Enzyme-linked immunosorbent assay for T-2 toxin metabolites in urine

A direct competitive enzyme-linked immunosorbent assay (ELISA) for determination of total T-2 toxin metabolites in urine was developed. The assay involves coating anti-3-acetyl-neosolaniol-hemisuccinate-bovine serum albumin conjugate (anti-3-Ac-NEOS-HS-BSA) antibody to the ELISA plate and using 3-Ac-NEOS-HS-peroxidase as the enzyme marker. Competitive ELISA revealed that the antibody had good cross-reactivity with acetyldiacetoxyscirpenol (Ac-DAS), T-2 tetraol tetraacetate, 3'-OH-Ac-T-2, 3-Ac-NEOS, and 3,4,15-triacetyl-12,13-epoxytrichothec-9-en-8-one (Ac-T-2-8-one), but less cross-reactivity with Ac-T-2 toxin and T-2 toxin. All metabolites of T-2 toxin in urine were converted to T-2 tetraol tetraacetate (T-2-4ol-4Ac) by acetylation of the sample extract before ELISA. To test the ELISA accuracy, a radioimmunoassay (RIA) was performed simultaneously. The linear portion of the standard curve of this direct ELISA for T-2-4ol-4Ac was 0.2-2.0 ng/mL, which was 10 times more sensitive than RIA. The minimum detection level for T-2-4ol-4Ac was 0.02 ng/mL (0.4 pg/assay) in the absence of urine sample. The overall analytical recoveries for T-2 toxin, HT-2, T-2-4ol, 3'-OH-HT-2, NEOS, and a mixture of these 5 toxins added to the urine samples in the ELISA at concentrations of 0.05 and 0.2 ng/mL were 87 and 94%, respectively.     

Development of qualitative and semiquantitative immunoassay-based rapid strip tests for the detection of T-2 toxin in wheat and oat

Novel qualitative as well as semiquantitative rapid strip tests for screening of T-2 mycotoxin in agricultural commodities were developed. Colloidal gold particles were coated with monoclonal anti-T-2 antibodies and used as detector reagent, indicating the strip test results by formation of up to two colored lines in a competitive assay format. The test line comprises a protein conjugate of the T-2 mycotoxin and the control line an antispecies-specific antibody to confirm the correct test development. To perform the test, 5 g of sample was extracted in a ratio of 1:5 with methanol/water (70:30) by shaking for 3 min and the extract directly used without further cleanup steps. The T-2 toxin lateral flow device (LFD) presented has a cutoff level around 100 microg/kg for naturally contaminated wheat and oat. The semiquantitative test may be used in the lower micrograms per kilogram range and allows for rapid semiquantitative photometric classification of the level of sample contamination. For both tests, results were obtained within 4 min. The developed LFDs therefore allow for the first time fast and on-site screening for the determination of T-2 toxin in cereals.     

Development of a microtiter plate ELISA and a dipstick ELISA for the determination of the organophosphorus insecticide fenthion

In previous studies, polyclonal antibodies against the organophosphorus insecticide fenthion were obtained and an indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed for this pesticide. In this study, using these antibodies and an enzyme tracer, direct competitive ELISAs for fenthion in microtiter plate and dipstick formats were developed. The microtiter plate ELISA showed an IC(50) value of 1.2 microg/L with a detection limit of 0.1 microg/L. The antibodies showed negligible cross-reactivity with other organophosphorus pesticides. The use of the dipstick format using Immunodyne as a support membrane allowed the quick visual detection of fenthion in concentrations >10 microg/L. The IC(50) value of the dipstick format using reflectance detection was 15 microg/L with a detection limit of 0.5 microg/L. The recoveries of fenthion from spiked vegetable samples using the two formats without any prior enrichment or cleanup steps were 87-116%.     

Simultaneous analysis of T-2 toxin and HT-2 toxin by an indirect enzyme-linked immunosorbent assay

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of HT-2 toxin in the presence or absence of T-2 toxin is described. In the indirect ELISA, the relative cross-reactivities of antibodies against T-2 toxin (anti-T-2) with T-2 toxin and HT-2 toxin were 1 and 0.1, whereas anti-HT-2 cross-reactivities with T-2 toxin and HT-2 toxin were 0.33 and 1, respectively. Using such relationships, a formula was established that could be used to calculate the individual toxin concentration in a mixed sample after experimentally analyzing for T-2 and HT-2 toxins in the 2 indirect ELISAs. This method was tested by analyzing urine samples spiked with HT-2 toxin alone and samples spiked with both T-2 toxin and HT-2 toxin. A cleanup protocol for treatment of urine samples before ELISA was also established. The overall analytical recovery of HT-2 toxin when it was added at concentrations of 0.1-10 parts per billion (ppb) to the urine samples was ca 89%. When both T-2 and HT-2 toxins were added to the urine samples at equal concentrations of 0.5 to 5.0 ppb, their recoveries were 112 and 109%, respectively.     

Determination of 12 type A and B trichothecenes in cereals by liquid chromatography-electrospray ionization tandem mass spectrometry

A new sensitive method for the simultaneous determination of 12 trichothecenes (deoxynivalenol, nivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, fusarenon X, T-2 toxin, HT-2 toxin, neosolaniol, monoacetoxyscirpenol, diacetoxyscirpenol, T-2 triol, and T-2 tetraol) by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) is presented. The development of the method and investigations on the matrix influence on the MS signal are described in particular. The matrix effect was thereby minimized by using an internal standard, a special mobile phase, and specific fragmentation parameters. The sample was extracted with acetonitrile/water (84:16, v/v), and the extract was cleaned up with a MycoSep 227 column. Quantification was based on the internal standard de-epoxy-deoxynivalenol. Calibration curves were linear between 16 and 1600 ng/g, and the limits of detection ranged from 0.18 to 5.0 ng/g. The developed method was applied for the determination of trichothecenes in 120 naturally contaminated wheat and oat samples.     

Effects of nitric oxide on fatty acid composition in peach fruits during storage

Peach (Prunus persica (L.) Batsch., cv. Feicheng) fruits were fumigated with 0, 5, 10, and 15 microL/L nitric oxide (NO), and the effects of NO on rot index, ion leakage, and the lipid compositions in peach fruits were studied. The results showed that the treatment with 10 microL/L NO significantly increased the contents of the palmitoleic (16:1), oleic (18:1), and linolenic (18:3) acid in phospholipid (PL), while it decreased the contents of linoleic (18:2) acid in PL. The content of monoacylglycerol (MG) in fruit fumigated with 10 microL/L NO were higher than in the control but lower in fruit fumigated with 15 microL/L NO. The treatment with 10 microL/L NO increased the content of MG, decreased the contents of diacylglycerol (DG) and triacylglycerol (TG). However, there was no significant difference in the contents of free fatty acid (FFA). The compositions of MG, DG, TG, and FFA in peach fruit were also changed by the treatment with 10 microL/L NO. It could preserve the content of 18:3, maintain the integrity of membrane, and prevent the softening and rotting of peach fruit. The content of 18:1 was detected in PL and FFA but not in MG, DG, and TG. This might be due to the different compositions of MG, DG, and TG.     

Gas chromatographic screening method for T-2 toxin, diacetoxyscirpenol, deoxynivalenol, and related trichothecenes in feeds

A gas chromatographic method for screening trichothecene mycotoxins in feeds is described. Feed is extracted with acetonitrile-water, and the toxins are purified with charcoal-alumina-Celite, Florisil, and silica mini-columns. Deoxynivalenol (DON), nivalenol (NIV), diacetoxyscirpenol (DAS), T-2 toxin, and their fungal metabolites are hydrolyzed to their corresponding parent alcohols (DON, NIV, scirpentriol, or T-2 tetraol) by alkaline hydrolysis. After derivatization to their pentafluoropropionyl analogs, they are quantitated by capillary gas chromatography with electron capture detection. Identity can be confirmed and sensitivity can be increased by using negative chemical ionization mass spectrometry with no additional sample workup. Recoveries of DAS, DON, and T-2 toxin averaged, respectively, 80, 65, and 85% in corn; 84, 65, and 88% in soybeans; and 70, 57, and 96% in mixed feeds at concentrations ranging from 0.1 to 2.0 ppm. Recoveries of 15-monoacetoxyscirpenol (MAS), HT-2, NIV, and T-2 tetraol were 97, 97, 86, and 56%, respectively, in corn at a concentration of 0.25 ppm: A detection limit of 0.02 ppm in corn, soybeans, and mixed feeds, and 0.05 ppm in silages is estimated.     

Gas-liquid chromatographic/mass spectrometric screening method for T-2 toxin in milk

A method for the analysis of T-2 toxin in milk is presented. Ethyl acetate extracts of milk samples which had been spiked with T-2 toxin were purified by thin layer chromatography and derivatized with N,O-bis(trimethylsilyl)acetamide to produce the T-2 toxin trimethylsilyl ether (T-2 toxin-TMS). N,O-bis(trimethylsilyl-d9)acetamide was used to make T-2 toxin d9-trimethylsilyl ether (T-2 toxin-d9 TMS) which was added to the derivatized milk extract as an internal standard. Samples were analyzed by combined gas-liquid chromatography/mass spectrometry using either electron impact ionization or chemical ionization mass spectrometry. In electron impact ionization analyses, simultaneous monitoring of the T-2 toxin-TMS fragment ion at m/z 436 and the T-2 toxin-d9TMS fragment ion at m/z 445 gave a T-2 toxin-TMS detectability estimated at 6 microgram/kg. In chemical ionization analyses, the T-2 toxin-TMS fragment ion at m/z 377 and the T-2 toxin-d9TMS fragment ion at m/z 386 were simultaneously monitored to give a T-2 toxin-TMS detectability estimated at 3 microgram/kg. Average recovery was 85% at 200 microgram/kg and 65% at 20 microgram/kg.     

Fluorescent excitation transfer immunoassay for the determination of spinosyn A in water.

A fluorescent excitation transfer immunoassay for spinosyn A, a fermentation derived insect control agent, has been developed and applied to the analysis of tap water and wastewater effluent from manufacturing plants. Fluorescein (F) and tetramethylrhodamine (TMR) were chosen as donor and quencher, respectively, for the excitation transfer. Fluorescence quenching was observed from the binding of F-labeled antigen to TMR-labeled antibody. By employing nonlabeled antigen in a competitive immunoassay format, we reversed fluorescence quenching. The assay provides a limit of detection of 0. 01 ppb and a working range of 0.05-1 ppb and allows for the rapid determination of spinosyn A in water with recovery values ranging from 96% to 120%. With the exploitation of the small size of optical fibers, fluorescence from an assay volume of 24 microL could be measured without special vessels.     

Characterization of the volatile pattern and antioxidant capacity of essential oils from different species of the genus Ocimum

The antioxidant capacity of essential oils obtained by steam hydrodistillation from five species of the genus Ocimum, namely Ocimum basilicum var. purpurascens, Ocimum basilicum, Ocimum gratissimum, Ocimum micranthum, and Ocimum tenuiflorum (syn. O. sanctum), were evaluated using a high-performance liquid chromatography-based hypoxanthine/xanthine oxidase and the DPPH assays. The yield of oils from the leaves of the five species was variable with the greater amount obtained from Ocimum gratissimum (3.5%) and the least from Ocimum basilicum var. purpurascens (0.5%). In the hypoxanthine/xanthine oxidase assay, strong antioxidant capacity was evident in all the oils but the greater was shown by that obtained from Ocimum tenuiflorum (syn. O. sanctum) (IC50 = 0.46 microL/mL) compared to Ocimum basilicum var. purpurascens (IC50 = 1.84 microL/mL). Antioxidant capacity was positively correlated (r = 0.92, p < 0.05) with a high proportion of compounds possessing a phenolic ring such as eugenol, while a strong negative correlation (r = -0.77, p > 0.1) with other major volatiles was observed. These correlations were confirmed to a large extent in the DPPH assay. The results of a 24 h experiment with Ocimum tenuiflorum (syn. O. sanctum) shows that the antioxidant capacity factor (amount of essential oil obtained x free radical scavenging capacity; mg x %/100) reaches a threshold between 10 and 12.00 h, corresponding to maximum sunlight intensity in Brasil and furthermore exhibits a clear diurnal variation. The data generated with Ocimum species indicates that essential oils obtained from various herbs and spices may have an important role to play in cancer chemoprevention, functional foods, and in the preservation of pharmacologic products.     

4-Pyridyl carbonyl compounds as thrips lures: effectiveness for Western flower thrips in y-tube bioassays

In a search for chemical lures to manage the cosmopolitan crop pest western flower thrips (WFT), Frankliniella occidentalis, a Y-tube olfactometer was used to screen 20 compounds, including 18 4-pyridyl compounds. Comparison of Y-tube results for New Zealand flower thrips (NZFT), Thrips obscuratus, with field trapping data for ethyl nicotinate and ethyl isonicotinate, suggested that the minimum attractive dose (MAD) of an odor compound, where significantly ( p < 0.05) more than 50% of thrips walked up the odor-laden arm, provided a measure for selecting compounds to evaluate for potential lure efficacy in the field. Eighteen synthetic 4-pyridyl compounds were tested on female WFT in a Y-tube olfactometer and four 4-pyridyl carbonyl compounds had MADs lower than the known WFT attractants p-anisaldehyde (MAD 10 (-3) microL) and ethyl nicotinate (10 (-2) microL): methyl isonicotinate (10 (-6) microL), ethyl-2-chloropyridine-4-carboxylate (10 (-6) microL), ethyl isonicotinate (10 (-4) microL) and methyl 4-pyridyl ketone (10 (-5) microL). The suitability of MAD for selecting compounds for further evaluation of practical lure efficacy is discussed. Comparisons of activities within homologous series of esters and ketones showed that attractant activity decreased with chain length. 4-Formyl pyridine was an attractant at a dose of 10 (-5) microL, but was repellent at higher doses (10 (-2)-10 degrees microL).     

Detection and quantitation of T-2 mycotoxin in rat organs by radioimmunoassay

A standard radioimmunoassay was compared with radiochromatography for the ability to detect unlabeled T-2 mycotoxin in organs from exposed animals. When 10% of HT-2, the only known metabolite that cross-reacts with T-2, was included and expressed as T-2 equivalents in the radiochromatographic detection, correlation between toxin detection in liver, spleen, and kidney by the 2 techniques was r = 0.98. An unknown metabolite was detected in heart extract by radiochromatography. Inclusion of this material in the T-2 equivalents detected by radiochromatography indicated a near-perfect correlation (r = 0.95; p greater than 0.05; slope = 0.82; y = intercept = 72) among all 4 tissues.     

Antifungal activity of strawberry fruit volatile compounds against Colletotrichum acutatum

Eight volatile products characterizing strawberry aroma, which is generated from the oxidative degradation of linoleic and linolenic acids by a lipoxygenase (LOX) pathway, were examined because of their antifungal activity against Colletotrichum acutatum, one of the causal agents of strawberry anthracnose. In this study, the effects of aldehydes, alcohols, and esters on mycelial growth and conidia development were evaluated. (E)-Hex-2-enal was found to be the best inhibitor of mycelial growth [MID (minimum inhibitory doses)=33.65 microL L(-1)] and of spore germination (MID=6.76 microL L(-1)), while hexyl acetate was the least effective of all volatile compounds tested (MID=6441.89 microL L(-1) for mycelial growth and MID=1351.35 microL L(-1) for spore germination). Furthermore, the antifungal activity of (E)-hex-2-enal on susceptibility of strawberry fruits to C. acutatum was also confirmed. The presence of these molecules in jars containing strawberry fruits inoculated with a suspension of spores inhibited the fungus growth and prevented the appearance of symptoms. Moreover, a study of the effects of (E)-hex-2-enal on conidial cells of C. acutatum was evaluated by transmission electron microscopy. This volatile compound altered the structures of the cell wall and plasma membrane, causing disorganization and lysis of organelles and, eventually, cell death.     

Growth characteristics,phytate contents,and coagulation properties of soymilk from a low-phytate Japanese soybean (Glycine max (L.) Merr.) line

The phytic acid (myo-inositol hexakisphosphate or InsP₆) content of seed crops is important to their nutritional quality. Since it represents 75?±?10% of the total seed phosphorus (P), phytic acid is also important regarding the management of P in agricultural production. A low-phytate F5 line, No. T-2-250-4-20, was selected from the progeny of a cross between the low-phytate soybean line CX1834 and the Japanese commercial cultivar Tanbakuro. This line and its parents were grown in a field nursery, and the growth characteristics, phytate accumulation, and processing suitability for tofu were evaluated. At full maturity, the weight of seeds per plant of line T-2-250-4-20 was 5.2- and 1.3-fold higher than that of CX1834 and Tanbakuro, respectively. The amount of phytate-phosphorus as a percentage of the total P content in seeds was 23% in line T-2-250-4-20-34, 30% in CX1834, and 69% in Tanbakuro. No significant difference was observed among the three cultivars/lines in their seed magnesium (Mg), potassium (K), crude protein, and sugar. However, the calcium (Ca), crude fat and ash contents in seeds of line T-2-250-4-20-34 and Tanbakuro was lowered compared to that of CX1834. The breaking stress of tofu was estimated employing a rheometer with a decreasing concentration of the coagulant magnesium chloride (MgCl₂), starting at 15.7?mmol?L^?1. In tofu made from Tanbakuro, the concentration of MgCl₂ required to achieve the maximum breaking stress was 12.6?mmol?L^?1; however, it was 9.5?mmol?L^?1 for tofu made from T-2-250-4-20-34 and CX1834. The tofu made from Tanbakuro was soft and broke at 6.3?mmol?L^?1 MgCl₂, but, in line T-2-250-4-20-34, harder tofu was made with lower MgCl₂ concentrations. No difference was observed among the cultivars/lines in the SDS-PAGE patterns of protein in soymilk. These results indicate that we have developed a low-phytate soybean with adequate productivity, and confirmed that tofu made from the low-phytate T-2-250-4-20-34 soybean becomes coagulated and harder at a lower MgCl₂ concentration than that from high-phytate soybean cultivars.     

Comparison of high pressure liquid chromatographic and chemical methods for vitamin D3 concentrates. II. Collaborative study.

A collaborative study was carried out which compared the official chemical method, 43.B14-43.B24, the official rat bioassay, 43.165, and the high pressure liquid chromatographic method for vitamin D3 resin, vitamin D3 resin in oil, and dry concentrate. A total of 340 samples were distributed to 17 collaborators for analysis. Five laboratories performed both the chemical and HPLC methods on 5 sets of blind duplicates. A 2-way analysis of variance comparing both methods for each sample showed a significant (P less than 0.01) difference between methods only for Sample 5. When the 2 methods were compared over all the samples, no significant (P less than 0.05) difference was found. Except for Sample 5, there were no differences in the repeatability of the methods. Per cent recoveries on Sample 3, which contained exactly 0.200 X 10(6) IU/g, showed 98.2% for the chemical method and 100.6% for the HPLC method for the 5 laboratories that performed both methods. The assay results of the HPLC and chemical methods are in good agreement with those found by the biological assay on Samples 1-4, but not for Sample 5. Evidence indicates that Sample 5 degraded partially to isotachysterol, and while the HPLC method yielded a reasonable value on this material, the chemical method erroneously showed full potency. An amendment is included for the collaboratively studied HPLC method which detects and eliminates 5,6-trans-vitamin D3, a possible interferant.     

Development of a solid-phase cleanup and portable rapid flow-through enzyme immunoassay for the detection of ochratoxin a in roasted coffee

A membrane-based flow-through enzyme immunoassay (patent application pending) for the detection of ochratoxin A (OA) in roasted coffee was developed. First, an extraction and solid-phase cleanup method was developed. A high partition coefficient for OA in the mobile phase was achieved by using methanol/5% aqueous NaHCO(3) as the sample extraction and cleanup solvent. The solid-phase (aminopropyl) cleanup was developed to chromatographically elute OA but retain cross-reacting compounds. Without using aminopropyl cleanup, cross-reacting compounds resulted in 100% false positives for both flow-through enzyme immunoassay and HPLC methods. However, after cleanup with aminopropyl, no false positives were observed. The flow-through results were visually evaluated. The sensitivity achieved for the flow-through was 4 microg kg(-1) in spiked roasted coffee. The assay was used to screen roasted coffee samples. Results were confirmed with HPLC with a detection limit of 1 microg kg(-1).     

谷蠹不同虫态蛀蚀对小麦成分及食用品质的影响

为研究谷蠹不同生长发育阶段侵害小麦后其成分及食用品质的变化,设定谷蠹在最适生长发育条件下（温度(32±1)℃、相对湿度75%±1%）对小麦进行侵害,测定受谷蠹卵期、幼虫期、蛹期和成虫期侵害后小麦的营养品质、流变学特性、食用品质等相关指标。结果表明：谷蠹感染小麦后,随着谷蠹在小麦籽粒内从卵生长发育到成虫阶段：灰分呈先降低后上升趋势,蛹期和成虫期与原始样间差异显著（P<0.05）;清蛋白和还原糖含量等营养指标呈现先升高后降低,清蛋白各虫期均与原始样间差异显著（P<0.05）,还原糖各虫期与原始样间无显著差异（P<0.05）;粗脂肪、谷蛋白含量逐渐降低,粗脂肪各虫期均与原始样间差异显著（P<0.05）,谷蛋白幼虫期、蛹期和成虫期与原始样间差异显著（P<0.05）;球蛋白、醇溶蛋白含量呈波动变化,球蛋白和醇溶蛋白均在幼虫期、蛹期和成虫期与原始样间差异显著（P<0.05）;另外,其微观结构显示随着谷蠹卵在小麦籽粒内生长发育,谷蠹侵害界面的质地变硬、表面粗糙、结构疏散、基质蛋白质断裂严重,且破损淀粉逐渐增多;流变学特性变化明显：出峰时间呈逐渐降低趋势,在蛹期和成虫期与原始样间差异显著（P<0.05）,峰高和峰面积先降低后升高,峰宽整体呈上升趋势,峰高、锋宽和峰值面积在各虫期间均与原始样间差异显著（P<0.05）,中线峰值右侧斜率呈先升高后降低,各虫期与原始样间差异显著（P<0.05）;馒头感官评价总分逐渐降低;全麦粉馒头质构参数中弹性、黏聚性和恢复性逐渐减小,硬度、咀嚼度、胶着性呈波动性变化。从谷蠹不同虫期感染小麦与其理化及应用品质指标间差异性分析可看出,不同虫期之间受谷蠹感染的小麦理化及应用品质指标变化显著（P<0.05）。谷蠹在幼虫期和成虫期侵害小麦后各项理化及应用品质指标变化更明显。结果表明不同虫期谷蠹对小麦蛀蚀不仅可造成理化指标的变化还可直接影响其食用品质,因此在储藏期间应注重防治不同生长期的谷蠹。     

Effect of 1-methylcyclopropene on volatile emission and aroma in cv. Anna apples

The rapidly ripening summer apple cultivar Anna was treated with 0.1 micro L(-1) and 1 microL L(-1) 1-methylcyclopropene (MCP) at harvest and kept at 20 degrees C, or stored for 5 weeks at 0 degrees C and then transferred to 20 degrees C. Total volatiles were not reduced by treatment with 0.1 microL L(-1) MCP, but were 70% lower in fruits treated with 1 microL L(-1) MCP than in untreated fruits. Ethylene production was 50% and 95% inhibited by 0.1 microL L(-1) and 1 microL L(-1) MCP, respectively. The volatiles produced by fruit at harvest were predominantly aldehydes and alcohols, with some acetate esters as well as 2-methyl butyl acetate and beta-damascenone. During ripening, the acetate and butyrate esters increased greatly and alcohols and aldehydes decreased. MCP-treated apples retained more alcohols, aldehydes, and beta-damascenone volatiles than did untreated apples. Sensory evaluation found that control and 0.1 microL L(-1) treated apples developed more fruity, ripe, and overall aromas, but the preference was for the 1 microL L(-1) treated apples with a less ripe aroma.     

Quantification of the biocontrol agent Pseudomonas fluorescens Pf153 in soil using a quantitative competitive PCR assay unaffected by variability in cell lysis- and DNA-extraction efficiency

Although often neglected, variability in cell lysis efficiency and DNA extraction yield represents the major hurdles of any polymerase chain reaction (PCR)-based quantification protocol in soil and other natural environments. In this study we developed a technique that minimizes the effects of these constraints, providing at the same time a reliable internal control to distinguish between PCR-inhibition and negative results. We used Pseudomonas fluorescens Pf153, a root-colonizing bacterium that shows biocontrol activity against tobacco and cucumber black root rot, as the target organism for PCR quantification. Prior to DNA extraction, the genetically engineered, cognate reference strain P. fluorescens CHA0/c2 was inoculated in a reference soil. CHA0/c2 in the reference soil and Pf153 in the soil sample were lysed in parallel and afterward the lysates were mixed in known proportions. CHA0/c2 carries the plasmid pME6031-cmp2 that contains an allelic variant (competitor) of the Pf153 specific sequence Pf153_2. In a quantitative competitive PCR (QC-PCR) assay the competitor allows the quantification of the target strain down to 0.66 Pf153 CFU/mg soil. Processing the reference strain in the same way as Pf153 enables the exact quantification of the target strain in biocontrol assays performed in natural soil, overcoming differences in DNA extraction efficiency and PCR amplification from different soil environments. This technique is easily adaptable to other Pseudomonas strains simply by replacing the competitor used here with one derived from a SCAR-marker which is specific for the strain of choice.     

Matrix effects in analysis of β-agonists with LC-MS/MS: influence of analyte concentration, sample source, and SPE type

The synergistic influences of analyte concentration, sample source, and solid-phase extraction (SPE) type on matrix effects in the multiresidue analyses of eight β-agonists with LC-ESI-MS/MS were evaluated. Porcine muscle and liver extracts and urine from diverse sources were purified by strong or mixed-mode cation exchange and molecularly imprinted polymer SPE cartridges, respectively. Three spiked concentrations (2, 10, and 20 ng/mL) of eight β-agonists in the purified matrices and the different sample sources were analyzed. The results show that for most β-agonists there are significant differences in matrix effects between analyte concentrations or sample sources (P < 0.05), whereas there is no significant difference in matrix effects between different SPE cartridges (P > 0.05). Results from main effects testing indicated that analyte concentration was the main effector.