农药残留的免疫分析及其进展

本文对农药残留免疫分析的原理、研究与应用进展和前景进行了评述，内容包括农药半抗原的设计与合成、免疫测定、免疫亲合色谱及其它联用技术。     

农产品中基质对酶联免疫分析法检测三唑磷残留的干扰及消除研究

基质干扰是免疫法检测食品中农药残留时经常碰到的问题。以胡萝卜、菠菜、香蕉、梨、稻米和花生等为试材,研究了不同农产品基质对酶联免疫分析法(ELISA)检测三唑磷的影响。结果表明:不同农产品基质对ELISA检测的干扰机制不同,香蕉主要是抑制了辣根过氧化物酶的活性,菠菜、梨和稻米则主要是干扰了抗原和抗体的结合反应,胡萝卜和花生不仅抑制酶的活性,同时还干扰了抗原和抗体的结合反应。将这些农产品的磷酸盐缓冲液(PBS)(含2.5%甲醇)提取液用含有质量分数为0.3%的脱脂奶粉、0.1%的乙二胺四乙酸(EDTA)和含2.5%甲醇的PBS稀释10倍,在12000r/min下离心10min,即可消除其对ELISA检测的干扰。研究结果表明, ELISA法是可用于农产品中三唑磷残留检测的一种简单、快速、有效的方法。     

农药残留检测中不同蔬菜的基质效应

基于QuEChERS前处理方法，在气相色谱-串联质谱 (GC-MS/MS) 和超高效液相色谱-串联质谱 (UPLC-MS/MS) 检测条件下，考察了200种农药在17种蔬菜基质中的基质效应，通过基质效应分析找到了合适的代表性基质用作配制检测过程中的基质匹配标准溶液。结果表明:由于基质干扰成分及农药性质的差异，导致不同蔬菜及农药品种之间的基质效应差异。在GC-MS/MS检测中，供试的150种农药中绝大部分表现为基质增强效应，其中葱、姜和大蒜表现为很强的基质效应，萝卜的基质效应较弱；菠菜、芹菜、豇豆和生菜4种蔬菜对其他蔬菜品种能够起到很好的基质校正效果，可作为代表性基质在日常检测中用于其他蔬菜基质的定量分析。在UPLC-MS/MS检测中，供试的105种农药中大部分表现为基质抑制效应，其中姜、大蒜、葱、芹菜、韭菜和菠菜的基质效应较强，西葫芦的基质效应弱；黄瓜、普通白菜、番茄和生菜4种蔬菜能较好地校正其他蔬菜的基质效应，但与GC-MS/MS相比校正能力稍弱。本研究结果认为代表性蔬菜基质可用于降低基质效应对检测结果的干扰。     

色谱法测定农产品中农药残留时的基质效应

在采用气相或液相色谱进行农药残留分析时,基质效应会严重影响对某些待测物的准确定量与定性,对基质效应进行考察评估并采取有效措施进行消除或补偿,是进行农药残留分析方法开发及验证过程中不可或缺的一个重要环节。通过总结相关文献,并结合自身的研究工作,综述了农药残留分析中基质效应的定义、类型、产生原因及影响因素,重点对基质效应的补偿与校正措施进行了阐述。     

生物检材中有机磷农药代谢物检测技术研究进展

有机磷农药代谢物检测分析在有机磷农药中毒、投毒案件定性、临床诊断和生物监测等方面发挥着重要作用。发展精确检测痕量甚至超痕量有机磷农药代谢物的技术,建立检测生物样本复杂基质中有机磷农药代谢物的方法,成为法庭科学领域亟待解决的问题之一。目前国内外常见的实验室检测方法有气相色谱、气相色谱-质谱联用、液相色谱-串联质谱等,本文重点综述了这些检测方法在有机磷农药代谢物检测方面的研究进展,以及血液、尿液等生物检材所需的液-液萃取、固相萃取、QuEChERS等前处理技术和毛发生物检材所需的固-液萃取等前处理技术。此外,本文还介绍了荧光探针法、免疫分析法、生物传感器法等快速检测技术的原理及应用进展,并对现有检测技术存在的问题进行了总结。     

对硫磷ELISA异源分析模式比较研究

为探讨方法模式对异源ELISA法各分析参数的影响情况及影响机理,设计并合成了4种与对硫磷结构相似的竞争半抗原,通过建立不同模式的同源与异源ELISA分析法,比较了不同模式下的抗原抗体用量、灵敏度、检测限及不同样品添加回收率。结果表明,异源分析对抗原和抗体的用量比同源分析有所增加,但异源分析可大大提高方法的检测灵敏度。在异源分析中,直接竞争包被抗原的ELISA(CC模式)方法重现性较差;直接竞争包被抗体的ELISA(AC模式)抗原抗体用量最多,检测灵敏度较低;间接竞争ELISA法(IC模式)具有较好的检测灵敏度,抗环境和基质干扰的能力较强,为异源ELISA分析中较优的模式。     

单克隆抗体及其在植物病毒上的应用

一、前言在植物病毒血清学诊断鉴定方面,目前所应用的技术几乎全部依赖于抗源—抗体反应这一基本原理。从常规的试管沉淀、免疫双扩散、乳胶凝集,反向间接血球凝集试验到较快速、灵敏的荧光抗体技术、免疫电镜和酶联免疫吸附技术(ELISA)等都是如此。由于传统方法制备的抗病毒抗体,都是含有     

拟除虫菊酯类农药酶免疫分析方法研究进展

对拟除虫菊酯类单一农药残留和多残留酶免疫分析方法的研究进展进行了综述。在介绍单一农药残留酶免疫分析方法中半抗原分子设计和ELISA方法建立的基础上,指出刚性连接臂是菊酯类农药免疫半抗原分子设计的一般原则,包被原和免疫原采用不同的载体蛋白和不同结构的半抗原有利于菊酯类农药ELISA方法的建立。在介绍菊酯类农药多残留酶免疫分析方法时,剖析了通用免疫半抗原的结构特点,阐述了宽谱特异性抗体的筛选方法,揭示了在菊酯类农药多残留酶免疫分析法中存在竞争半抗原的选择具有盲目性、宽谱特异性抗体对不同农药的特异性差异较大的问题。并对拟除虫菊酯类农药通用免疫半抗原设计及多残留酶免疫分析技术的发展趋势进行了探讨。     

三种基质中15种农药及其代谢物的不确定度评估

依据GB 23200.113—2018《食品安全国家标准 植物源性食品中208种农药及其代谢物残留量的测定 气相色谱-质谱联用法》中的QuEChERS方法，建立了一种气相色谱-串联质谱法 (GC-MS/MS) 测定茶叶、普通白菜和苹果3种基质中15种农药及其代谢物残留量的数学模型，通过分析测定过程的主要不确定度来源，对各个分量进行评估。结果表明:工作曲线拟合和回收率所引入的不确定度较大，其次为标准溶液配制和样品制备，而测量重复性和仪器引入的不确定度相对较小；不同基质对工作曲线拟合、回收率和测量重复性所引入的不确定度存在一定差异；不匹配基质校正曲线会对部分农药造成一定程度的基质增强或抑制效应。该方法适用于GC-MS/MS法测定植物源性食品中农药残留量的不确定度评估，可为农药残留测量结果的准确性提供科学可靠的依据。     

纳米标记免疫层析法在农药残留检测中的应用研究进展

农药残留超标已成为影响农产品质量安全的重要问题,迫切需要探寻开发灵敏、准确、可靠、便捷且适用性强的农药残留快速检测方法。免疫层析法是将抗原抗体特异性免疫反应和色谱层析分离技术相结合的一种快速检测方法,其中,基于胶体金标记的免疫层析技术以其便捷、成本低、可视化等优点而受到普遍欢迎。近年来随着量子点、时间分辨荧光微球、上转换发光纳米粒子等新型纳米标记材料的出现,免疫层析技术得到了广泛发展。文章从标记类型（非共价作用标记及共价作用标记）及标记材料（胶体金、纳米碳、量子点、上转换发光纳米粒子、磁性纳米颗粒、时间分辨荧光微球及荧光乳胶颗粒）等方面,综述了不同纳米材料标记的免疫层析技术及其在农药残留检测领域的研究及应用进展,可为深入开展农药残留免疫层析技术研究提供参考。     

Characterization of antixenosis to Dichelops melacanthus (Hemiptera: Pentatomidae) in soybean genotypes

Soybeans are of great importance in the world agricultural landscape, and their productive potential is significantly reduced by attacks from insect pests. Factors such as the expansion of national agricultural regions, together with no-tillage management and “off-season” maize cultivation, have favored the increase of secondary species such as Dichelops melacanthus (Dallas) (Hemiptera: Pentatomidae), intensifying the damage caused by the soybean stink bug complex. The use of resistant genotypes may be a valuable strategy as an alternative to the excessive use of chemical control in crops. This study evaluated the attractiveness and feeding preferences of the green-belly stink bug in 17 soybean genotypes in different maturity groups (early, semiearly, and late) to characterize the expression of antixenosis resistance. To this end, free-choice tests of attractiveness and food preference were performed under laboratory conditions. The early genotypes PI 171451 and D 75-10169, the semiearly genotypes IAC 78-2318, “IAC 100”, IAC 74-2832, PI 227687, and “IAC 24” and the late genotypes PI 274454, PI 274453, and L 1-1-01 expressed significant levels of antixenosis against adult D. melacanthus. These results will be useful for soybean breeding programs focusing on the stink bug resistance complex.     

A Multiplex Real-Time Polymerase Chain Reaction (TaqMan) Assay for the Simultaneous Detection of Meloidogyne chitwoodi and M. fallax

ABSTRACT This study describes a multiplex real-time polymerase chain reaction (PCR) approach for the simultaneous detection of Meloidogyne chitwoodi and M. fallax in a single assay. The approach uses three fluorogenic minor groove binding (MGB) TaqMan probes: one FAM-labeled to detect M. chitwoodi, one VIC-labeled to detect M. fallax, and one NED-labeled to detect the internal amplification control (IAC) to monitor false negative results. One common primer set is used for the amplification of part of the internal transcribed spacer (ITS) region of M. chitwoodi and M. fallax and one primer set for the amplification of the IAC. The test enabled detection of M. chitwoodi and/or M. fallax in DNA samples extracted from batches of juveniles, from single juveniles, and from infected plant material. Compared with current assays to detect M. chitwoodi and M. fallax, the multiplex real-time PCR offers the following advantages: it is faster because the test can simultaneously detect both quarantine species without the need for post-PCR processing; and it is at least 10 times more sensitive than a comparable regular PCR also targeting the ITS sequence. Inclusion of the IAC facilitates the interpretation of the FAM and VIC cycle threshold (Ct) values and can prevent the scoring of false negative results when FAM, VIC, and NED Ct values are high. The test allows precise quantification when only one of the two species is present in the sample. However, experiments with mixtures of genomic DNA of M. chitwoodi and M. fallax revealed that the ability of the multiplex real-time PCR assay to detect small quantities of DNA of one species is reduced when large quantities of DNA of the other species are present.     

Adult attractiveness and oviposition preference of <Emphasis Type="Italic">Zabrotes subfasciatus</Emphasis> toward genotypes of common bean <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis>

The Mexican bean weevil Zabrotes subfasciatus (Coleoptera: Chrysomelidae: Bruchinae) is among the key pests of common bean Phaseolus vulgaris L. around the world. Identifying resistance sources and the resistance types involved is important for development of pest-resistant bean varieties. This study assessed the adult attractiveness and oviposition preference of Z. subfasciatus toward eight genotypes of common bean, including seven pre-selected resistant genotypes and one standard for susceptibility. Attraction to the genotypes was assessed at one and four days after infestation. Oviposition preference was tested under free- and no-choice (confinement) conditions. We observed that IAC 853 was the least oviposited genotype in the free-choice test. However, this finding was not confirmed by the no-choice test. Therefore, IAC 853 could not be characterized as resistant to Z. subfasciatus. Presence of the toxic protein arcelin does not appear to influence host selection by Z. subfasciatus.     

Evaluation of antixenosis in soybean against Spodoptera litura by dual-choice assay aided by a statistical analysis model: Discovery of a novel antixenosis in Peking

The method for evaluating soybean (Glycine max) antixenosis against the common cutworm (Spodoptera litura) was developed based on a dual-choice assay aided by a statistical analysis model. This model was constructed from the results of a dual-choice assay in which Enrei, a soybean cultivar susceptible to S. litura, was used as both a standard and a test leaf disc for 2nd–5th instar larvae. The statistical criterion created by this model enabled the evaluation of the presence of antixenosis. This method was applied to four soybean varieties, including Tamahomare (susceptible), Himeshirazu (resistant), IAC100 (resistant), and Peking (unknown), as well as Enrei. Subsequently, the degrees of antixenosis were also compared by F-test, followed by maximum likelihood estimation (MLE). According to the results, the antixenosis of Tamahomare, Himeshirazu, and IAC100 was statistically reevaluated and Peking exhibited a novel antixenosis, which was stronger for 3rd–5th instar larvae than for 2nd instar.     

Evaluation of rice allelopathy in hydroponics

The inhibitory activity of water extracts from the shoots and roots of three rice cultivars, Taichung native 1 (TN1) and IAC165 (both allelopathic rice) and AUS196 (non-allelopathic rice), grown in hydroponics was evaluated. The release of germination inhibitors by allelopathic rice plants into hydroponic solution was also determined with freshly collected solution and XAD-4 resin desorbate. The degree of the inhibition was quantified in terms of root growth in Echinochloa colona, Echinochloa crus-galli, Echinochloa crus-galli var. oryzicola, Triantema portulacastrum and Lactuca sativa. The allelopathic activity of rice was species specific, and depended on source and concentration. Root length of all test species was inhibited by the different concentrations of shoot extract of allelopathic and non-allelopathic rice. However, of the three cultivars, TN1 showed higher inhibition than IAC165 and AUS196 in all test species. Water extracts of shoots and roots significantly inhibited root growth in E. crus-galli but the shoot extract gave a greater inhibitory effect on E. crus-galli than the root extract. Root exudate of TN1 inhibited root elongation of E. crus-galli from 2 weeks after transplanting (WAT) and the inhibition continued for 4 WAT. The results confirmed the previous finding of a laboratory bioassay that the TN1 had allelopathic activity and produced allelochemicals that inhibit growth of some weed species.     

Screening tomato genotypes for resistance and tolerance to Tomato chlorosis virus

Tomato chlorosis virus (ToCV) is an emerging crinivirus in Brazil that causes an economically important disease in tomato (Solanum lycopersicum) and other solanaceous species. ToCV is transmitted predominantly by the whitefly Bemisia tabaci Middle East‐Asia Minor 1 (MEAM1, formerly biotype B), in a semipersistent manner. As all cultivated tomato varieties and hybrids are susceptible to this crinivirus, the main alternatives for the control of the disease are the use of healthy seedlings for transplanting and the chemical control of the insect vector. The objective of this work was to evaluate the responses of tomato genotypes to infection with this crinivirus and their tolerance to the disease in order to support the development of other alternatives for disease control. Resistance to infection was evaluated by ToCV inoculation with viruliferous B. tabaciMEAM1 followed by virus detection by RT‐PCR and RT‐qPCR. To measure tolerance to the disease, plant development and fruit yield of ToCV‐infected and healthy plants were compared. Among 56 genotypes, only the lineage IAC‐CN‐RT (S. lycopersicum ‘Angela Gigante’ × S. peruvianum ‘LA 444‐1’) was highly resistant to infection with ToCV. Tolerance to the disease over two trials with different genotypes showed variable results. The effect of ToCV on plant development varied from 2.9% to 71.9% reduction, while yield loss varied from 0.2% to 51.8%. The highly ToCV‐resistant lineage IAC‐CN‐RT, which is also resistant to a Spanish isolate of ToCV, might be useful for tomato breeding programmes.     

Genetic variability of Meloidogyne paranaensis populations and their aggressiveness to susceptible coffee genotypes

Meloidogyne paranaensis is one of the most destructive root‐knot nematode (RKN) species parasitizing coffee in Brazil and in the Americas generally. The objectives of this study were to assess the genetic variability, aggressiveness and virulence of seven different M. paranaensis populations on susceptible and resistant Coffea spp. All seven RKN populations were identified by biochemical and molecular methods. Coffee seedlings were inoculated in the greenhouse, and the nematode reproduction factor was used to infer their reproduction on coffee genotypes. Phylogenetic studies showed a low genetic variability in M. paranaensis populations, regardless of the existence of three esterase phenotypes (Est P1, P2 and P2a), except for the population Est P2a from Guatemala, which is genetically different from other M. paranaensis populations from Brazil. The Est P2a and Est P2 (Herculândia, SP, Brazil) populations were the most aggressive on two susceptible C. arabica cultivars under greenhouse conditions. None of the M. paranaensis populations were virulent on resistant coffee genotypes, confirming their resistance to the seven M. paranaensis populations tested. The resistant coffee cultivars, namely Clone 14 INCAPER, Catuaí Vermelho × Amphillo MR2161 (E1 16‐5 III), Apoatã IAC 2258, Timor Hybrid UFV 408‐01 (E1 6‐6 II) and IPR 100, exhibited segregation for resistance in the ratio of 0%, 2.4%, 12%, 26% and 29%, respectively. These are promising results, because they validate resistance against several M. paranaensis populations in different Coffea spp. genetic resources, which can be used in breeding programmes or as rootstocks, such as Apoatã IAC 2258 and Clone 14 INCAPER.     

马铃薯斑纹片病菌含扩增内标的双重实时荧光PCR检测

马铃薯斑纹片病菌(Candidatus Liberibacter solanacearum)是一种主要为害伞形花科和茄科植物的重要有害生物,目前无法人工培养且可随种子远距离传播。准确灵敏的PCR检测方法对病害预警和防止扩散具有重要意义。本文通过复合引物法构建扩增内标(internal amplification control,IAC),建立了马铃薯斑纹片病菌含扩增内标的双重实时荧光PCR检测体系,有效指示了PCR检测过程中的假阴性现象。试验结果表明:双重与单重实时荧光PCR的检测灵敏度一致,当扩增内标添加量为3.4×10^-5ng时,样品DNA的检测低限为0.4 ng。40个样品的检测结果显示,与单重实时荧光PCR相比,双重荧光PCR方法的阳性检出率由25.00%提高到27.50%,提高了检测结果的准确性。