Establishment Of The Multiplex PCR With An Internal Amplification Control For The Detection Of Foodborne Pathogens

Salmonella,Escherichia coli 0157:H7,Shigella,Listeria monocytogenes,Bacillus cereusy Cronobacter sakazakii,Vibrio parahaemolyticus and Staphylococcus aureus are common foodborme pathogens.In this study,PCR detection methods with internal amplification control(IAC)and a two-tube synchronous multiplex PCR detection method with IAC were established with newly designed primers and two pairs of reported primers targeting Salmonella,E.coli 0157:H7,Shigella,L.monocytogenes,B.cereus,C sakazakii,V.parahaemolyticus and S.aureus.PCR detection methods for single species of bacteria each were evaluated by the specificity,sensitivity and detection limit of artificially contaminated food samples.Specificity test results showed good specificity.The sensitivity test and artificial contaminated test results showed that,the sensitivity for detection Salmonella genomic DNA and pure culture were 61.11 fg/?L and 2×102 CFU/mL,respectively,and the detection limit for Salmonella in artificially contaminated eggs was 2 CFU/25 mL by the Salmonella PCR method with IAC.The sensitivity for detection Escherichia coli 0157:H7 genomic DNA and pure culture were 3.18×102 fg/?L and 2.2×102 CFU/mL,respectively,and the detection limit fox Escherichia coli o157:H7 in artificially contaminated milk was 2.2 CFU/mL by the Escherichia coli O157:H7 PCR method with IAC.The sensitivity for detection Shigella genomic DNA and pure culture were 7.55×102 fg/?L and 31 CFU/mL,respectively,and the detection limit for Shigella in artificially contaminated pork was 0.31 CFU/g by the Shigella PCR method with IAC.The sensitivity for detecting Listeria monocytogenes genome DNA and pure culture were were 1.80×102fg/?L and 1.21×102CFU/mL,and the detection limit for Listeria monocytogenes in artificially contaminated milk was 0.48 CFU/mL by the Listeria monocytogenes PCR method with IAC.The sensitivity for detecting Bacillus cereus genome DNA and pure culture were 2.68×102 fg/uL and 7.97×103 CFU/mL,and the detection limit for Bacillus cereus in artificially contaminated milk was 8 CFU/mL by the Bacillus cereus PCR method with IAC.The sensitivity for detecting C.sakazakii genome DNA and pure culture were 2.15×102 fg/uL and 9.4×103 CFU/mL,and the detection limit for C.sakazakii in artificially contaminated milk powder was 0.94 CFU/g by the C.sakazakii PCR method with IAC.Multiplex PCR reaction is carried out synchronously in two tubes.Vibrio parahaemolyticus,Escherichia coli O157:H7,Salmonella and Shigella were tested in the reaction tube 1.Listeria monocytogenes,Bacillus cereus,Cronobacter sakazakii and Stophyloeoccus aureus were tested in the reaction tube 2.In this study,the concentration of Taq DNA polymerase,primers,Mg2+,dNTPs and the annealing temperature,extension time,cycle times of PCR conditions were optimized to determined the best reaction conditions.Twenty-four strains of bacteria were detected which showed that the multiplex PCR method has a favorable specificity.Sensitivity test results showed that,the genomic DNA sensitivity of V.parahaemolyticus,E.coli O157:H7,Salmonella,Shigella,L,monocytogenes,B.cereus,C.sakazakii and S.aureus were 104,104,103,102,103,103,102,102 fg/?L,respectively,and the pure cultures sensitivity were 103,103,104,103,104,104,10,10 CFU/mL,respectively.The pork that artificial contaminated with eight pathogenic bacterias were detected after 12 h enrichment culture and the result showed that,the detection limit of V.parahaemolyticus was 102 CFU/mL,E.coli 0157:H7 and Salmonella were 10 CFU/g,Shigella,L.monocytogenes,B.cereus,C.sakazakii and S.aureus were 1 CFU/g.