Study On The Rapid Detection Technology Based On Nucleic Acid In Donkey-hide Gelatin Adulteration And Bacteria Detection

In recent years,food safety problems have surfaced with the rapid development of economy,such as the problem of high-value food adulteration,drug residues in farmed animals and so on.As an important technical means of food safety supervision,continuous innovation and progress of detection technology is an important guarantee for effective supervision.Food safety detection technology is divided into "routine detection technology" and "rapid detection technology" two categories."Routine detection technologies" are accurate and reliable,but with poor timeliness,long detection time,high cost,and the need for special testing instruments and professional operators."Rapid detection technologies" as a screening and the law enforcement support technology,such as chemical colorimetric analysis technology,biosensor technology,biochip technology,etc.,based on the guarantee of the accuracy of the test results,can shorten the testing time,simplify the operation process,reduce the testing cost,and especially not the restriction of the environment and equipment.Any operator can carry out testing at any time and any place by applying the relevant technology of rapid detection,which greatly improves the efficiency of food safety supervisionThis study focuses on the research and practice of biological rapid detection technology based on nucleic acid detection.In this study,a rapid and facile technology for donkey-hide gelatin adulteration was developed based on single-copy housekeeping nuclear reference primers.In addition,a recombinant polymerase amplification based lateral flow strip was also developed for S.pullorum identification.This study includes the following contents:(1)Donkey-hide gelatin is highly recognized for its high nutritional value,especially the medicinal value.However,it is also a potential candidate for adulteration because of its low yield and high price.In order to quantitatively detect adulterated donkey-hide gelatin with all possible adulterated animal species,a real-time PCR approach based on single-copy housekeeping nuclear reference primers was proposed in this study.For the system establishment,mixtures containing designated contents of pig hide with donkey-hide were employed to generate a calibration curve based on the ratio of Ct(Specificity/Reference)with reasonable linearity(5%-100%).Then,a set of experiments were performed on practical samples.The proposed PCR approach could specifically identify donkey-hide from mixed animal products and quantify the content of donkey-hide gelatin,thus facilitating the control over this novel form of donkey-hide gelatin adulteration.The results suggested that the proposed method is a rapid and facile technology for donkey-hide gelatin adulteration detection.The assay showed high specificity and sensitivity for donkey-hide admixed with pig hide up to 5%-100%.As the deliberate adulteration for seeking economic profit is generally more than 5%,the proposed method is helpful for economically motivated detection of donkey-hide gelatin adulteration.(2)Pullorosis is a bacterial disease threatening the poultry industry and has been listed as the bacterial disease to be eliminated by the government.Traditional serotyping for Salmonella detection is costly and labor-intensive,while other commonly used plate agglutination test methods often cause physical injury of chickens.Therefore,a rapid and non-damaging detection method is of great significance for early diagnosis,which is the key step in accurate medication and elimination of pullorosis.In this study,we propose a novel lateral flow nucleic acid assay(LFNAA)system combining recombinase polymerase amplification for the detection of S.pullorum.In this method,the DNA of S.pullorum strains was quickly amplified by RPA.Reverse primers were modified using spacer C3 and tail nucleotide sequences,which could be combined with gold nanoparticle probe,while forward primers labeled with biotin were bound to streptavidin at the test line,resulting in chromogenic reaction on the test line after amplification of target DNA by RPA on the lateral flow nucleic strips.The proposed assay is fast,ultra-sensitive and delivers visible results to naked eyes in field test.The sensitivity and specificity of the proposed LFNAA system were evaluated using agarose gel electrophoresis method and traditional serotyping tests.The limit of detection for genomic DNA was 5×10-3 ng/μL,indicating high sensitivity.Additionally,this method can be employed for identification of S.pullorum in chicken farms.The proposed LFNAA system is cost effective,efficient and non-damaging to chicks in field test.This system provides technical support for early diagnosis of S.pullorum in the poultry and paves a way for future precision medicine to avoid the global drug resistance issues.Then,A summary of breakthroughs and innovations in international cooperative project management have been taken.The project is the key special program of intergovernmental international innovative cooperation in science and technology,a National Key Research and Development(R&D)Programs,based on Interim Regulations on the Management of the National Key R&D Program supported by National Scientific Research Fund(2017)No.152.This program has realized innovative cooperation between mixed scientific research teams,flat management,comprehensive and omnidirectional publicity,and extension from a "small program circle" to a "large academic circle" in drug resistance and animal welfare during the program implementation process.These novel methods and ideas can be promoted for management and implementation of subsequent international cooperative programs.