A PCR-based method was developed to distinguish between durum/common wheat and common wheat by leveraging slight differences of DNA sequence in Starch Synthase II (SS II) coded on wheat A, B and D genomes. A primer pair, SS II ex7-U/L, was designed to hybridize with a conserved DNA sequence region found in SS II-A, B and D genes. Another primer pair, SS II-D 1769U/1889L, was constructed to recognize a unique sequence in the SS II-D gene. The target region of SS II ex7-U/L with the size of 114 bp was amplified from durum and common wheat DNA, while no amplification was observed from any cereals other than those in the wheat genus. A DNA fragment with the size of 121 bp was specifically amplified from common wheat with SS II-D 1769U/1889L. In blended flour prepared from wheat and other cereals, the developed PCR system composed of two primer pairs effectively detected durum/common wheat and common wheat. These results suggested that PCR using two primer pairs is useful for detecting common and/or durum wheat in blended flour and could be utilized to ensure accurate food labeling.
To develop a method for detecting GM wheat that may be marketed in the near future, we evaluated the proline-rich protein (PRP) gene as an endogenous reference gene of common wheat (Triticum aestivum L.) and durum wheat (Triticum durum L.). Real-time PCR analysis showed that only DNA of wheat was amplified and no amplification product was observed for phylogenetically related cereals, indicating that the PRP detection system is specific to wheat. The intensities of the amplification products and Ct values among all wheat samples used in this study were very similar, with no nonspecific or additional amplification, indicating that the PRP detection system has high sequence stability. The limit of detection was estimated at 5 haploid genome copies. The PRP region was demonstrated to be present as a single or double copy in the common wheat haploid genome. Furthermore, the PRP detection system showed a highly linear relationship between Ct values and the amount of plasmid DNA, indicating that an appropriate calibration curve could be constructed for quantitative detection of GM wheat. All these results indicate that the PRP gene is a suitable endogenous reference gene for PCR-based detection of GM wheat.
This study examined the distribution of aflatoxigenic fungi in 25 imported Indonesian nutmeg samples contaminated with aflatoxins Bs or Bs and Gs. The incidence of aflatoxigenic fungi in the samples contaminated with high levels of aflatoxin was significantly higher than that in the samples with low levels of the toxins(r=0.752). The aflatoxin production of isolates from the samples in cultures of YES broth was examined by means of TLC and HPLC analyses. The ability of isolates to produce aflatoxins did not necessarily correlate with the contamination levels of aflatoxin in the samples. We isolated aflatoxins B and G-producing fungi from 3 samples contaminated with the high levels of aflatoxins B and G. The aflatoxigenic isolates were identified as Aspergillus nomius and A. bombycis based on morphological characters, growth rates at 37°C and 42°C and also molecular-genetic methods. Our results indicate that these two species are mainly responsible for aflatoxin G contamination in nutmeg products.
A method using liquid chromatography with tandem mass spectrometry has been developed for the determination of clomeprop and its metabolite clomeprop acid in livestock and seafood products. Clomeprop and clomeprop acid were extracted with acetone–n-hexane mixture under acidic conditions, and were defatted by liquid–liquid separation using acetonitrile and n-hexane, followed cleanup with SAX and PSA cartridges. The average recoveries from 10 kinds of food (bovine muscle, bovine fat, bovine liver, milk, yellowtail, salmon, eel, fresh water clam, egg and honey) spiked at the level of the MRLs or at uniform limits (0.01 ppm) were in the range of 81–97% for clomeprop and 93–101% for clomeprop acid. Repeatability was in the range of 2.1–14% for clomeprop and 1.3–4.0% for clomeprop acid. The quantitation limits were 0.002 mg/kg for clomeprop and 0.00154 mg/kg (0.002 mg/kg as clomeprop) for clomeprop acid.
The broth microdilution (BMD) method is an antimicrobial susceptibility testing method defined as a guideline by the Clinical and Laboratory Standards Institute (CLSI). To date, the Japanese Veterinary Antimicrobial Resistance Monitoring System (JVARM), has adopted the agar dilution (AD) method for monitoring antimicrobial resistances targeting isolates of Enterococcus spp., found in the fecal flora of food-producing animals, as indicator bacteria. However, the AD method is tedious, and time-consuming. In order to examine whether it could be replaced with the BMD method, the two methods were compared in terms of the correlation of MICs. In this study, the BMD results agreed with the AD results within ±1 log2 dilutions in 72.3% of cases, except for the antimicrobial feed additive, Nosiheptide (NHT). Similarly, except for NHT, the MIC50 and MIC90 values obtained by the two methods were well correlated. In conclusion, our results indicate that the BMD method might be suitable for antimicrobial susceptibility testing targeting Enterococcus spp..
In Japan, maximum residue limits for pesticides (MRL) in coffee are set on green coffee beans, but not roasted coffee beans, although roasted beans are actually used to prepare coffee for drinking. Little is known about the behavior of pesticides during the roasting process. In the present study, we examined the changes in the concentration of pesticide (organochlorine: γ-BHC, chlordane and heptachlor) residues in coffee beans during the roasting process. We prepared green coffee beans spiked with these pesticides (0.2 and 1.0 μg/g), and the residue levels in the beans were measured before and after the roasting process. We determined the residual rate after the roasting process. γ-BHC was not detectable at all, and more than 90% of chlordane was lost after the roasting (3.1 and 5.1% of chlordane remained in the beans spiked with 0.2 and 1.0 μg/g of chlordane, respectively). A low level of heptachlor (0.72%) was left in the coffee beans spiked with 1 μg/g of heptachlor. Disappearance of γ-BHC during the roasting process may be due to the high vapor pressure of γ-BHC, while chlordane has a lower vapor pressure. We also examined the behavior of piperonyl butoxide and atrazine during the roasting process. Piperonyl butoxide behaved similarly to chlordane, but atrazine disappeared after the roasting process, because it is unstable to heat.
In this study, the identification of mushrooms by using DNA analysis was investigated. Our analysis of internal transcribed spacer (ITS) regions revealed that a DNA-based method could be applicable for samples that are difficult to distinguish in terms of the morphological characteristics. PCR amplification using templates extracted from cooked samples gave sufficient fragments to analyze the sequence. However, treatment with simulated gastric fluid (SGF) for more than 30 min affected the analysis of the ITS region. Application to samples of vomit is also discussed.
A method for rapid analysis of multi-class residual veterinary drugs in livestock products was developed and validated in accordance with the Japanese guideline for pesticides. Using LC-MS/MS, 43 multi-class veterinary drugs, including sulfonamides, quinolones, coccidiostats and antiparasites, could be analyzed simultaneously in only 18 minutes. The extraction process was developed by modifying the QuEChERS approach to provide faster and less expensive extraction. The samples were extracted by using two kinds of solvent, acetonitrile and acetonitrile including 1 vol% formic acid, and salted out with magnesium sulfate, trisodium citrate and sodium chloride. Using these two extractants, 40 out of 43 drugs satisfied the guideline criteria in bovine muscle and swine muscle, 39 drugs were found in chicken muscle, and 37 drugs were found in eggs. The limit of quantification was less than the MRL for all analytes.