Molecular techniques for the detection of granary weevil (Sitophilus granarius L.) in wheat and flour

The granary weevil (Sitophilus granarius L.) is a stored grain pest that causes major economic losses. It reduces the quantity and quality of the grain by its feeding and excretion. Sequences of S. granarius mitochondrial cytochrome oxidase subunits genes mtCOI and mtCOII were analysed and compared with mtCOI/II sequences available in GenBank. The analysed genes displayed a high level of homology between corresponding subunits. Attempts were undertaken to develop detection methods for contamination by S. granarius in wheat and wheat flour based on the molecular biology techniques: standard and real-time polymerase chain reaction (PCR) with a TaqMan molecular probe. (TaqMan probes are dual-labelled hydrolysis probes) Specific primers designed based on available sequences for mtCOI and mtCOII genes were applied and optimal reaction conditions established. The specificity of both methods was studied by using a species closely related to S. granarius: S. oryzae and S. zeamais. It is shown that the sensitivity threshold was very high - we were able to detect the equivalent of one beetle per 100 kg of flour when the real-time PCR with TaqMan probe method was applied to model samples. The primer sets used turned out to be species specific, and the technique was rapid, reliable and very sensitive.     

Some factors affecting egg-laying of the granary weevil (Sitophilus granarius L.)

The effect of some factors affecting egg laying of the granary weevil Sitophilus granarius was studied. In choice-tests, females laid less eggs in grains previously infested, but in no-choice tests, there was no statistical difference between the number of eggs in sound grain (59 per female) and previously infested grain (66 per female). There was a negative correlation between the number of egg plugs deposited previously and the additional number of plugs: the greater the number of plugs initially, the fewer were added. Chemical analyses of plugs and female abdomens showed a compound appearing in both extracts. It was tentatively identified as a glycosidic derivative of hexose. The kernels from which cuticular waxes had been washed with n-hexane were less attractive for egg-laying females than the sound ones. Chemical analyses of grain cuticular lipids revealed alkanes, wax esters, triacylglycerols, free fatty acids, alcohols, sterols and unknown compounds. Grain fungal volatile metabolites such as 1-octanol and 2-methyl-1-butanol added to sterilized kernels increased egg laying.     

Highly sensitive PCR-based detection method specific for Aspergillus flavus in wheat flour

Aspergillus flavus is frequently found in food, producing a wide variety of toxins, aflatoxins being the most relevant in food safety. A specific PCR-based protocol for this species is described which allowed discrimination from other closely related species having different profiles of secondary metabolites from the Aspergillus Section Flavi, particularly A. parasiticus. The specific primers were designed on the multi-copy internal transcribed region of the rDNA unit (ITS1-5.8S-ITS2 rDNA) and were tested in a wide sample of related species and other fungal species commonly found in food. The PCR assay was coupled with a fungal enrichment and a DNA extraction method for wheat flour to enhance the sensitivity of the diagnostic protocol. The results indicated that the critical PCR amplification product was clearly observed for wheat flour contaminated by 10² spores after 16 h of incubation.     

A study of the behaviour of the grain weevil Sitophilus granarius (L.) at the Pitfall Cone trap using a method to identify individuals

The effect of marking individual grain weevils (Sitophilus granarius) was examined by observing the behaviour of marked and unmarked beetles at the Pitfall Cone trap in wheat. The handling and marking of the beetles did not result in significant differences in behaviour when compared with unmarked beetles. Observations with marked beetles showed that most individuals made more than one visit to the trap. The technique allows greater accuracy in behavioural studies on grain weevils at traps.     

Detection of granary weevil Sitophilus granarius (L.) eggs and internal stages in wheat grain using soft X-ray and image analysis

In order to prevent grain mass and quality losses, rapid methods for early detection of insect infestation of cereal grain during trade and storage are urgently needed. Amongst many options, the soft X-ray method using roentgenograms is one of the most frequently applied. It has been shown that when some corrections for working parameters of the equipment used are made and some modification of the digital image analysis introduced, the soft X-ray method is suitable for accurate detection of granary weevil eggs laid in wheat kernels if at least 5 days after oviposition have elapsed.     

3种染色剂检测大米中米象虫卵感染效果比较

比较了改进后的高锰酸钾、酸性品红、碘-碘化钾溶液处理感染米象虫卵大米的染色效果和检出结果,研究了高锰酸钾对新陈大米卵斑的检测效果.试验结果显示,相对于感染虫卵的大米中幼虫达43.3％的情况,高锰酸钾溶液染色的百粒大米卵斑检出率最大,可达29.0％;新大米感染虫卵的第1天卵斑检出率最高,为26.0％,且不同感染时间的卵斑检出率差异显著;陈大米感染虫卵的第1天卵斑检出率也最高,为22.0％,不同感染时间的卵斑检出率差异不显著.结果表明,采用质量分数为1％的高锰酸钾溶液染色处理大米10s,可较为快速、准确地检测大米中米象虫卵感染情况;新大米感染虫卵的时间对染色检出卵斑效果有显著影响,而陈大米感染虫卵的时间对染色检出卵斑效果影响不显著.     

The effect of wheat grain composition, cuticular lipids and kernel surface microstructure on feeding, egg-laying, and the development of the granary weevil, Sitophilus granarius (L.)

Adult feeding intensity, oviposition, and larval development of Sitophilus granarius (Coleoptera: Curculionidae) were observed on grain from three Polish wheat varieties (Begra, Korweta, and LGR 896/64a) washed with petroleum ether to remove cuticular lipids. Extraction of lipids did not cause any statistically significant changes in the physicochemical, biochemical and technological (milling, rheological and baking) properties of the wheat grain studied. Wheat grain washed with petroleum ether did not show any visible changes in the surface and morphology of the outer layer of the wheat grain. However, differences were noted in the microstructure of the kernel surface. Grain with a thicker seed coat (LGR 896/64a) was infested at a lower rate than the other varieties. Of the 18 hydrocarbons extracted from the grain surface, three compounds - n-heptacosane (C₂₇), n-nonacosane (C₂₉) and n-hentriacontane (C₃₁) - were found in significant amounts. In general, beetles produced 64-95% less dust and laid 7-16% fewer eggs in kernels from which cuticular lipids had been removed. This implies that these compounds have a major role in food selection and the search for an oviposition site prior to grain infestation.     

鱼肉制品中巴沙鱼源性成分的实时荧光聚合酶链式反应快速检测法

目的 建立巴沙鱼源性成分的实时荧光聚合酶链式反应(PCR)快速检测手段.方法 根据巴沙鱼的线粒体cytb基因序列设计引物,使用实时荧光PCR进行扩增,从而达到快速检测产物的目的.结果 此方法特异性良好,巴沙鱼基因组DNA灵敏度可达到10-4 ng,在与婴幼儿米粉、儿童副食芝麻粉、鸡肉粉和大西洋鳕鱼粉混合的鱼肉制品中均可...     

The detection of foodborne pathogens by the polymerase chain reaction (PCR)

Nucleic acid probes are being used increasingly in the food industry. These can be relatively insensitive but a new technique for amplification of a target sequence of nucleic acid has the potential of being rapid, sensitive and specific. The method, termed polymerase chain reaction or PCR, utilises a thermostable DNA polymerase from the bacterium Thermus aquaticus. Following thermal denaturation of double stranded DNA, small oligonucleotides, termed primers, are annealed to the single strands of DNA and these determine the starting point for replication of the DNA by the polymerase enzyme. Applications for food microbiology are being researched but problems have been encountered due to the complex nature of many foodstuffs.     

不同破壁方法提取绍兴黄酒麦曲中的微生物总DNA

分别采用超声波、Yatalase酶法、氯化苄法对黄酒麦曲样品进行破壁,提取微生物总DNA,并通过细胞裂解率、DNA的纯度、片段的分布情况、ITS(内转录间隔区)序列扩增产物的多态性来评估不同的破壁方法对麦曲样品总DNA提取效果的影响。实验结果表明,采用氯化苄法能裂解86%的孢子,OD260/OD280在1.5左右,所得DNA片段分布在100bp～20kbI,TS-PCR扩增出的条带最多,而且简单、经济、提取效果好,可以作为麦曲中微生物总DNA的提取方法。     

多重PCR技术在食源性病原细菌检测中的应用

食品中污染的病原细菌是引起食源性疾病的主要因素之一,快速检测食品中病原细菌是及时有效预防病原细菌传播及食物中毒的重要前提。多重PCR作为一种可同时检测多种病原细菌的方法应用日益广泛。本文介绍了多重PCR技术在食源性病原细菌检测中的应用现状及其多重PCR反应条件的优化,同时提出了存在的问题并对应用前景作了展望。     

Real-time polymerase chain reaction for quantitative detection of histamine-producing bacteria: use in cheese production

Biogenic amines are toxic substances that appear in foods and beverages as a result of AA decarboxylation. The enzyme histidine decarboxylase catalyzes the decarboxylation of histidine to histamine, the biogenic amine most frequently involved in food poisoning. The aim of the present work was to develop a real-time quantitative PCR assay for the direct detection and quantification of histamine-producing strains in milk and cheese. A set of primers was designed, based on the histidine decarboxylase gene sequence of different gram-positive bacteria. The results show the proposed procedure to be a rapid (total processing time <2 h), specific and highly sensitive technique for detecting potential histamine-producing strains. Chromatographic methods (HPLC) verified the capacity of real-time quantitative PCR to correctly quantify histamine accumulation.     

Susceptibility to contact insecticides of granary weevil Sitophilus granarius (L.) (Coleoptera: Curculionidae) originating from different locations in the former Yugoslavia

The insecticides dichlorvos, malathion, chlorpyrifos-methyl, pirimiphos-methyl, deltamethrin and cypermethrin were applied to filter paper to assess their toxicity to granary weevil Sitophilus granarius adults. Based on discriminating doses obtained from tests on a susceptible laboratory population, the susceptibility of 12 populations originating from different storage facilities (11 in Serbia and 1 in Bosnia-Herzegovina) was tested. The facilities included silos, floor stockpiles and attic stockpiles. Weevils originating from Apatin, Belgrade Port, Bijeljina and Kikinda were submitted to toxicity testing, and determination of ld-p lines, LD values and levels of susceptibility/resistance. Chlorpyrifos-methyl proved the most toxic and cypermethrin the least toxic insecticides against all populations. Dichlorvos, malathion and pirimiphos-methyl had the least toxic effect on weevils originating from Belgrade Port and Kikinda, While deltamethrin was most toxic to weevils from Belgrade Port, and least toxic to weevils from Kikinda. The resistance ratios (RR) for deltamethrin at the LD₅₀ and LD₉₅ levels 48 h after exposure to treated filter paper were 11.2 and 14.5 for Bijeljina weevils, and 20.9 and 25.5 for Kikinda weevils.     

副溶血弧菌PCR检测方法研究进展

副溶血弧菌是引起包括我国在内的世界各地沿海地区食物中毒的重要食源性致病菌,患者有典型的肠胃炎症状。及时准确地对食品中的副溶血弧菌进行检测是预防该菌引起的食物中毒的关键。分子生物学检测方法在副溶血弧菌检测中具有许多优势,现已得到广泛的应用。本文对PCR检测方法中的多重PCR、有扩增内标的PCR、实时荧光PCR(包括荧光染料法和荧光探针法)、基于DNA染料叠氮溴化乙锭和叠氮溴化丙锭的PCR、纳米粒子PCR、免疫捕获PCR、PCR-变性高效液相色谱、PCR-酶联免疫吸附等方法的国内外研究情况进行了综述,并对其检测效率、灵敏度、优点和缺点等进行了分析比较,环介导等温扩增(loop-mediated isothermal amplification,LAMP)——包括常规LAMP和原位LAMP因与PCR方法有相似之处,故一并进行了综述,为副溶血弧菌PCR检测方法的应用与开发提供参考。     

Altered susceptibility of granary weevil Sitophilus granarius (L.) (Coleoptera: Curculionidae) populations to insecticides after selection with pirimiphos-methyl and deltamethrin

Toxicity of the contact insecticides dichlorvos, malathion, chlorpyrifos-methyl, pirimiphos-methyl, deltamethrin and cypermethrin to granary weevil Sitophilus granarius (L.) adults from five populations previously selected with deltamethrin and pirimiphos-methyl, and two populations that had no contact with pesticides over an interval of 12 generations, was investigated in the laboratory by application to filter paper. The populations originated from storage facilities situated in different parts of the former Yugoslavia.A population originating from Apatin and one from Belgrade Port were selected three times each at the LD₅₀ level with deltamethrin and pirimiphos-methyl, respectively. In separate experiments, weevils from Belgrade Port were selected once at the pirimiphos-methyl LD₇₀ level and Bijeljina and Kikinda populations both once at the deltamethrin LD₇₀ level. All selection doses were at a level obtained from determining weevil mortality after 24 h of exposure to treated filter paper.Compared with the toxicity to laboratory weevils, the weevils from Apatin after the third selection were found to be 32.1 and 51.9 times less susceptible to deltamethrin at the LD₅₀ and LD₉₅ levels, respectively, while those from Belgrade Port were 2.7 and 3.2 times less susceptible to pirimiphos-methyl. Selection of Belgrade Port weevils at the pirimiphos-methyl LD₇₀ did not significantly affect their susceptibility to that insecticide but it caused a significant decrease in deltamethrin toxicity, which is indicative of a cross-resistance between the two compounds. Selection of Bijeljina weevils caused an increased resistance to deltamethrin, while selection of Kikinda weevils had little effect on their susceptibility to dichlorvos, but it caused a significant decrease in malathion and cypermethrin toxicity, and resistance to deltamethrin markedly increased (RR=238.8 at LD₅₀ and 660.8 at LD₉₅ levels, compared with laboratory weevils). Chlorpyrifos-methyl was the most toxic insecticide to all populations, while cypermethrin was the least toxic compound.     

Truncation of oligonucleotide primers confers specificity on real-time polymerase chain reaction assays for food authentication

The advent of real-time polymerase chain reaction has revolutionized the field of molecular biology, but the design and optimization of these assays has been largely overlooked in the literature. This dearth of information is probably in response to the provision of assay design software and robust guidelines issued by the leading manufacturer. However, many applications require highly specific assays with no cross-amplification of non-target DNA and it has been found that the software and guidelines, whilst producing assays of great sensitivity, do not necessarily produce specific assays. Two complementary strategies were used to confer specificity on a real-time assay, first by placing the 3' end of the primers on a point of sequence heterogeneity and, second, by truncating the primers at the 5' end to lower the calculated melting temperature. Using these strategies in concert, a specific assay for a conserved region of the mitochondrial cytochrome b gene was developed that can be used for the unambiguous detection of the target species in a meat mixture. This approach can be used for any real-time polymerase chain reaction assay to increase assay selectivity and specificity.     

Molecular characterization of atoxigenic strains for biological control of aflatoxins in Nigeria

Aflatoxins are highly toxic carcinogens produced by several species in Aspergillus section Flavi. Strains of A. flavus that do not produce aflatoxins, called atoxigenic strains, have been used commercially in North America as tools for limiting aflatoxin contamination. A similar aflatoxin management strategy is being pursued in Nigeria. In the current study, loci across the 68 kb aflatoxin biosynthesis gene cluster were compared among 18 atoxigenic and two aflatoxin-producing vegetative compatibility groups (VCGs) from Nigeria and an atoxigenic VCG used commercially in North America. Five of the atoxigenic VCGs had large deletions (37–65 kb) extending from the teleomeric side of the aflatoxin biosynthesis cluster. In one VCG (AV0222) the deletion extended through the cluster to the adjacent sugar cluster. The remaining twelve atoxigenic VCGs, including the VCG used for aflatoxin management in North America, contained all the aflatoxin pathway genes, but with defects. Two observations support the long-term persistence of atoxigenicity within A. flavus: first, a comparison of pathway genes revealed more changes in atoxigenic than in aflatoxin-producing isolates relative to the aflatoxin-producing strain NRRL 3357; and second, several non-synonymous changes are unique to atoxigenics. Atoxigenic VCG diversity was assessed with phylogenetic analyses. Although some atoxigenics share relatively recent ancestry, several are more closely related to aflatoxin producers than to other atoxigenics. The current study demonstrates VCGs of A. flavus in West Africa with diverse mechanisms of atoxigenicity and potential value in aflatoxin management programmes.     

转基因鲑鱼AquAdvantage品系特异性实时荧光聚合酶链式反应检测方法的建立

目的实现转基因鲑鱼AquAdvantage的标识管理,建立其品系特异性实时荧光聚合酶链式反应(PCR)检测方法。方法针对转基因鲑鱼的品系特异性序列设计引物和TaqMan探针,建立转基因鲑鱼实时荧光PCR检测方法,并对该方法的特异性、灵敏度和重复性进行检测。结果建立的转基因鲑鱼实时荧光PCR方法特异性强,在600 000~60拷贝范围内呈良好的线性关系,其线性回归方程为y=-3.2194x+40.805,R~2=0.997,检测限为60拷贝,检测重复性良好。结论建立的品系特异性实时荧光PCR方法可应用于转基因鲑鱼AquAdvantage的鉴定。