Picrate paper kits for determination of total cyanogens in cassava roots and all forms of cyanogens in cassava products

The simple semiquantitative picrate method for the determination of total cyanogens in cassava flour has been modified by increasing the concentration of the picrate solution used to make up the picrate papers, such that a linear Beer's Law relation between absorbance and cyanogen content is obtained over the range 0–800 mg HCN equivalents kg⁻¹ cassava. The method has been adapted to determine the total cyanogen content of cassava roots and the results compared using the picrate method and the acid hydrolysis method for six different roots from five cultivars. The agreement between the results is satisfactory. The simple method for determination of total cyanogens in cassava roots in the field is available in kit form. The methodology has been modified to allow determination of the three different forms of cyanogens present in cassava flour, viz HCN/CN⁻, acetone cyanohydrin and linamarin. HCN/CN⁻ is determined by the picrate method in which cassava flour is reacted with 0.1 M sulphuric acid for 3 h at room temperature. HCN/CN⁻ plus acetone cyanohydrin is also determined by the picrate method after treating cassava flour with 4.2 M guanidine hydrochloride at pH 8 for 3 h at room temperature. A comparison has been made of the amounts of the three cyanogens present in six cassava flour samples using the semiquantitative picrate and the acid hydrolysis methods. The agreement between the two methods is satisfactory, which shows that the new methodology works well. The picrate method for determination of the three cyanogens in cassava flour is also available as a kit. © 1999 Society of Chemical Industry     

Cyanogen content of cassava roots and flour in Indonesia

A survey has been made of the total cyanogen content of cassava roots and products from the cassava growing provinces of Lampung and East, Central and West Java, in Indonesia. Twenty five samples of cassava products were analysed for cyanogens by the acid hydrolysis method and also by the simple picrate kit method. The mean percentage difference between the results was 17%. Thirty samples of cassava starch and other specialised products had a mean cyanogen content of only 5 ppm, whereas 29 samples of cassava flour, chip and gaplek gave a much higher mean cyanogen content of 54 ppm (SD 51). The WHO safe value for cassava flour is 10 ppm and the Indonesian level is 40 ppm. There are four outliers of cyanogen content 140–200 ppm, which would be dangerous to human health. The cyanogen content of starch/chips/gaplek needs to be reduced by using cultivars of lower cyanogen content and by using improved processing methods. Twenty seven samples of cassava roots gave a mean cyanogen content of 19 ppm (SD 14). ©     

Prediction of Colloidal Stability of Highly Stabilized Beers by a Modified Chapon Tannoid Content Test

The tannoid content test, introduced by Chapon, is accepted as a recommended MEBAK analytical method for the detection of haze‐forming polyphenols in beer and other intermediates. Under certain conditions, the results can be used directly for predicting the colloidal stability of beer. Its disadvantage is that it does not provide detectable results for beers stabilized by high doses of polyvinylpolypyrrolidone (PVPP). A test modification is proposed that increases the test sensitivity and gives non‐zero results, even for highly stabilized samples that are not measurable by the standard test. The modification is based on adding saturated ammonium sulphate solution (SASS) to the sample to reach 9% saturation (0.5 mL of SASS to 5 mL sample volume) before the actual test performance. The results of the modified test can simply be converted to results obtained by the standard method. Tannoid content correlates linearly with the content of haze‐forming polyphenols and with chill haze produced after defined accelerated aging. In a set of beer samples of one brand having different degrees of stabilization, a high correlation was determined between test results and long‐term colloidal stability. The modified test can be used as a rapid and simple test for checking the stabilization process. It is particularly suitable for Pilsner type beer of long (more than 6 months) colloidal stability controlled by stabilization on the side of polyphenols.     

A model freed from endogenous reference gene for quantification of genetically modified DNA by real-time PCR. 1. Quantification of DNA from genetically modified organisms in haplo-species materials

This paper is the first part of a serial study investigating a quantification model freed from endogenous reference gene for genetically modified (GM) content by real-time polymerase chain reaction (PCR). The serial study involves two parts: (1) quantitative determination of GM DNA in haplo-species plant samples; (2) quantitative determination of GM DNA in multi-species plant samples. The paper describes a methodology to quantify the GM content in a DNA extract using on one hand real-time PCR to determine the amount of GM targets present and on the other hand absorbance reading at 260 nm to measure the total DNA present in sample. The ratio of both values is expressed as GM percentage. The most prominent dominance of the novel model is that the direct quantitative relation between the initial amount of target template in real-time PCR reaction (X ₀) and the content of GM DNA in tested material (X _SD) is established. Theoretical analysis indicates that the developed quantitative model relieved from the dependence on endogenous reference genes is suitable to quantify the GM content in haplo-species plant sample, in addition, it has the applicability in the quantitative detection of GM content in multi-species GM plant sample. A trail, in which 75 haplo-species GM plant samples were involved, was conducted to validate the suitability of the novel quantification model. The bias varied from 0.00 to 24.00% except a tested sample with lower level of GM content, and the precision expressed as coefficient of variation (CV) was from 2.81 to 25.00%. The limit of quantitation (LOQ) of the quantitative assay was as low as 0.1%. Compared with the previous papers and the performance requirements raised by European Network of GMO Laboratories (ENGL) for analytical methods of GMO testing, the results demonstrated that the established quantification model is a suitable alternative to the more traditional endogenous reference assay in the quantification of GM content in haplo-species plant sample.     

Development of nanocolloidal gold based immunochromatographic assay for rapid detection of transgenic vegetative insecticidal protein in genetically modified crops

Vegetative insecticidal protein (Vip) is now being used for transgenic expression in several crops; conferring resistance against lepidopteron pests. A rapid, single step, sensitive and specific immunochromatographic (IC) strip test for the detection of recombinant Vip-S protein in the transgenic samples was developed. Polyclonal rabbit anti-Vip-S IgG conjugated to nanocolloidal gold served as a probe to detect Vip protein in test samples. The detection limit for the developed IC strip was 100 ng/ml (100 ppb) and on addition of gold enhancer the sensitivity increased to 1 ng/ml (1 ppb) of Vip-S protein. The assay was validated with transgenic brinjal samples. The assay time was less than 10 min, suitable for rapid on-site testing. No cross-reactivity was observed with other transgenic plant proteins employed for pest and weed management, i.e. Cry1Ac, Cry1Ab, and CP4-EPSPS. This on-site test offers rapid screening for a genetically modified crops having relatively new transgene (vip) entering the global market.     

VARIED ASSAYS FOR PSP TOXINS IN HEAT SHOCKED PHILIPPINE GREEN MUSSELS (PERNA VIRIDIS)

Toxin assays namely: the mouse bioassay, the receptor binding assay (RBA) and, the immuno-chromatography assay using MIST Alert™ rapid test kit were used to determine the concentrations of Paralytic Shellfish Poisoning (PSP) toxins from untreated and heat shocked Philippine green mussels, Perna viridis contaminated with Pyrodinium bahamense var. compressum. Toxin levels ranging from 4–15 μg STXeq/100 g sample were quantified in the mussel samples analyzed using RBA. Higher levels of PSP toxins at about 30 μg STX eq/100 g sample were recorded using mouse bioassay, which was attributed to interfering factors that could induce mouse death resulting in false positive reactions. The MIST Alert™ test kit showed positive reaction in the samples evaluated based on the reported average profile of PSP toxin analogues at about 40 μg STX eq/100 g sample. The test heat treatments did not elicit definitive change in the PSP toxin profiles of heat shocked mussels relative to the untreated samples.     

基于SVM方法的猪肉新鲜度分类问题研究

文中采取减压贮藏方式对新鲜猪肉进行了贮藏实验,测定了不同贮藏时间样品的挥发性盐基氮含量(TVB-N)、细菌总数、pH值及感官评价数据,并运用支持向量机(support vector machine,SVM)对这些样本数据进行训练,选取不同的核函数,得到SVM神经网络模型,随后利用此模型对测试数据进行猪肉新鲜度分类预测。实验表明,根据样本特性进行数据预处理,且选取合适的核函数后,SVM神经网络能得到极高的猪肉新鲜度正确分类率。     

Assessment of a new lateral flow immunochromatographic (LFIC) assay for the okadaic acid group of toxins using naturally contaminated bivalve shellfish from the Portuguese coast

A new rapid assay for the okadaic acid group of toxins, based on lateral flow immunochromatographic (LFIC) test strips developed by Jellett Rapid Testing Ltd., was assessed on naturally contaminated bivalves from the Portuguese coast. One prototype was evaluated using samples harvested during 2005, extracted with 80% methanol, followed by dilution with the running buffer of a methanolic extract after alkaline hydrolysis for esters. The second prototype was assessed using samples harvested during 2006, extracted with 100% methanol and, after alkaline hydrolysis, the methanol was evaporated by a nitrogen stream followed by re-suspension with the running buffer. The first prototype failed to detect 20% of samples that were positive by LC–MS in the range 160–480 µg kg^?1, and were classified as negative or trace level by LFIC. The presence of methanol in the extracts made correct detection of toxins more difficult. The second prototype classified as positive all samples above 160 µg kg^?1, as confirmed by LC-MS. However, in the second prototype, matrix effects were a major drawback and led to 45% false positives, particularly for mussels, due to compounds in shellfish extracts interfering with the antibodies and reducing the test line intensity. Extraction with a higher percentage of methanol was thought responsible for these matrix effects. Regarding sample migration, both prototypes needed one hour before reading. In an attempt to speed-up sample preparation, a direct digestion of bivalve tissues with sodium hydroxide was evaluated. Low recoveries for esters were found by LC–MS with this hydrolysis technique compared to conventional hydrolysis of methanolic extracts. While prototype A was not sensitive enough, prototype B had too many false positives to be of use to the shellfish industry or in a monitoring program.     

Development of a sensitivity‐improved immunoassay for the determination of carbaryl in food samples

BACKGROUND: With the aim of developing a highly sensitive immunoassay for carbaryl, a hapten which had high similarity to carbaryl was synthesised using a safer and more practical approach. After it was conjugated to horseradish peroxidase, direct competitive heterologous enzyme‐linked immunosorbent assay (CD‐ELISA) was optimised and characterised. The assay performance conditions were investigated in details. Enhanced chemiluminescence ELISA (ECL‐ELISA) was also used in preliminary studies. RESULTS: The assay obtained an IC₅₀ value (the concentration causing 50% inhibition) of 2 µg kg^?1, which was 12‐fold more sensitive than previous results of homologous CD‐ELISA. In ECL‐ELISA, the IC₅₀ was further decreased 10‐fold to 0.2 µg kg^?1. The CD‐ELISA developed was applicable for broad conditions, and could be applied on various food samples with a more convenient pre‐treatment. Average recoveries were in a range of 88.3–101.7%. The results correlated well with those obtained using high‐performance liquid chromatography (HPLC) analysis (R² = 0.989). CONCLUSION: The ELISA developed was a great improvement in the determination of carbaryl, showing that the immunoassay developed was a simple, rapid and efficient method that was reliable for the detection of carbaryl and suitable for rapid quantitative or qualitative determination in food samples. Copyright © 2010 Society of Chemical Industry     

Development of a quantitative lateral flow immunoassay for the detection of aflatoxins in maize

An immunoassay-based lateral flow device for the quantitative determination of four major aflatoxins in maize has been developed. The one-step assay has performance comparably with that of other screening methods, as confirmed by the intra- and the inter-day precision of the data (RSD 10–22%), and can be completed in 10?min. Quantification was obtained by acquiring images of the strip and correlating intensities of the coloured lines with analyte concentration by means of a stored calibration curve carried out by diluting aflatoxins in the extract from a blank maize sample. Limit of detection (1?µg?kg^–1) and dynamic range (2–40?µg?kg^?1) allows the direct assessment of aflatoxin contamination in maize at all levels of regulatory relevance. All reagents are immobilized on the lateral flow device. In addition, very simple sample preparation, using an aqueous buffered solution, has been demonstrated to allow the quantitative extraction of aflatoxins. Twenty-five maize samples were extracted with the aqueous medium and analyzed by the developed assay. A good correlation was observed (y?=?0.97x?+?0.07, r ^2?=?0.980) when data was compared with that obtained through an official method. The developed method is reliable, rapid and allows for application outside the laboratory as a point-of-use test for screening purposes.     

COMPARISON OF A REAL-TIME POLYMERASE CHAIN REACTION ASSAY WITH A CULTURE METHOD FOR THE DETECTION OF SALMONELLA IN RETAIL MEAT SAMPLES

A real‐time polymerase chain reaction (PCR) method developed in this study was compared with a conventional International Organization for Standardization culture method for the detection of Salmonella in Irish beef, chicken, pork and turkey. This also provided a snapshot of the incidence of Salmonella in such products. After selective enrichment, all meat samples (n = 100) were: (1) subjected to DNA extraction and real‐time PCR analysis using primers against Salmonella spp. specific gene (16S rRNA) or the invA virulence gene; and (2) plated on differential media and identified as Salmonella using immunological and sub‐typing methods. Retail samples (5/100) were shown to contain Salmonella using the 16S rRNA gene‐based real‐time PCR assay and the standard culture method. However, only two (of five) samples were shown to contain Salmonella using the invA gene‐based real‐time PCR assay. For the sample set examined, the developed 16S rRNA gene‐based real‐time PCR assay demonstrated comparable specificity and sensitivity to the currently used standard culture method but was considerably more rapid.     

Proficiency study for the determination of nitrofuran metabolites in shrimps

A proficiency test for the determination of nitrofuran metabolites in shrimp tissue was organized in the first half of 2003. This test was intended to allow the participants to use their routine method and to assess their competence on this specific analysis. The participation in this proficiency test was offered to all the National Reference Laboratories (NRLs) of the European Union (EU) in charge of the analysis of nitrofurans, to Official Laboratories of the then 10 Candidate Countries for entry in EU and to some countries exporting food to the EU. The participants (20) analysed nitrofuran metabolites in eight randomly coded frozen samples including three blank samples. All participants performed a confirmatory method using liquid chromatography/mass spectrometry to detect total nitrofuran metabolite residues. Both qualitative and quantitative analyses of the results were investigated. Qualitatively, 16 laboratories out of 20 gave the correct interpretation of the results in term of compliant/non-compliant sample. Quantitatively, laboratory performance was evaluated by calculating the z-scores.     

Validation of a commercial ELISA for the analysis of the insecticide dinotefuran in a variety of analytically challenging vegetables

The main objective of this paper was to assess the suitability of an enzyme-linked immunosorbent assay (ELISA) as a rapid and simple screening method for the detection of neonicotinoid insecticide dinotefuran residues in various vegetable samples including carrot, cabbage, green pepper, leek, Japanese mustard spinach (komatsuna), and spinach. Simple dilution of crude sample extracts was effective to circumvent or reduce matrix interference derived from samples. Consequently, ELISA was useful to determine dinotefuran accurately and directly, only requiring methanol extraction and dilution with water. The theoretically estimated limit of detection of dinotefuran for tested samples ranged from 0.06 to 0.12?mg?kg^?1. The ELISA gave recovery values close to 100% for all samples except komatsuna, which contained lower concentration levels, and the results of the ELISA agreed well with those obtained with conventional HPLC (r?>?0.99). Results show that the ELISA evaluated in this study is suitable for the rapid and simple screening method of dinotefuran residue in agricultural samples even without sample pre-treatment.     

Interlaboratory Proficiency Test Using MALDI-TOF MS for Identification of Food-Associated Bacteria

An interlaboratory proficiency test using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify food-associated bacteria was performed with 16 food microbiological laboratories, comprising ten authorities, four companies, and two academic institutions using either the Bruker Daltonics MALDI Biotyper system, the VITEK MS Ruo SARAMIS, VITEK MS IVD Ruo (bioMérieux, France) or PAPMID (Mabritec AG, Switzerland). A blinded set of 15 reference strains was selected from the Bavarian Health and Food Safety Authority strain collection, comprising common food-associated bacteria. Depending on the presence in the databases and appropriate interpretation of data, mean accuracy rates between 34 and 100% were achieved for the respective bacterial species. Percentages of correct identifications were compared independence of the platform and the databases used. The influence of the sample preparation method and interpretation of results were also considered. Our results showed that the platform itself had no significant impact on credit points achieved. The use of an adequate sample preparation method, as well as correct interpretation of results, was much more decisive for the results achieved. In-house databases improved correct identifications when the demanded species was not mapped in the commercial databases.     

Performance of several Cr and Yb analytical techniques applied to samples of different biological origin (digesta or faeces)

This paper compares the performance, in terms of Cr (liquid phase marker) and Yb (solid phase marker) recovery from spiked samples, of five mineralization procedures applied to faeces, whole duodenal digesta, particulate and liquid phases of duodenal digesta, whole ruminal digesta and ruminal fluid from cattle. Two Holstein–Friesian heifers, fitted with permanent ruminal and duodenal cannulae, and fed twice daily on barley straw and a concentrate (15:85), were used as sample donors. Cr and Yb were added to the samples at 5, 25, 125 and 625 µg g^?1 dry matter, and analyzed by atomic absorption spectrometry. There were differences in marker recovery (p = 0.0481) depending on the mineralization procedure utilized but their performance was not affected by marker type (p = 0.6143). The interaction between type of sample and digestion procedure was statistically significant (p = 0.0290) and the results showed that homogeneous samples (whole duodenal digesta, the particulate phase of duodenal digesta and faeces) allowed the more even distribution of the marker and an easier and more complete mineralization. In contrast, heterogeneous samples (whole rumen digesta and rumen fluid) resulted in either a lesser attachment of markers to the particles and/or incomplete mineralization of the samples, which resulted in a higher variability between procedures. The interaction between type of sample and marker was also significant (p = 0.0426), with Cr recovery being more affected by type of sample than Yb. Copyright © 2004 Society of Chemical Industry     

Development of an Ultrasensitive Immunochromatographic Assay (ICA) Strip for the Rapid Detection of Phenylethanolamine A in Urine and Pork Samples

In this study a one‐step immunochromatographic assay based on competitive format was developed for the rapid detection of phenylethanolamine A (PEAA) residues in urine and pork samples. A monoclonal antibody against PEAA was produced from BALB/c mice immunized with the PEAA‐BSA conjugate. The results of this qualitative test strip were to be interpreted visually. The visual detection limit (VDL) and threshold level of the optimized immunochromatographic assay for PEAA were 0.1 ng/mL and 0.5 ng/mL, respectively. Cross‐reactions with other β‐agonists were not significant inhibitions to the performance of the test strip assay. The results from the test strip were in a good agreement with those obtained using a high performance liquid chromatography‐tandem mass spectrometry (HPLC‐MS/MS) assay. The immunochromatographic assay developed here was a useful on‐site screening tool that is rapid to use, low in cost, and extremely convenient for the detection of PEAA in urine samples and pork samples.     

Isolation and Identification of Lactobacillus plantarum from Vegetable Samples

Lactobacillus plantarum, a non-pathogenic species of lactic acid bacteria, has considerable industrial and medical interest. It is used as starter culture in food and feed fermentation. For developing new starter culture usually needs identification from natural sources. Many literatures are available for its definite identification. But ambiguous results of phenotypic characterization and application of laborious and expensive molecular approaches has generated the need of a rapid molecular assay. Therefore, a specific PCR assay is described which exploits PCR amplification of conserved gene sequences encoding for plantaricin biosynthesis protein by designed species specific primer pair plnX (F/R). This has generated 228 bp amplicons with test and positive controls. This assay was established by six genotypically identified test strains of vegetable samples, two positive and four negative controls. The described assay could be used for preliminary screening of L. plantarum from large samples before performing a battery of phenotypic and molecular methods.     

高通量微生物显色法快速检测动物源性食品中抗生素残留

建立一种采用高通量微生物显色法对动物源性食品中抗生素残留进行筛检,再结合高效液相色谱-串联质谱(high performance liquid chromatography-tandem mass spectrometry,HPLC-MS/MS)法对阳性样品进行复测的分析检验方法。本方法利用嗜热脂肪芽孢杆菌(Bacillus sterothermophilus)作为指示菌,制备96微孔板,可同时对不同类别多种抗生素进行联合检测。结果表明:该微生物显色法操作简便、快捷,成本低,结果准确且易判断,选取pH 7.4磷酸盐缓冲液进行提取,以反应液150μL、菌液50μL(初始OD_(600 nm)为0.4左右)、样品提取液100μL作为检测体系,检测效果最佳,对动物源性食品中氨基糖苷类检出限为60μg/kg,β-内酰胺类检出限为20~40μg/kg,大环内酯类检出限为60~80μg/kg,四环素类检出限为40~60μg/kg,符合国内外对抗生素残留限量的要求。对60份动物源性样品进行检测,其结果与HPLC-MS/MS复测结果一致,说明该微生物法稳定性良好,可用于动物源性食品中抗生素残留的初筛检测。     

Milk progesterone enzyme immunoassay: modifications and a field trial for pregnancy detection in dairy cows

A milk progesterone enzyme immunoassay was modified to shorten the antibody:antigen incubation time and tested in a field trial. Coupling the antibody to paper fibers at pH 7 increased the binding activity of the paper-antibody conjugate to allow incubation for 3 h at room temperature with no significant loss of competitive binding of progesterone. The relationship between progesterone concentrations measured by the modified and original enzyme immunoassays was r = .94 (n = 80 pairs). Milk samples (n = 67) collected 21 d after artificial insemination were classified by the original and modified methods as being from pregnant or nonpregnant cows using both spectrophotometric and visual evaluations. Comparison to subsequent rectal palpation or return to estrus showed that the two methods were comparable. A field trial was conducted involving 622 cows and 40 producers using the modified enzyme immunoassay method. The overall field trial accuracy of the enzyme immunoassay in diagnosing the cow's reproductive status for a single sample on d 21 after breeding was 71% for correct diagnosis of pregnancy and 81% for correct diagnosis of nonpregnancy. It was concluded that the modified method is a valuable technique for rapid monitoring of milk progesterone concentrations.