Study On Identification Of Duck-derived Components In Mutton By QSRCA And Visual SRCA

Adulteration of mutton is an important problem in the field of food quality and safety.Driven by economic interests,the phenomenon of duck meat mixed with mutton has been reported many times.It not only infringes the interests of consumers,affects the operation of the market economy,violates relevant laws and regulations,but also easily causes allergies and health emergencies.Therefore,it is of great practical significance to develop an accurate,sensitive and rapid method for the identification of adulterated duck meat.In this study,the saltatory circle isothermal amplification(SRCA)technique was combined with the fluorescence technique to develop a real-time quantitative SRCA(qSRCA)quantitative detection method and a visual SRCA qualitative detection method,used for identification of duck meat components in mutton.The growth hormone(GH)gene was used as the target gene to design the specific primers for goat and duck,selected the suitable primers and optimized the reaction system.Finally,qSRCA quantitative detection and visualized SRCA qualitative detection method were established to identify the components of duck meat in mutton.(1)The established methods for quantitative detection of goat and duck by qSRCA were tested for specificity,absolute sensitivity,relative sensitivity,artificial imitation adulteration and real samples respectively,and were compared with qPCR quantitative methods,to evaluate the qSRCA quantitative detection method.The results showed that:In the specificity test,both goat and sheep samples were positive,10 non-mutton samples were negative,duck sample was positive,and 11 non-duck samples were negative.The results suggested that the method had good specificity.The absolute sensitivity of qSRCA for detecting goat DNA was 2.0×101 fg/μL,which was 1000 times more than qPCR.The absolute sensitivity of qSRCA for detecting duck DNA was 2.0×101 fg/μL,which was 1000 times more than qPCR.The relative sensitivity of qSRCA for detecting goat DNA in binary mixture was 0.001%(v/v),which was 1000 times more than qPCR.The relative sensitivity of qSRCA for detecting duck DNA in the binary mixture was 0.001%(v/v),which was 1000 times more than qPCR.Therefore,the qSRCA detection method has strong specificity and high sensitivity.In the artificial simulation adulteration detection test,different proportions of duck meat were artificially contaminated with goat,and the qSRCA quantitative detection test was performed.The relative error of the qSRCA detection method was between-20.00%and 13.00%(<±25%),and the coefficient of variation was between 0.96%and 4.63%.Therefore,the method has high accuracy and precision.Twenty-nine commercially available mutton samples were detected using three methods including industry standard(NY/T 3309-2018),qPCR and qSRCA.The results showed that the detection rate of duck meat ingredients of industry standard(NY/T 3309-2018)and qPCR were both 10.34%.The detection rate of duck meat ingredients in qSRCA was 17.24%,which was significantly higher than the other two methods.(2)The established visual SRCA qualitative detection methods for goat and duck were tested for specificity,sensitivity and detection limit to evaluate its qualitative detection performance.The test results showed that the goat and sheep samples emerged visible green fluorescence by the naked eye,which was judged as positive results,and the 13 non-goat samples all presented visible orange fluorescence,which was judged as negative results.The duck sample exhibited visible green fluorescence by the naked eye,which was judged as positive result.The 14 kinds of non-duck samples all demonstrated visible orange fluorescence by the naked eye,which was judged as negative results.Therefore,the specificity of the detection method was good.The sensitivities of visual SRCA for detecting DNA of goat and duck were both 2.0×101 fg/μL.The detection limit of visual SRCA for detecting duck meat components in mutton was 0.001%(w/w).In the detection of commercially available commercial samples,visual SRCA was used to detect duck meat ingredients in mutton samples,and compared with the current industry standard(NY/T 3309-2018).The detection rate was 17.24%,which was 6.9%higher than the industry standard.In summary,the qSRCA and visual SRCA established in this study can identify the duck meat components in mutton.The above two methods have the advantages of accurate,rapid and sensitive detection results,the visual method of qualitative detection of SRCA has the advantages of simplicity and visible results.Therefore,qSRCA quantitative detection method can be applied to the relatively good conditions of the laboratory or testing institutions for quantitative detection,visual SRCA qualitative detection is more suitable for the relatively backward conditions of the laboratory or basic-level testing institutions with relatively scarce resources.