The Saltatory Rolling Circle Amplification For Sensitive Visual Detection Of Pork And Horsemeat Adulteration In Beef And Its Products

Meat adulteration remains a global challenge.In recent years,meat adulteration events emerge in endlessly,and the public has become more and more concerned about the quality and safety of the meat consumed,and aware of the harm caused by the contamination of exotic meat.Meat adulteration not only has a negative impact on the economic interests of consumers,but also causes harm to people with allergies constitution and religious believers.The traditional detection method has complicated steps,time-consuming and low sensitivity,and cannot realize rapid detection of meat adulteration.Therefore,it is of great significance to develop a rapid and sensitive detection method for adulteration of meat products.This study established a Saltatory rolling circle amplification(SRCA)method for the detection of adulterated horsemeat and pork in beef.SRCA method can accomplish in vitro isothermal amplification with a pair of primers,and the test results can be directly visualized by observing fluorescence with the naked eye.This method is reliable,sensitive,specific,rapid and low cost.In this study,the cytochrome b(cyt b)gene of beef,horsemeat and pork was used as the target gene to design primers,and the SRCA reaction system and conditions of beef,horsemeat and pork were optimized,and the corresponding primers.Optimal reaction system and reaction conditions of beef,horsemeat and pork were finally determined.The optimal SRCA reaction conditions and system for beef are:reaction temperature(63℃),reaction time(60 min),10 mM dNTPs(5.0 μL),20 mM Mg2+(2.5 μL),10×ThermoPol Reaction Buffer(2.0 μL),10 μM forward and reverse primers(2.0 μL),template DNA(1.5μL,negative not added),8 000 U/mL Bst DNA polymerase(3.0 μL),the reaction system was supplemented with sterile deionized water to 20.0 μL.The optimal SRCA reaction conditions and system for horsemeat are:reaction temperature(64℃),reaction time(65 min),10 mM dNTPs(5.0 μL),20 mM Mg2+(2.5 μL),10×ThermoPol Reaction Buffer(2.0μL),10 μM forward and reverse primers(1.0 μL),template DNA(2.5 μL,negative not added),8 000 U/mL Bst DNA polymerase(1.0 μL),the system was supplemented with sterile deionized water to 20.0 μL.The optimal SRCA reaction conditions and system for pork are:reaction temperature(62℃),reaction time(65 min),10 mM dNTPs(3.0 μL),20 mM Mg2+(1.0 μL),10×ThermoPol Reaction Buffer(0.5 μL),10 μM forward and reverse primers(1.5 μL),template DNA(0.5 μL,negative not added),8 000 U/mL Bst DNA polymerase(2.0 μL),the reaction system was supplemented with sterile deionized water to 20.0 μL.Fresh muscle tissue from nine different animals was selected to verify the specificity of the method.The results showed that the three pairs of primers corresponding to beef,horsemeat and pork had high specificity.Template DNA was extracted from pure beef,horsemeat,pork and their binary mixtures by kit method,respectively,for SRCA reaction,and compared with PCR method.The results showed that the sensitivity of beef,horse and pork SRCA gel electrophoresis were 7.1×101 fg/μL,6.3×101 fg/μL and 6.5×101 fg/μL,respectively.The sensitivity of SRCA fluorescence visual method were 7.1 × 100 fg/μL,6.3 × 100 fg/μL and 6.5 × 100 fg/μL,respectively.The sensitivity of PCR was 7.1 × 103 fg/μL,6.3 × 103 fg/μL and 6.5 ×103 fg/μL,respectively.SRCA method was used to detect adulterated horsemeat in artificially contaminated beef,the detection limit was 0.01%by gel electrophoresis and fluorescence visualization,and 0.1%by PCR.SRCA method was used to detect adulterated pork in artificially contaminated beef,the detection limit was 0.01%by gel electrophoresis and fluorescence visualization,and 0.1%by PCR.Therefore,SRCA gel electrophoresis is 100 times more sensitive than PCR.Fluorescence visualization is 1000 times more sensitive than PCR.SRCA fluorescence visualization is 10 times more sensitive than gel electrophoresis.The detection limits of SRCA fluorescence visualization and gel electrophoresis were 10 times that of PCR.In this study,horsemeat and pork in 66 beef products were tested by industry standard methods(NY/T3309-2018),the sensitivity,specificity and coincidence rate of the SRCA method were 100%,98.41%and 98.48%for detecting horsemeat in beef products,and 100%,96.66%and 98.48%for detecting pork in beef products,respectively.In conclusion,it is feasible to use SRCA method to detect the adulteration of horsemeat and pork in beef,and SRCA method has the advantages of high specificity,high sensitivity,low cost and fast detection speed,which is particularly suitable for promotion in grass-roots testing institutions.