PCR Detection Of Several Exogenous Vegetative Components In Cocoa Powder

Cocoa powder was a kind of nutrient-rich deep-processing food made of cocoa bean directly. It was the important raw material of chocolate and one of the three hobby drinks in the world. In the domestic market, its high price led to the fake and shoddy's flooding. The main fake was plant waste such as soybean meal, sesame meal, peanut shell and chestnut shell et al. But the existing test standards of cocoa powder and cocoa products were limited to fat content test and protein content test et al. These could not determine whether exogenous vegetative components adulterate or not. The paper based on PCR technique and established a rapid identification technology of detecting soybean, sesame, peanut and chestnut-derived ingredients in cocoa powder.Two modified CTAB methods and SDS method were contrasted with effects on extracting DNA from cocoa powder in this paper. Results showed that: the DNA yields of all of the three methods were high, and the DNA purity was only little difference between the two CTAB methods, but the SDS method was lower. 18S rDNA gene in high plants was used as an internal control to identify DNA quality. And it could be amplified with template DNA extracted by the two CTAB methods, but not by SDS method sometimes. It indicated that these two modified CTAB methods were competent for DNA extracting from cocoa powder.PCR primers were designed by software Primer Premier 5.0 against genes those announced in GenBank database, such as soybean's kti-s, sesame's oleosin, peanut's 2S protein and chestnut's leafy-like et al. The species-specific primers were screened by PCR amplifications which template DNA was extracted from soybean meal, sesame meal, peanut shell and chestnut shell individually and using the corresponding genome DNA as positive control, cocoa genome DNA as negative control. The species-specific PCR primers were as following: soybean, 5'-GAAACGGCGGCACATACT-3', 5'-GGCAGACCCTTGAGAATA -3'; sesame, 5'-CCTTCCTTGGCTGTGCTCTTA-3', 5'-TTCGTCGCTTTCTTGGGTTC-3'; peanut, 5'-TAAGGCAAAGGGTGGAGC-3', 5'-GCAGTTCTGGGGCAAGTT-3'; chestnut, 5'-TAGGTTTGCGAAGAAGGC-3', 5'-ACGGATAGACGGGGATGT-3'. The length of each amplicon was 266 bp, 153 bp, 267 bp and 167 bp respectively.Primers above were used to PCR detect corresponding objective DNA. The PCR system was following: 10×PCR buffer 5μL, MgCl2 ( 25 mmol/L) 4μL, dNTPs ( 5 mmol/L) 4μL, upstream and downstream primers ( 10μmol/L) each 2μL, Ex-Taq ( 5 U/μL) 0.3μL, template DNA 2μL ( 100 ng-200 ng), ddH2O up to 50μL. The PCR conditions were following: predegenerated at 95℃for 5 min; then at 94℃, 50 s→Ta ( annealing temperature of each primer pairs), 50 s→72℃,45 s,43 cycles; at last 72℃extended for 8 min. The PCR products were examined by agarose gel electrophoresis. Target bands'occurring illustrated there would be corresponding material adulterations. In this way, a PCR method was established for detecting exogenous vegetative components of soybean, sesame, peanut and chestnut in cocoa powder.This PCR detection method could detect less than 50 mg submitted under test samples. And also could detect DNA solutions extracted from soybean meal, sesame meal, peanut shell and chestnut shell each in no more than 82.5 pg/μL, 32.5 pg/μL, 124 pg/μL and 157 pg/μL. The detection limits of these four materials in cocoa powder (w/w) were 0.5%, 0.5%, 5% and 5% respectively. This method had a good repeatability and suitable to detect these exogenous vegetative components in cocoa powder qualitatively.