluidic LAMP Technology

ObjectivesChinese medicinal decocting pieces（CMDP）work as clinical drugs in Traditional Chinese medicine,and its market demand is increasingly ris-ing which is in the pillar postion of industry of Chinese materia medica.Once CMDP occur security issues,the healing medicine could instantly become hazardous drugs,which seriously endangers national health.Alt-hough the safety standards of the national GMP,Chinese Pharmacopoeia（Version 2020）and local production regulations have become stricter,pathogenic contamination of CMDP still happens from time to time,and it is difficult to completely eliminate.Currently,the prevalently used meth-ods for monitoring pathogenic bacteria in CMDP mainly include the gold standard method culture-based identification and the conventional PCR-based methods.These methods are still time-consuming,laboratory-or bulky equipment-dependent,high cost,and low throughput,so these meth-ods fail to meet the in-field,low-cost,and rapid detection requirements for pathogenic bacteria in CMDP.Loop-mediated isothermal amplification（LAMP）is proposed as an alternative to PCR for nucleic acid amplification,because it is thermo cycling-independent,high sensitivity,short amplifica-tion time,and good tolerance to inhibitors.Based on above,we believed that the application of microfluidic chips loaded with LAMP could exhibit several prominent advantages including operational automation,low cost and high efficiency.Until now,there is no such technology in development or application for detecting phathogenic bacteria in CMDP.In this study,we aimed to develop a rapid detection technique based on microfluidic LAMP technology for simple,rapid,accurate,sensitive,multi-target de-tection of pathogenic microorganisms in CMDP.Methods1.Targeting these four common pathogenic bacteria in CMDP（Salmo-nella,Escherichia coli O157:H7,Staphylococcus aureus and Shigella）,we investigated relevant literatures and bioinformatically analyzed rel-evant genomes.We specifically searched for the virulence gene se-quence with specificity,and then we designed,screened,and validatedLAMP primer groups for these four pathogens based on virulence genes.2.We established the LAMP reaction system and figured out the opti-mized conditions including temperature,amplification time and rea-gent concentrations.3.We further selected the LAMP primer group with good specificity afterthe specificity test of all the designed primers.During the specificity test,bacteria with various taxonomic relativity to the target bacteria were used as the negative samples.4.Based on the optimal reaction conditions and the screened primers,we next validated the amplification efficiency and detection limit of pri-mers by using artificial plasmids and targeting genomes.5.Taking Staphylococcus aureus as an example,we compared the detec-tion limit and response time of LAMP to PCR.We clarified the ad-vantages of LAMP over PCR.6.We designed and prepared a microfluidic chip with 10 channels,and we tested the specificity,sensitivity,limit of detection of the proposed microfluidic LAMP platform by loading LAMP system on the micro-fluidic chips.After these tests,we evaluated the practicability of this microfluidic LAMP platform.7.Then,biological samples with complex matrices were used as samples to verify the recovery and sensitivity of the microfluidic LAMP detec-tion system.The complex samples were prepared by mixing different bacteria into different biological matrices such as milk,apple,yogurt and pork.8.Finally,we prepared bacterial contaminated CMDP including Lycium chinense,Glycyrrhiza uralensis,Rehmannia glutinosa,and Cyathula officinalis.We analyzed the specificity and limit of detection during the pathogenic monitoring of these four contaminated CMDP.Results1.In the beginning,we designed 4 sets of primers targeting gene sir A in Salmonella,6 sets of primers targeting gene sp A for S.aureus,4 sets of primers targeting gene ipa H for Shigella,and 2 sets of primers against gene fli C for E.coli O157:H7.2.The LAMP reaction system was optimized and confirmed the optimal temperature（65°C）,concentration of Mg²⁺（6 m M）and betaine（0.6M）.3.At least one specific LAMP primer set was screened for every pathogen,and the target genes could be detected in 20 min under optimal reaction conditions.4.The detection of limit examination demonstrated that the designed pri-mers could detect as low as 10~3 CFU/m L pathogens for genomic sam-ples,and achieve less than 10~2 copies/reaction for the plasmid samples.5.During a certain detection time（45 min）,we confirmed that the LOD of LAMP was lower by an order of magnitude than that of PCR after comparing the electrophoresis results of products from PCR and LAMP reaction.6.On the microfluidic LAMP platform,we verified the specificity and detection limit of the targeting genomes and plasmids of the four path-ogenic bacteria.The results proved the feasibility of the microfluidic LAMP platform.7.We prepared simulated samples（such as milk,apple,yogurt,pork）spiked with Staphylococcus aureus and Shigella.And we extracted the bacterial genomic DNA by simple methods,and the genomes were ex-amined on the microfluidic chips.The results demonstrated the favor-able anti-interference capability with appropriate recovery rate（86.1%–110%）and relative standard deviation（<9%）.8.By targeting bacteria in the contaminated CMDP,the proposed micro-fluidic LAMP platform can specifically assay four pathogenic bacteria with LODs as low as 50-100 CFU/g,which is highly consistent with the result in tube system.The work fully supported the feasibility of our microfluidic LAMP platform on determining bacterial pathogens in CMDP.Conclusions1.In this study,we obtained LAMP primer sets with a moderate targeting range,which can be used for multi-target pathogenic bacteria detection.2.This microfluidic LAMP platform could be used for specific detection of pathogenic bacteria in complex-matrix samples.The reaction results could be identified by both visible and fluorescent signals,which can facilitate the development of fluorescent-independent equipment.3.This study preliminarily demonstrated the feasibility of detection of pathogenic bacteria in CMDP by LAMP technology,and also provided a research basis for its microfluidic application.4.The microfluidic LAMP technique developed in this study has several advantages,including rapid response,low LOD,multiple target,and high automation,which is highly potential for application in on-site detection of bacterial contamination on CMDP.This study provides a solid pre-study for on-site and rapid visualizing detection of pathogenic bacteria in CMDP.