Polymerase chain reaction-based methods for detection of Listeria monocytogenes: toward real-time screening for food and environmental samples

A review is presented of nucleic acid amplification-based methodology, specifically polymerase chain reaction (PCR)-based assays, for the detection of Listeria monocytogenes in food and environmental samples. Until recently, developmental challenges including poor sensitivity, due in part to reaction inhibition by components of the sample matrix, and the potential for false-positive reactions have limited routine application of PCR-based screening assays. Commercial assays address these challenges while offering convenient, standardized protocols, a high level of automation, and results within 2 days after the sampling date. Although sample enrichment is necessary to achieve desired detection limits, continued efforts toward template purification will facilitate the development of assays offering real-time, quantitative results. The development of ribonucleic acid (RNA) amplification-based assays may increase in importance, particularly if end-product testing is prioritized by regulatory agencies, as messenger RNA appears to serve as an accurate indicator of cell viability. Further, the increase in target copy number may improve assay sensitivity. PCR-based screening methods offer efficient, reliable results and are ideal for monitoring the presence of L. monocytogenes in foods and in the food processing environment.     

基于分子信标荧光纳米探针的李斯特菌DNA均相检测方法

基于分子信标(MB)识别和荧光纳米粒子探针技术,建立了均相体系中李斯特菌目标DNA的高灵敏检测新方法.首先以羊抗人免疫球蛋白(IgG)标记的异硫氰酸荧光素(FITC)为核材料,成功制备了FITC-IgG@SiO2核壳荧光纳米粒子,有效防止了传统方法中采用单一FITC制备纳米颗粒时泄露严重的问题.随后以FITC-IgG@SiO2荧光纳米粒子和纳米金分别标记单核细胞增生李斯特菌序列特异性分子信标探针5'端和3'端,成功构建了单核细胞增生李斯特菌序列特异性分子信标荧光纳米探针.在实验优化条件下,α(令α=F/F0,F代表MB和目标DNA杂交以后的荧光强度,F0代表MB完全闭合时的荧光强度)与目标DNA浓度在1～200pmol/L浓度范围内呈良好的线性关系,检出下限为0.3pmol/L,相对标准偏差为2.6%(50pmol/L,n=11).将该方法应用于食品样品中单核细胞增生李斯特菌的检测,结果与国标法一致.     

Pesticide analysis by micellar electrokinetic capillary chromatography

On-capillary sample concentration using sample stacking for the improvement of detection limits for various pesticides separated by micellar electrokinetic capillary chromatography was examined. The dependence of the stacking on different parameters was investigated. An approximately 30-fold preconcentration was achieved by applying sample stacking. Employing a two-step enrichment process (solid-phase extraction combined with sample stacking), detection limits were improved and the sample volume for SPE was reduced. In addition, the total time for the analysis was considerably reduced. Detection limits were between 0.01 and 0.1 ng/ml under these enrichment conditions.     

液相微萃取-高效液相色谱联用分析化妆品中痕量的雌激素

建立了液相微萃取-高效液相色谱联用(LPME-HPLC)测定爽肤水中痕量的雌三醇、雌二醇、炔雌醇和雌酮的分析方法,研究了萃取溶剂种类、接受相体积、搅拌速度、萃取时间等对萃取效率的影响。结果表明,该方法对4种雌激素的富集倍数可达到247~343倍,方法的线性范围为1~200 μg/L,检出限为0.4~1.0 μg/L,6次平行测定的相对标准偏差为3.6%~7.3%,爽肤水中的加标回收率为101.2%~114.9%。方法简单快速、灵敏度高、环境友好,满足痕量雌激素分析的要求。     

Determination of estrogens in wastewater using three-phase hollow fiber-mediated liquid-phase microextraction followed by HPLC

Three-phase hollow fiber-mediated liquid-phase microextraction followed by HPLC was used for the determination of three synthetic estrogens, namely diethylstilbestrol, dienestrol, and hexestrol, in wastewater. Extraction conditions including organic solvent, volume ratio between donor solution and acceptor phase, extraction time, stirring rate, donor phase and acceptor phase were optimized. The target compounds were extracted from a 10 mL aqueous sample at pH 1.5 (donor solution) through a 45 mm in length hollow polypropylene fiber that was immersed in 1-octanol in advance, and then the hollow fiber was filled with 10 microL 0.5 mol/L sodium hydroxide solution (acceptor phase). After a 40 min extraction, the acceptor phase was directly injected into an HPLC system for detection. Under the optimized extraction conditions, a large enrichment factor (more than 300-fold) was achieved for the three estrogens. The determination limit at an S/N of 3 ranged from 0.25 to 0.5 microg/L for the estrogens. The recovery ratio was more than 86% in the determination of these estrogens in wastewater.     

Spiral stir bar sorptive extraction with polyaniline‐polydimethylsiloxane sol‐gel packings for the analysis of trace estrogens in environmental water and animal‐derived food samples

A spiral stir bar was proposed by using stainless steel spring as the extraction phase carrier to avoid the extraction phase friction and increase the amount of extraction phase for improving extraction efficiency. The extraction phase is filled in the cavity of the spring, resulting in a larger amount of the extraction phase than that conventionally coated on glass stir bar or stainless steel wire. Polyaniline‐polydimethylsiloxane sol‐gel packed spiral stir bar was prepared and evaluated for the extraction of five estrogens. The prepared spiral stir bar presented good extraction efficiency/preparation reproducibility and long lifetime (more than 150 reused times) for target estrogens. Based on it, a method of spiral stir bar sorptive extraction combined with high performance liquid chromatography coupled with ultra‐violet detection was developed for the analysis of trace estrogens in environmental and food samples. The detection limit for five estrogens was 0.11–.31 µg/L, with the enrichment factors of 83.0–118‐fold (maximal enrichment factor: 200‐fold). The reproducibility evaluated with each estrogen of 5 µg/L (n = 5) was 5.8–8.9%. The method was successfully applied for the determination of estrogens in environmental water and animal‐derived food samples.     

Evaluation of applied biosystems MicroSeq real-time PCR system for detection of Listeria monocytogenes in food. Performance Tested Method 011002

Increasingly, more food companies are relying on molecular methods, such as PCR, for pathogen detection due to their improved simplicity, sensitivity, and rapid time to results. This report describes the validation of a new Real-Time PCR method to detect Listeria monocytogenes in nine different food matrixes. The complete system consists of the MicroSEQ L. monocytogenes Detection Kit, sample preparation, the Applied Biosystems 7500 Fast Real-Time PCR instrument, and RapidFinder Express software. Two sample preparation methods were validated: the PrepSEQ Nucleic Acid extraction kit and the PrepSEQ Rapid Spin sample preparation kit. The test method was compared to the ISO 11290-1 reference method using an unpaired-study design to detect L. monocytogenes in roast beef, cured bacon, lox (smoked salmon), lettuce, whole cow's milk, dry infant formula, ice cream, salad dressing, and mayonnaise. The MicroSEQ L. monocytogenes Detection Kit and the ISO 11290-1 reference method showed equivalent detection based on Chi-square analysis for all food matrixes when the samples were prepared using either of the two sample preparation methods. An independent validation confirmed these findings on smoked salmon and whole cow's milk. The MicroSEQ kit detected all 50 L. monocytogenes strains tested, and none of the 30 nontargeted bacteria strains.     

Evaluation of dispersive liquid-liquid microextraction for the simultaneous determination of chlorophenols and haloanisoles in wines and cork stoppers using gas chromatography-mass spectrometry

Dispersive liquid-liquid microextraction (DLLME) coupled with gas chromatography-mass spectrometry (GC-MS) was evaluated for the simultaneous determination of five chlorophenols and seven haloanisoles in wines and cork stoppers. Parameters, such as the nature and volume of the extracting and disperser solvents, extraction time, salt addition, centrifugation time and sample volume or mass, affecting the DLLME were carefully optimized to extract and preconcentrate chlorophenols, in the form of their acetylated derivatives, and haloanisoles. In this extraction method, 1mL of acetone (disperser solvent) containing 30μL of carbon tetrachloride (extraction solvent) was rapidly injected by a syringe into 5mL of sample solution containing 200μL of acetic anhydride (derivatizing reagent) and 0.5mL of phosphate buffer solution, thereby forming a cloudy solution. After extraction, phase separation was performed by centrifugation, and a volume of 4μL of the sedimented phase was analyzed by GC-MS. The wine samples were directly used for the DLLME extraction (red wines required a 1:1 dilution with water). For cork samples, the target analytes were first extracted with pentane, the solvent was evaporated and the residue reconstituted with acetone before DLLME. The use of an internal standard (2,4-dibromoanisole) notably improved the repeatability of the procedure. Under the optimized conditions, detection limits ranged from 0.004 to 0.108ngmL(-1) in wine samples (24-220pgg(-1) in corks), depending on the compound and the sample analyzed. The enrichment factors for haloanisoles were in the 380-700-fold range.     

应用高场非对称离子迁移谱技术检测食品接触材料中甲基丙烯酸甲酯的迁移量

应用高场非对称离子迁移谱(FAIMS)技术, 无需经过萃取、 富集等过程, 可直接进样分析甲基丙烯酸甲酯在水基食品模拟物中的迁移量. 通过考察扫描次数、 样品温度、 取样体积、 载气流速和溶剂掺杂对离子特征信号的影响, 确定甲基丙烯酸甲酯的检出限为10 μg/L, 并建立了FAIMS检测甲基丙烯酸甲酯的离子流强度与浓度关系曲线. 该方法操作便捷、 灵敏度高、 分析速度快, 能满足实际工作的要求.     

Core-shell magnetic porous organic polymer for magnetic solid-phase extraction of fluoroquinolone antibiotics in honey samples followed by high-performance liquid chromatography with fluorescence detection

By monomer-mediated in-situ growth synthesis strategy, with hydroquinone and 1,3,5-tris(4-aminophenyl)benzene as monomers, a core-shell magnetic porous organic polymer was synthesized through a simple azo reaction. Based on this, a magnetic solid-phase extraction–high-performance liquid chromatography–fluorescence detection method was proposed for the analysis of fluoroquinolones in a honey sample. With ofloxacin, ciprofloxacin, enrofloxacin, lomefloxacin, and difloxacin as target analytes, factors affecting the extraction efficiency had been optimized. The LODs were 1.5–5.4 ng/L (corresponding to 0.23–0.81 μg/kg in honey). The linear range was 0.005–20 μg/L for difloxacin, 0.01–20 μg/L for ofloxacin, ciprofloxacin and lomefloxacin, and 0.02–20 μg/L for enrofloxacin. The enrichment factor was 84.4–91.7-fold with a high extraction efficiency of 84.4–91.7%. The method was assessed by the analysis of target fluoroquinolones in honey samples, and the recoveries for the spiked samples were 79.3–95.8%. The results indicated that the established magnetic solid-phase extraction–high-performance liquid chromatography–fluorescence detection method is efficient for the analysis of trace fluoroquinolones in honey.     

Ionic liquid for single‐drop microextraction followed by high‐performance liquid chromatography‐ultraviolet detection to determine carbonyl compounds in environmental waters

A sensitive method based on ionic liquid for single‐drop liquid microextraction coupled with HPLC‐UV was developed for the determination of carbonyl compounds in environmental waters using 1‐octyl‐3‐methylimidazolium hexafluorophosphate [C₈min][PF₆] as extraction solvent and 2,4‐dinitrophenylhydrazine as derivatizing agent. The extraction parameters affecting the enrichment factors such as solvent volume, pH, extraction time and salt concentration were investigated. A homemade funnel form polytetrafluoroethylene sleeve was fixed at the tip of the syringe needle and this allowed the use of 10 μL drop of ionic liquid for direct immersion extraction. Under the optimal conditions, the remarkable enrichment factors up to 150‐fold were obtained depending on the target analytes. The method has been validated when rectilinear relationship was obtained between the concentrations of analytes and peak area in the range of 5–100 ng/mL, the correlation coefficients were from 0.995 to 0.998, and the limit of detection was in the range of 0.04–2.03 ng/mL. The method was applied to monitor the concentration of carbonyl compounds in environmental waters with spiked recovery in the range of 84.2–106.9%.     

Development of a novel stirrerliquid/solid microextraction method for the separation and enrichment of trace levels of active compounds in traditional Chinese medicine

A novel stirrer‐liquid/solid microextraction method was developed for the separation and enrichment of trace levels of curcumin, bisdemethoxycurcumin, and demethoxycurcumin in Rhizoma Curcumae Longae, Radix Curcumae, and Rhizoma Curcumae before their analysis by high‐performance liquid chromatography with ultraviolet detection. In the proposed approach, a magnetic stirrer was immersed in decanol to coat its surface completely with decanol, which was used as an extraction platform. The stirrer coated with decanol is not only a power to agitate the sample solution to constantly update the sample on the stirrer surface but also it can adsorb and extract the target analytes. Some effective parameters, including suitable superficial area of stirrer, extraction solvent, sample phase pH, NaCl concentration, stirring rate, extraction time, sample phase volume, were analyzed and selected. Under the optimal conditions, the linearities are 0.0044–2.20 μg/mL, detection limits are 0.3–0.6 ng/mL, and the extraction content per unit length and enrichment factors of the target analytes are 6.24–9.71/mm and 589–917, respectively. Also, the stirrer‐liquid/solid microextraction mechanism for the extraction and enrichment of the target analytes was analyzed and expounded. The results showed that stirrer‐liquid/solid microextraction is a simple, rapid sample pretreatment approach with a high enrichment factor.     

Single drop liquid-liquid-liquid microextraction of methamphetamine and amphetamine in urine

Single drop liquid-liquid-liquid microextraction (LLLME) combined with high performance liquid chromatography (HPLC)-UV detection was investigated for the determination of a popular drug of abuse, methamphetamine (MAP), and its major metabolite, amphetamine (AP), in urine samples. The target compounds were extracted from NaOH modified sample solution to a thin layer of organic solvent membrane, and back-extracted to an acidic acceptor drop suspended on the tip of a 50-microL HPLC syringe in the aforementioned organic layer. This syringe was also used for direct injection after extraction. Factors affecting extraction efficiency were studied. At optimal conditions, the overall enrichment factor (EF) was 500-fold for AP and 730-fold for MAP, respectively. The method exhibited a wide linear range (1.0-1500 microg/L), low detection limit (0.5 microg/L), and good repeatability (RSD<5.0%) for both analytes. The feasibility of the method was demonstrated by the analysis of human urine samples.     

Sensitive determination of amide herbicides in environmental water samples by a combination of solid‐phase extraction and dispersive liquid–liquid microextraction prior to GC–MS

In this paper, solid‐phase extraction (SPE) in combination with dispersive liquid–liquid microextraction (DLLME) has been developed as a sample pretreatment method with high enrichment factors for the sensitive determination of amide herbicides in water samples. In SPE–DLLME, amide herbicides were adsorbed quantitatively from a large volume of aqueous samples (100 mL) onto a multiwalled carbon nanotube adsorbent (100 mg). After elution of the target compounds from the adsorbent with acetone, the DLLME technique was performed on the resulting solution. Finally, the analytes in the extraction solvent were determined by gas chromatography–mass spectrometry. Some important extraction parameters, such as flow rate of sample, breakthrough volume, sample pH, type and volume of the elution solvent, as well as salt addition, were studied and optimized in detail. Under optimum conditions, high enrichment factors ranging from 6593 to 7873 were achieved in less than 10 min. There was linearity over the range of 0.01–10 μg/L with relative standard deviations of 2.6–8.7%. The limits of detection ranged from 0.002 to 0.006 μg/L. The proposed method was used for the analysis of water samples, and satisfactory results were achieved.     

Evaluation of a chromogenic medium for identification and differentiation of Listeria monocytogenes in selected foods

Listeria monocytogenes continues to be a threat to food safety in the United States despite a "zero tolerance" policy. When Listeria species are identified by standard cultural methods, confirmation of L. monocytogenes takes days to complete. RAPID'L.Mono agar, developed by Bio-Rad Laboratories, is a chromogenic medium that differentiates L. monocytogenes from other species of Listeria by a simple color change reaction. Differentiation is based on the specific detection of phosphatidylinositol phospholipase C activity, resulting in a blue colony, and the inability of L. monocytogenes to metabolize xylose, resulting in the absence of a yellow halo. Detection principles of standard method agars, Oxford and PALCAM, are based on the ability of all species of Listeria to hydrolyze esculin. Thus, all species of Listeria have similar colony morphology on these agars, making differentiation of pathogenic L. monocytogenes from other nonhuman pathogens difficult. RAPID'L.Mono agar has been validated with surimi, mixed salad, brie, and processed deli turkey because of the prevalence of L. monocytogenes in these foods. Sensitivity and specificity for this medium was determined to be 99.4 and 100%, respectively. Overall method agreement of RAPID'L.Mono with standard culture methods (U.S. Department of Agriculture/Food Safety and Inspection Service; U.S. Food and Drug Administration/Bacteriological Analytical Manual; and AOAC INTERNATIONAL) was excellent, with enrichment protocols 24 h shorter than those of standard methods.     

Optimized, one-step, recovery-enrichment broth for enhanced detection of Listeria monocytogenes in pasteurized milk and hot dogs

A one-step, recovery-enrichment broth, optimized Penn State University (oPSU) broth, was developed to consistently detect low levels of injured and uninjured Listeria monocytogenes cells in ready-to-eat foods. The oPSU broth contains special selective agents that inhibit growth of background flora without inhibiting recovery of injured Listeria cells. After recovery in the anaerobic section of oPSU broth, Listeria cells migrated to the surface, forming a black zone. This migration separated viable from nonviable cells and the food matrix, thereby reducing inhibitors that prevent detection by molecular methods. The high Listeria-to-background ratio in the black zone resulted in consistent detection of low levels of L. monocytogenes in pasteurized foods by both cultural and molecular methods, and greatly reduced both false-negative and false-positive results. oPSU broth does not require transfer to a secondary enrichment broth, making it less laborious and less subject to external contamination than 2-step enrichment protocols. Addition of 150mM D-serine prevented germination of Bacillus spores, but not the growth of vegetative cells. Replacement of D-serine with 12 mg/L acriflavin inhibited growth of vegetative cells of Bacillus spp. without inhibiting recovery of injured Listeria cells. oPSU broth may allow consistent detection of low levels of injured and uninjured cells of L. monocytogenes in pasteurized foods containing various background microflora.     

Determination of four heterocyclic insecticides by ionic liquid dispersive liquid–liquid microextraction in water samples

A novel microextraction method termed ionic liquid dispersive liquid–liquid microextraction (IL-DLLME) combining high-performance liquid chromatography with diode array detection (HPLC-DAD) was developed for the determination of insecticides in water samples. Four heterocyclic insecticides (fipronil, chlorfenapyr, buprofezin, and hexythiazox) were selected as the model compounds for validating this new method. This technique combines extraction and concentration of the analytes into one step, and the ionic liquid was used instead of a volatile organic solvent as the extraction solvent. Several important parameters influencing the IL-DLLME extraction efficiency such as the volume of extraction solvent, the type and volume of disperser solvent, extraction time, centrifugation time, salt effect as well as acid addition were investigated. Under the optimized conditions, good enrichment factors (209–276) and accepted recoveries (79–110%) were obtained for the extraction of the target analytes in water samples. The calibration curves were linear with correlation coefficient ranged from 0.9947 to 0.9973 in the concentration level of 2–100 μg/L, and the relative standard deviations (RSDs, n = 5) were 4.5–10.7%. The limits of detection for the four insecticides were 0.53–1.28 μg/L at a signal-to-noise ratio (S/N) of 3.     

LC Determination of Chloramphenicol in Honey Using Dispersive Liquid–Liquid Microextraction

A novel method, dispersive liquid–liquid microextraction coupled with liquid chromatography-variable wavelength detector (LC-VWD), has been developed for the determination of chloramphenicol (CAP) in honey. A mixture of extraction solvent (30 μL 1,1,2,2-tetrachloroethane) and dispersive solvent (1.00 mL acetonitrile) were rapidly injected by syringe into a 5.0 mL real sample for the formation of cloudy solution, the analyte in the sample was extracted into the fine droplets of C₂H₂Cl₄. After extraction, phase separation was performed by centrifugation and the enriched analyte in the sedimented phase was determined by LC-VWD. Some important parameters, such as the kind and volume of extraction solvent and dispersive solvent, extraction time, sample solution pH, sample volume and salt effect were investigated and optimized. Under the optimum extraction condition, the method yields a linear calibration curve in the concentration range from 3 to 2,000 μg kg^?1 for target analyte. The enrichment factor for CAP was 68.2, and the limit of detection (S/N = 3) were 0.6 μg kg^?1. The relative standard deviation (RSD) for the extraction of 10 μg kg^?1 of CAP was 4.3% (n = 6). The main advantages of method are high speed, high enrichment factor, high recovery, good repeatability and extraction solvent volume at μL level. Honey samples were successfully analyzed using the proposed method.     

Dynamic hollow fiber protected liquid phase microextraction and quantification using gas chromatography combined with electron capture detection of organochlorine pesticides in green tea leaves and ready-to-drink tea

The dynamic hollow fiber protected liquid phase microextraction (DHFP-LPME) technique was evaluated for the extraction of organochlorine pesticides (OCPs) in green tea leaves and ready-to-drink tea prior to gas chromatography combined-electron capture detection (GC-ECD) analysis. A conventional microsyringe with a 1.5 cm length of hollow fiber attached to its needle was connected to a syringe pump to perform the extraction. The microsyringe was used as both the microextraction device and the sample introduction device for GC-ECD analysis. In this work, the organochlorine pesticides were extracted and condensed to a volume of 3 microl of organic extracting solvent (1-octanol) confined within a 1.5 cm length of hollow fiber. The effects of extraction solvent, extraction time, sample agitation, plunger speed, and extraction temperature and salt concentration content on the extraction performance were also investigated. Good enrichments were achieved (34-297-fold) with this method, and good repeatabilities of extraction were obtained, with full name (RSDs) below 12.57%. Detection limits were much below 1 microg l(-1) for ready-to-drink tea and much below 1 microg g(-1) for green tea leaves.     

气相色谱法同时测定粮食中14种有机磷残留量

建立了气相色谱法同时测定粮食中14种有机磷农药残留的方法。样品经乙腈溶解,高速匀浆,振荡提取,离心浓缩定容,气相色谱外标法定性定量。敌敌畏等14种有机磷农药在DB-1701毛细管柱上得到了很好的分离,在浓度为0.05～2.00mg/L范围内线性关系良好,相关系数r为0.99～1.00,最低检出限为0.01～0.08mg/kg;当添加水平为0.100、0.500、1.000mg/kg 时,回收率为71.2%～113.1% ,相对标准偏差为2.2%～6.9%。方法简单、快速、准确、灵敏,适用于一般检验机构的日常检验需要。