Development Of Nucleic Acid Amplification Technique For The Analysis Of Food Samples

Nucleic acid amplification test(NAAT)is a sensitive molecular diagnostic technique and has been widely applied for food safety detection.Though there are various mature and stable protocols for each unit of NAAT,current NAAT is developing towards two major tendencies.On the one hand,the NAAT technique should be easy to operate,cost-effective,and able to deliver the results on-the-spot with no requirement of any complicated instruments.On the other hand,the technique should be automatic enough so that high-throughput could be reached with less labor force involved.Therefore,some novel methods for NAAT are in urgent demand.To develop simple and rapid food safety detection methods,herein we have proposed some alternative methods against different units of NAAT.The main contents and realated conclusions are as followed:1)Conventional methods for nucleic acid extraction are cumbersome and may not apply to samples of large volume.To solve this problem,we have proposed a genomic DNA extraction method based on magnetic beads.Silica-coated magnetic beads(Fe3O4@SiO2)are synthesized and used for genomic DNA extraction from rice.The obtained DNA is evaluated by agarose gel electrophoresis and real-time PCR.The results indicate that the highest DNA yield is obtained with 2%SLS as lysis agent and 5 M GITC as binding agent.The obtained DNA show better performance for real-time PCR than the commercial Mag-Bind(?)Plant DNA kit.The developed method based on magnetic beads is used for DNA extraction from rice powder contaminated with different levels of transgenic ingredients.And the limit of detection is 0.01%for uncooked rice and 0.1%for cooked rice.The Ct values show a very good linear relationship to the logarithm value of the transgenic ingredients,indicating that DNA extracted with magnetic beads meets the requirement of real-time quantitative PCR.Overall,the developed method based on magnetic beads is easy to operate and does not need the centrifuge,which is expected for automatic extraction in the future.2)Taking advantage of the strong tolerance to the inhibitors and the capability of being performed at a wide temperature range,a powerless method based on LAMP for detection of transgenic crops in the field has been developed.The method employs phosphate(Pi)-induced coloration reaction for analysis of the amplification products and does not need the DNA purification process.Results show that after 10-fold dilution,the lysis obtained from rice leaves with 0.5 M NaOH treated for 4 min can be used for LAMP reaction directly.When the temperature falls between 51.9 ? and 66.6 ?,the positive samples can be amplified in 40 min by the real-time LAMP.The best amplification performance is obtained when the reaction temperature falls between 58.0 ? and 63.0 ?,at which the plateau of LAMP can be reached in 20 min.Three kinds of dewar flasks are compared as the incubator for LAMP reaction,and with the best dewar flask,the templates can be detected by phosphate(Pi)-induced coloration reaction after 20 min amplification.The method is employed for detection of transgenic crops and the results are consistent with the real-time PCR analysis.Overall,DNA extracted from the transgenic crops with 0.5 M NaOH can be used for LAMP amplification without purification.With dewar flasks as the incubator and phosphate(Pi)-induced coloration reaction for analysis of the amplicons,the method does not need any power-dependent instrument during the 30 min detection duration and is easy to operate.3)Due to the severe non-specific amplification,the NEAA has been optimized with synthetic oligonucleotides as the template.Results show that the specific amplification could be inhibited by the severe non-specific amplification.The non-specific amplification is caused by the co-existence of DNA polymerase,dNTP,and primers.The highly active nicking enzyme directs and accelerates the non-specific amplification in a way which favors nicking.To suppress the non-specific amplification,a manual hot-start operation is recommended.And the nicking enzyme concentration,reaction temperature,and magnesium ion concentration are optimized.The compatibility of Bst polymerase and the concentration of monovalent cation is also crucial.Besides,the sensitivity could be enhanced by shortening the target sequences and priming the 3' end of the target.However,none of these methods can completely suppress the non-specific amplification.To avoid the interference of non-specific amplification in NEAA,a highly specific detection method has been developed with GTS 40-3-2 as the target.The sequences adjacent to the restriction site of nicking enzyme on CaMV 35S gene in GTS 40-3-2 are amplified by asymmetric NEAA for 10 min.And the generated products are captured by two antigen-labeled probes so that they can be detected by the lateral flow dipstick(LFD).The optimum concentrations of the primers are 100 nM and 400 nM,respectively.And the outer primer concentration is 100 nM.The optimum concentration of the probes is 80 nM.As low as 3 copies of the target gene and 0.1%transgenic contamination can be detected by LFD.To study the robustness of the method,DNA extracted from leaves,stem,roots,and seeds with 0.5 M NaOH are detected after 10-fold dilution without purification.All of the samples show positive amplification except the roots,and the results are consistent with real-time PCR,indicating the strong robustness of NEAA-LFD.The whole detection process can be integrated into a disposable cassette to prevent the cross-contamination.Characterized by the high reaction rate,high specificity,high sensitivity and strong robustness,NEAA-LFD has an extensive application prospect.4)Since real-time PCR is expensive and gel electrophoresis is cumbersome,we have developed a visual detection method for PCR based on the DNA binding dye.And nanomaterials are introduced to enhance the detection signal.Results show that graphene oxide(GO),reduced GO(rGO),molybdenum disulfide(MoS2),and tungsten disulfide(WS2)are effective for suppressing the background signal of PCR.The main reason for nanomaterials enhancing the signal is the adsorption of the primers and the SYBR Green ?.Besides,nanomaterials can eliminate the non-specific amplification caused by excess Taq polymerase.We have also confirmed that the sensitivity of visual detection with nanomaterials is consistent with real-time PCR.Overall,the method is very suitable for rapid evaluating the PCR amplification results due to the property of low cost,easy to operate,and high sensitivity.