| In 2008,the outbreak of the "Sanlu milk powder" incident makes dairy safety received extensive attention of society.For the high nutritional dairy products which means higher nutrition and higher market price,such as camel milk,donkey milk and yak milk for the special groups,some illegal businessmen adulterate high-value milk products with low-cost dairy product under the drive of interests,which not only harm the rights of consumers,but perhaps produce some health problems,so it is of great significance to establish a fast and sensitive method to detect dairy products adulteration.At present,the identification of adulterated dairy products faces two major problems: first,the absence of on-site rapid detection technology;Second,the lack of quantitative determination technology.To solve these two problems,the rapid extraction method of DNA from liquid milk products was studied based on liquid milk for six species commonly available in the market;And a rapid identification method based on colloidal gold strip was developed to identify yak milk;In addition,the single-copy nuclear genes were chosen as species-specific primers of camel and donkey and reference primers,and used fluorescent quantitative PCR to establish a quantitative method to determinate the content of camel milk and donkey milk in adulterated samples,respectively.The main research deteails and consequences are as follows:(1)A rapid extraction method of genomic DNA from liquid milk was developed,and the best pyrolysis conditions were optimized.We added 300 ?L 0.5 mol/L of Na OH lysis buffer to 1 mL liquid milk products and boiled the mixture at 100 ? for 5 min,after centrifuged for 10 min at 13400 r/min,the DNA of liquid milk products of different species was extracted,and the detection limit was up to 1%cow milk content by agarose gel electrophoresis after PCR amplification.(2)A lateral flow nucleic acid assay(LFNAA)based on RPA technology was developed,which could be used for the identification of yak milk.The use of special tail primers can produce a special product with a single stranded nucleotide tail that can be hybridized with colloidal gold labeled capture probe.At the same time,the biotin modified by forward primer can be captured by the streptavidin on the test line,forming a "sandwich structure",and showing a visible red band on the test line,while the excess gold-labeled capture probe continues to flow to the control line and is captured to show a red color.The method avoids the use of expensive antibodies and greatly reduces the cost of lateral flow assay based on immunoassay technique.After 20 min amplification at 37~42 ?,the results can be directly observed with the naked eyes within 5min,and as low as 5% yak milk can be detected in the mixture of yak milk and cow milk.The whole amplification and detection process can be completed in40 minutes without the use of complex heating instrument,which is very suitable for on-site testing.(3)A fluorescence quantitative PCR method for the determination of camel milk and donkey milk was developed.Single-copy nuclear genes HEG1 and GHR were selected as the species-specific gene of camel and donkey respectively,and the gene MSTN was selected as the reference gene,and then based on the Ct ratio(specific/reference genes)and the content of camel milk or donkey milk(specific/totalmilk),quantitative standard curves were developed for 5~100% camel milk and donkey milk.The linear formulas were Y =-0.0017 x + 1.1319(camel milk)and Y =-0.0023 x + 1.2288(donkey milk),respectively,and the correlation coefficients were 0.9615 and 0.9650,respectively.The recovery rates of the simulated samples ranged from 90% to 120%,CV<10%,and the CV value of inter-assay analysis<5% and the CV value of intra-assay analysis <6%.This method reduced the deviation of different DNA extraction method and PCR amplification efficiency,and was proved as a simple and fast method for the determination of camel and donkey milk content in adulterated milk samples. |
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