Detection of cashew allergen in food with PCR

Objective To develop a PCR method for detection of cashewa llergen in food.Methods Nut DNA was extracted with alkyol trmie thylammon iumbromide(CTAB)and magnetic micro spheres.Nine pairs of prmiers were designed according to cashe wallergenic prote in gene sequences from NCBI GenBank and a pair of specific prmiers was screened o ut by am plify ing results.Th irteen nut sam ples were determ ined using do uble PCR with the cashew specific prmier pair AnaO3F3R3 and plant universal prmier pair Plant159.The sensitivity of the PCR method was detected by DNA gradient dilution expermient and the detection of cashew allergen in food was smiulated by stmiulated sample expermient.Results A₂₆₀/A₂₈₀ of DNA extracted by CTAB method was lower than 1.8,which meant there was prote in contamination.While A₂₆₀/A₂₈₀ of DNA extracted by magnetic bead method was higher than 1.8,which meant the prote in was cleaned.The specificity of prmier pair AnaO3F3R3 was best among the 9 prmier pairs and the best anneal temperature was 55.4.Double PCR method was established to detect cashew and the other 12 kinds of nuts with prmier plant 159 and AnaO3F3R3.Only for cashew the 312bp specific band was amplified.The sensitivity of the method was 0.1pg/l.And 0.001% cashew allergen in food could be detected.Conclusion APCR method for detection of cashew allergen was deve loped and the method can be used to detect cashew allergen effectively,rapidly,differentially,and sensitively.