Rapid Nucleic Acid Amplification And Visual Detection Strategies For The Detection Of Food-Borne Pathogen And Genetically Modified Organisms

Nucleic acid amplification and detection technology is very sensitive through amplifying the template for million times.It plays an important role in the field of food safety detection.However,it is mostly used in laboratory work and not suitable for practical applications.In this study,we analyzed nucleic acid extraction,amplification and detection separately,developed several rapid,sensitive and visual detection methods for foodborne pathogen and genetically modified organism(GMO)testing.Moreover,we proposed a new strategy to count the number of DNA molecules in each sample with naked eye observasion.Main contents and results are summarized as following:(1)In this work,loop mediated nucleic acid amplification(LAMP)combined with phosphate-based visual detection was used for Vibrio parahaemolyticus detection from spiked shrimp samples.Vibro parahaemolyticus collected from shrimp samples by disposable Q-tips was treated with sodium hydroxide(NaOH)and the rough cell lysate was used as DNA template for LAMP amplification after 10-fold dilution.The amplification products were tested through phosphate induced coloration reaction and colorimetric results were observed with naked eye.Results indicated that the detection sensitivity was 2.63×10~3 CFU/mL for pure cultured Vibrio parahaemolyticus and 3.00×10~3 CFU/g for Vibrio parahaemolyticus spiked shrimp samples,respectively.Then,151 shrimp samples in total were tested with this visual detection method.In parallel,all shrimp samples were tested with traditional clony counting method as standard.Results indicated that the positive detection rate of shrimp samples was 94.7%(143/151).The detection specificity and sensitivity of the visual detection method were 100%(65/65)and90.7%(78/86),respectively.The proposed visual detection method could be accomplished within 1 h,avoided cross-contamination during sample preparation and only a simple thermo block was required.Thus,it demonstrated great potential for rapid and in situ foodborne pathogen detection.(2)In this work,we found that pullulan could be used as cross-priming amplification(CPA)acceleration additive.Results indicated that 1%(w/v)concentration of pullulan worked best.It could accelerate CPA reaction by 7 min,which was equal to one third of the time for CPA to reach the cycling threshod(Ct).With 1%(w/v)concentration of pullulan as CPA acceleration additive,genetically modified(GM)rice DNA could be detected with lateral flow dipstick(LFD)in 20 min.In order to prevent amplicon contamination caused by uncapping operation,three types of enclosed and portable LFD cartridges were developed.After DNA amplification,LFD detection could be easily achieved with a simple reversed shake or gentle press.Rapid,sensitive and specific DNA detection could be realized with pullulan as CPA acceleration additive coupled to LFD detection.With LFD cartridges,DNA amplification and LFD detection were carried out in enclosed space,thus totally avoid amplicon contamination from the source.(3)In this work,a simple,rapid and visual detection method was developed for GTS 40-3-2soybean screening.The unpured DNA template was directly used for body temperature triggered RPA amplification.With the help of a mini UV-touch,fluoresent results were observed after adding SYBR Green I to the amplicon products.It could be finished within 10min from sampling to results,with thermo block abandoned.This visual detection method demonstrated high sensitivity and specificity,shown high robust to different length of primers(22~30 bp),unpurified DNA template and various incubation temperatures(30~40°C).Results illustrated that the positive detection rate of GTS 40-3-2 soybean samples was 100%by 20volunteers tested under different ambient temperature.This body temperature triggered-RPA amplification and visual detection method was rapid,sensitive,high robust and thermo block-free,which was suitable for rapid and in-the-field GM crops screening.(4)In this work,a simple approach was proposed to count the number of DNA molecules via amplicon cluster-based readouts in minutes,without any pre-modification or physical separation process.This method was generally based on ultrafast nucleic acid amplification,extremely weak convection and diffusion effect of amplicon molecules in the thin flexible plastic tube.In this paper,a home-made device was built to shuttle the thin flexible plastic tube between two water baths for rapid PCR amplification within 5 min.After amplification,DNA amplicon was combined with pre-added fluorescent dye and generated fluorescent amplicon cluster.By counting the number of amplicon clusters with naked eye,the initial amount of DNA molecules in each sample could be known easily.Moreover,a diffusion and amplification model during rapid PCR amplification was proposed based on the diffusion process of amplicon cluster in the thin flesible plastic tube.This visualized DNA molecules counting method was direct,ultrafast and easy to be implemented,omited complex microfluidic device and precise optical equipment,providing a new strategy for counting DNA molecules in while-you-wait testing and injecting distinctive ideas into the fields of single molecule detections.