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1.
N Z Dent J ; 110(2): 65-73, 2014 Jun.
Article in English | MEDLINE | ID: mdl-25000809

ABSTRACT

OBJECTIVE: To provide a snapshot of the New Zealand dental technology industry and influencing factors. BACKGROUND: Developing an understanding of the commercial dental laboratory environment in New Zealand can provide insight into the entire dental industry. METHODS: A web-based survey was the primary method for data collection, with separate questionnaires used for dental laboratory owners and dental technician employees. RESULTS: The mean net income for dental laboratory owners in New Zealand was similar to that of the United Kingdom, at $40.50 per hour. Clinical dental technicians are the highest paid employees, with a mean of $33.49 per hour. The mean technical charge for complete dentures was $632.59; including clinical services, it was $1907.00. The mean charge for a porcelain-fused-to-metal (PFM) crown was $290.27. Dental laboratory owners expressed fear about the possibility of losing dental clients to overseas laboratories due to the availability and cheap charge of offshore work. Only 25.4% of dental laboratories surveyed had computer-aided design (CAD) facilities, and even fewer (7.9%) had computer-aided manufacturing (CAM) systems. CONCLUSION: Clinical dental technology appears to be prospering. The dental technology industry appears to be adapting and remains viable, despite facing many challenges.


Subject(s)
Laboratories, Dental/statistics & numerical data , Technology, Dental/statistics & numerical data , Adult , Computer-Aided Design , Crowns/economics , Crowns/statistics & numerical data , Dental Porcelain/economics , Dental Technicians/economics , Dental Technicians/statistics & numerical data , Denture, Complete/economics , Denture, Complete/statistics & numerical data , Denture, Partial, Removable/economics , Denture, Partial, Removable/statistics & numerical data , Employment , Ethnicity , Fees and Charges , Female , Forecasting , Humans , Income , Job Satisfaction , Laboratories, Dental/economics , Laboratories, Dental/trends , Male , Metal Ceramic Alloys/economics , New Zealand , Ownership , Practice Management/economics , Technology, Dental/economics , Technology, Dental/trends
2.
N Z Dent J ; 106(2): 50-4, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20608308

ABSTRACT

OBJECTIVES: To identify any potentially toxic elements in porcelain-fused-to-metal (PFM) crowns and a bridge manufactured in China. MATERIALS AND METHODS: Eight PFM crowns and part of a bridge were sourced from China for testing. They were given a typical glaze firing cycle prior to scanning electron microscope analysis. Electron dispersive spectroscopic spot and mapping analysis was carried out on the porcelain/metal collar interface areas in order to determine their elemental composition and distribution. RESULTS: No toxic elements were detected. The alloy used in the crowns was a nickel-chromium base metal, and that in the bridge was a gold-palladium noble alloy. The veneering porcelain was similar to a standard dental veneering porcelain. CONCLUSIONS: Within the limitations of the testing method and small sample size, no toxic elements were detected.


Subject(s)
Metal Ceramic Alloys/chemistry , Adult , China , Crowns , Denture, Partial, Fixed , Elements , Humans , Male , Microscopy, Electron, Scanning , New Zealand , Pilot Projects , Spectrometry, X-Ray Emission
3.
Article in English | MEDLINE | ID: mdl-18484306

ABSTRACT

The aryl hydrocarbon receptor (AhR) receives much attention for its role in the toxicity of dioxins and dioxin-like polychlorinated biphenyls. However, many other compounds have also been reported to bind and activate AhR, of which natural food components are of special interest from a human health perspective. Using the dioxin receptor-chemical-activated luciferase gene expression (DR CALUX) bioassay, extracts from many food items frequently consumed in the Netherlands were screened to estimate the intake of natural AhR agonists (NAhRAs). Using the prototypical AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as standard, it was estimated that the daily intake of NAhRAs might be considerably higher than the reported intake of dioxins and dioxin-like polychlorinated biphenyls. Potatoes, cruciferous vegetables, bread, hamburgers, and grapefruit juice contained most NAhRAs. Food preparation and acid treatment can show a significant effect on AhR activation. The interaction of natural and xenobiotic AhR agonists should be taken into account when performing risk-benefit analysis of both types of compounds.


Subject(s)
Food Contamination/analysis , Polychlorinated Dibenzodioxins/analysis , Receptors, Aryl Hydrocarbon/agonists , Animals , Biological Assay/methods , Environmental Pollutants/analysis , Feeding Behavior , Food Analysis/methods , Humans , Rats , Tumor Cells, Cultured , Vegetables/chemistry
4.
Toxicol In Vitro ; 22(2): 396-410, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18061397

ABSTRACT

Cruciferous vegetables and citrus fruits are reported to possess health-beneficial properties, but also have been shown to contain natural aryl hydrocarbon receptor (AhR) agonists (NAhRAs). Binding to the AhR is widely assumed to activate the main pathway by which dioxins, like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exert their toxicity. To establish whether or not activation of the AhR pathway by NAhRAs and dioxin-like substances results in similar cellular responses, gene expression profiles induced in Caco-2 cells were studied using microarray analysis. Cells were exposed to indolo[3,2-b]carbazole (ICZ), an acid reaction product from cruciferous vegetables, and to extracts of citrus pulp and grapefruit juice. Gene expression profiles induced by these NAhRAs were compared to those of the xenobiotic AhR agonists TCDD and benzo[a]pyrene (B[a]P). Over 20 genes were found more than 1.5 times up- or down-regulated by TCDD, and the expression of most of these genes was modulated in the same direction and to a similar extent by B[a]P and the NAhRAs. Results were confirmed by RT-PCR, and many of these genes may be involved in dioxin-related toxic effects. In conclusion, this in vitro study showed similar effects induced by NAhRAs, TCDD and B[a]P at the transcriptome level in a human intestinal cell line.


Subject(s)
Benzo(a)pyrene/toxicity , Citrus/chemistry , Environmental Pollutants/toxicity , Gene Expression Profiling , Polychlorinated Dibenzodioxins/toxicity , Receptors, Aryl Hydrocarbon/agonists , Vegetables/chemistry , Caco-2 Cells , Chromatography, High Pressure Liquid , Cytochrome P-450 CYP1A1/metabolism , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , Gene Expression/drug effects , Genes, Reporter , Humans , Luciferases/genetics , Oligonucleotide Array Sequence Analysis , Plant Extracts/chemistry , RNA/biosynthesis , RNA/genetics , Reverse Transcriptase Polymerase Chain Reaction , Xenobiotics/toxicity
5.
Anal Biochem ; 298(1): 93-102, 2001 Nov 01.
Article in English | MEDLINE | ID: mdl-11673900

ABSTRACT

The aim of the work described in this report is to develop and characterize a cell-based androgen reporter assay. For this purpose, the androgen receptor (AR) expressing human breast cancer cell line T47D was stably transfected with a luciferase gene under transcriptional control of the PB-ARE-2 androgen response element. The application of this cell line in an endogenous Androgen Receptor-mediated LUciferase eXpression assay (AR-LUX) was validated. An EC50 value of 86 pM was determined for the standard androgen R1881 with a detection limit of 46 pM. Other androgens like dihydrotestosterone, 17beta-trenbolone, and bolasterone also induced luciferase expression, while anti-androgens suppressed these responses. As expected, AR-mediated responses were also elicited by high concentrations of the steroids progesterone, 17beta-estradiol, d-aldosterone, and dexamethasone, with observed EC50 values 10 to 350,000 times higher than that for R1881. A unique feature of the AR-LUX assay is that effects on modulation of active endogenous AR-levels are reliably reflected in the luciferase induction response, as exemplified by vitamin D, all-trans-retinoic acid, epigallocatechin gallate, and forskolin. This feature is especially useful when assessing complex mixtures, e.g., environmental samples or natural compound libraries. From these data it is concluded that the AR-LUX assay is a reliable in vitro test system for the detection and quantification of AR-mediated biological effects. The 96-well plate format makes the assay particularly suitable for high-throughput screening.


Subject(s)
Androgens/metabolism , Breast Neoplasms/metabolism , Genes, Reporter/physiology , Luciferases/analysis , Luciferases/metabolism , Receptors, Androgen/metabolism , Aldosterone/metabolism , Dexamethasone/metabolism , Down-Regulation , Estradiol/metabolism , Genes, Regulator/genetics , Genes, Reporter/genetics , Humans , Luciferases/genetics , Metribolone/metabolism , Transfection/methods , Tumor Cells, Cultured/cytology , Tumor Cells, Cultured/metabolism
6.
Food Addit Contam ; 15(8): 863-75, 1998.
Article in English | MEDLINE | ID: mdl-10366995

ABSTRACT

There is a strong need for the development of relatively cheap and rapid bioassays for the determination of dioxins and related compounds in food. A newly developed CALUX (Chemical-Activated LUciferase gene eXpression) bioassay was tested for its possible use to determine low levels of dioxins in bovine milk. Data show that this mammalian cell-based test is very sensitive for 2,3,7,8-substituted dioxins and related PCBs, thereby reflecting the relative potencies of these compounds in comparison to TCDD (TEF-values). The limit of detection was about 50 fg of TCDD. Furthermore, the response obtained with a mixture of dioxins was additive, in accordance with the TEF-principle. Milk fat was isolated by centrifugation followed by clean-up of the fat with n-pentane, removal of the fat on a 33% H2SO4 silica column, and determination of Ah receptor agonist activity with the CALUX-bioassay. An equivalent of 67 mg fat was tested per experimental unit, resulting in a limit of quantification around 1 pg i-TEQ/g fat. To investigate the performance of the method, butter fat was cleaned and spiked with a mixture of 17 different 2,3,7,8-substituted PCDD and PCDF congeners at 1, 3, 6, 9, 12 and 15 pg TEQ/g fat, as confirmed by GC/MS. In this concentration range, the method showed a recovery of TEQs around 67% (58-87%). The reproducibility, determined in three independent series showed a CV varying between 4% and 54%, with the exception of the sample spiked at 1 pg i-TEQ (CV 97%). The repeatability determined with the sample spiked at 6 pg i-TEQ/g showed a CV of 10%. Testing of 22 bovine milk samples, taken at different sites in The Netherlands, in the CALUX-assay showed combined dioxin and dioxin-like PCB levels equivalent to 1.6 pg TCDD/g fat (range 0.2-4.6). GC/MS analysis of these samples revealed an average level of 1.7 pg i-TEQ/g fat, varying between 0.5 and 4.7 pg i-TEQ/g fat. All five samples showing a GC/MS determined dioxin content of more than 2 pg i-TEQ/g fat gave a response in the CALUX-assay corresponding to more than 2 pg TCDD/g fat. These data clearly show that the CALUX-bioassay is a promising method for the rapid and low cost screening of dioxins in bovine milk.


Subject(s)
Biological Assay/methods , Dioxins/analysis , Milk/chemistry , Polychlorinated Biphenyls/analysis , Animals , Cattle , Dose-Response Relationship, Drug , Gas Chromatography-Mass Spectrometry , Luciferases/drug effects , Luciferases/genetics , Luciferases/metabolism , Polychlorinated Dibenzodioxins/pharmacology , Rats , Reproducibility of Results , Tumor Cells, Cultured
7.
Fundam Appl Toxicol ; 30(2): 194-203, 1996 Apr.
Article in English | MEDLINE | ID: mdl-8812265

ABSTRACT

Exposure to specific polychlorinated diaromatic hydrocarbons (PCDH), such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), produces a wide variety of species- and tissue-specific toxic and biological effects. Many of these responses are mediated by the Ah receptor (AhR) and are modulated by the interaction of the PCDH:AhR complex with its DNA recognition sequence (the dioxin-responsive element (DRE)). We have constructed a recombinant expression plasmid which contains the luciferase gene under TCDD-inducible control of several DREs and responds to TCDD-like chemicals with the induction of firefly luciferase. Stable transfection of this vector into various cell lines has produced a series of species-specific cell bioassay systems that respond to TCDD-like chemicals with the induction of luciferase in a time-, dose-, and AhR-dependent manner. In addition, these cell lines have been used to demonstrate that 2,2',5,5'-tetrachlorobiphenyl can act as a species-specific AhR antagonist. Overall, these recombinant cell lines can be used for the detection and relative quantitation of AhR agonists/antagonists in complex mixtures of environmental and biological samples, for identification and characterization of novel AhR agonists, and for examination of species differences in PCDH responsiveness.


Subject(s)
Polychlorinated Dibenzodioxins/analogs & derivatives , Polychlorinated Dibenzodioxins/analysis , Receptors, Aryl Hydrocarbon/drug effects , Animals , Biological Assay/methods , Cell Line , Humans , Luciferases/biosynthesis , Luciferases/genetics , Mice , Polychlorinated Biphenyls/pharmacology , Receptors, Aryl Hydrocarbon/genetics , Receptors, Aryl Hydrocarbon/metabolism , Recombination, Genetic , Species Specificity , Transfection
8.
Toxicol Appl Pharmacol ; 137(2): 316-25, 1996 Apr.
Article in English | MEDLINE | ID: mdl-8661358

ABSTRACT

A recombinant H4IIE rat hepatoma cell line (H4L1.1c4, H4IIE-luc), containing a luciferase reporter gene under control of dioxin-responsive enhancers, was examined for responsiveness to several polyhalogenated aromatic hydrocarbons (PHAHs). The recombinant cell system was compared with the widely used wild-type cell line (H4IIE-wt), which expresses Ah receptor-mediated cytochrome P450 1A induction. We also report an improved and down-scaled method for the H4IIE-wt bioassay which allows for the rapid screening of environmental samples for Ah-active PhAHs. This method employs 96-well plates, a plate-reading spectrofluorometer, and a fluorescence-based protein assay that enables the simultaneous measurement of resorufin and protein. Both cell lines demonstrated a dose-dependent increase in Ah receptor-mediated response upon exposure to a number of known Ah receptor agonists, including Halowax 1014. H4IIE-luc cells were 3-fold more sensitive than H4IIE-wt cells to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The detection limit and ED50 for EROD induction by TCDD were 0.6 and 4.9 fmol/well (2,4 and 20 pM), respectively; for luciferase induction they were 0.2 and 1.4 fmol/well (0.8 and 5.6 pM). The detection limit for EROD induction in H4IIE-wt cells was a 50-fold improvement over that reported previously (Tillitt et al., Environ. Sci. Technol. 25, 87-92, 1991) and comparable to that of a chicken embryo primary hepatocyte bioassay (Kennedy et al., Anal. Biochem. 211, 102-112, 1993). The tested PHAHs exhibited a similar structure-activity relationship in H4IIE-luc as in H4IIE-wt cells. Binary mixtures of TCDD, PCB-126, and PCB-77 showed no departure from additivity in their combined responses when tested in H4IIE-wt cells. PCB-153 at the highest tested dose of 14 nmol/well (56 microM) significantly reduced the potency of TCDD and PCB-126 without affecting their efficacy in both H4IIE-wt and H4IIE-luc cells. These findings support the use of H4IIE-luc cells as an alternative bioanalytical tool to the wild-type cells for the detection of Ah agonists in environmental samples.


Subject(s)
Cytochrome P-450 Enzyme System/biosynthesis , Liver Neoplasms, Experimental/enzymology , Luciferases/biosynthesis , Oxidoreductases/biosynthesis , Polychlorinated Biphenyls/analysis , Polychlorinated Dibenzodioxins/analysis , Receptors, Aryl Hydrocarbon/physiology , Animals , Cytochrome P-450 CYP1A1 , Enzyme Induction/drug effects , Fluorescamine , Liver Neoplasms, Experimental/pathology , Liver Neoplasms, Experimental/ultrastructure , Rats , Sensitivity and Specificity , Serum Albumin, Bovine , Structure-Activity Relationship , Tumor Cells, Cultured
9.
Eur J Pharmacol ; 293(4): 463-74, 1995 Dec 07.
Article in English | MEDLINE | ID: mdl-8748700

ABSTRACT

Using recombinant cell lines showing Ah receptor-controlled expression of a luciferase reporter gene, the interaction of di-ortho-substitute polychlorinated biphenyls (PCBs) with Ah receptor agonists was studied. In the recombinant Hepa1c1c7 mouse hepatoma (H1L1.1c7) cells strong antagonistic interaction of 2,2',5,5'-tetrachlorobiphenyl (PCB52) with luciferase expression induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 3,3',4,4'-tetrachlorobiphenyl (PCB77) was observed, and similarly, between 2,2',3,3',4,4'-hexachlorobiphenyl (PCB128) and PCB77. Accordingly, PCB52 was found to inhibit ethoxyresorufin-O-deethylase (EROD) induction by PCB77 in wild-type Hepa1c1c7 cells. In contrast, the antagonistic effect of PCB52 on TCDD-induced luciferase expression was only minor in recombinant guinea pig GPC16 colon adenocarcinoma (G16L1.1c8) and human HepG2 hepatoma (HG2L1.1c3) cells, and intermediate in recombinant H4IIE rat hepatoma (H4L1.1c4) cells. Gel retardation studies using a 32 P-labelled dioxin responsive element (DRE)-containing oligonucleotide, and ligand binding studies using [3H]TCDD, demonstrated that the species-specific antagonistic activity of PCB52 on Ah receptor-controlled luciferase expression is due to inhibition of Ah receptor ligand and DNA binding. We conclude, that Ah-mediated luciferase expression provides a useful tool to study the species specificity of Ah receptor (ant)agonists.


Subject(s)
Polychlorinated Biphenyls/pharmacology , Receptors, Aryl Hydrocarbon/antagonists & inhibitors , Animals , Base Sequence , Carcinoma, Hepatocellular , Cytochrome P-450 CYP1A1 , Cytochrome P-450 Enzyme System/biosynthesis , Enzyme Induction , Guinea Pigs , Humans , Luciferases/biosynthesis , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Oxidoreductases/biosynthesis , Polychlorinated Dibenzodioxins/pharmacology , Rats , Rats, Sprague-Dawley , Tumor Cells, Cultured
10.
Obstet Gynecol Surv ; 50(6): 459-69, 1995 Jun.
Article in English | MEDLINE | ID: mdl-7659394

ABSTRACT

The treatment of patients with cervical incompetence presenting with advanced cervical changes in the second trimester remains a challenge to every obstetrician. Cerclage operation may be the only hope for prolonging gestation until fetal viability is reached. A retrospective study on so-called emergency cervical cerclage in 20 patients with supposed cervical incompetence in the late second trimester is presented, together with a review of comparable studies published between 1980 and 1992. It is concluded that emergency cerclage can be of benefit, and that the pregnancy is saved in the majority of cases, although the incidence of complications, often due to infection, is high. Many patients require prolonged hospitalization or bed rest and few pregnancies reach full term. There is a particularly high rate of infectious complications and attention must be focused on preventing chorioamnionitis to improve the outcome of the procedure in the future.


Subject(s)
Cervix Uteri/surgery , Emergencies , Uterine Cervical Incompetence/surgery , Adolescent , Adult , Female , Gestational Age , Humans , Infant, Newborn , Pregnancy , Pregnancy Outcome , Retrospective Studies , Suture Techniques , Uterine Cervical Incompetence/etiology
11.
Toxicol Appl Pharmacol ; 129(2): 283-93, 1994 Dec.
Article in English | MEDLINE | ID: mdl-7992318

ABSTRACT

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) and the coplanar 3,3',4,4'-tetrachlorobiphenyl (3,3',4,4'-TCB) and 3,3',4,4',5,5'-hexachlorobiphenyl (3,3',4,4',5,5'-HCB) on intercellular communication (IC) were determined in order to investigate the in vitro tumor promoting potency of these compounds. 2,3,7,8-TCDD and the two coplanar PCBs tested caused a rapid (2 hr) and a sustained inhibition (48 hr) of IC in the mouse hepatoma cell line (Hepa1c1c7) to 20 and 50% of the unexposed control, respectively. Inhibition of IC was dose dependent with an EC50 range of about 50-100 pM for 2,3,7,8-TCDD, 2-5 nM for 3,3',4,4'-TCB, and 10-15 nM for 3,3',4,4',5,5'-HCB, respectively. A comparison of the IC inhibitory effects of 2,3,7,8-TCDD and PCBs with a well-known aryl hydrocarbon receptor (AhR)-mediated response, the induction of ethoxyresorufin O-deethylase (EROD) activity, in the same cells revealed EROD induction by 2,3,7,8-TCDD, 3,3',4,4'-TCB, and 3,3',4,4',5,5'-HCB with EC50 ranges of 100-200 pM, 20-70 nM, and 5-10 nM, respectively. The time course of IC inhibition was paralleled by EROD induction, although the time of onset of the response was earlier for IC (1 hr) than for EROD (2.5 hr). A role of the AhR in the inhibition of IC by 2,3,7,8-TCDD and PCBs was demonstrated by the lack of inhibition in AhR-defective Hepa1c1c7 cells. Transient inhibition of IC was observed in the mutant cells only at early time points (within 2 hr of exposure). These results and the observation that alpha-naphtoflavone (an AhR antagonist) greatly reduced the 2,3,7,8-TCDD-dependent sustained inhibition of IC strongly support a role of the AhR in the sustained inhibition of IC by these compounds. Furthermore, these data suggest that the mouse Hepa1c1c7 cells may be a good model in which to study in vitro tumor promoting capacity of dioxins, PCBs, and related compounds.


Subject(s)
Carcinogens/toxicity , Cell Communication/drug effects , Liver Neoplasms, Experimental/physiopathology , Polychlorinated Biphenyls/toxicity , Polychlorinated Dibenzodioxins/toxicity , Receptors, Aryl Hydrocarbon/drug effects , Animals , Cytochrome P-450 CYP1A1 , Cytochrome P-450 Enzyme System/metabolism , Dose-Response Relationship, Drug , Liver Neoplasms, Experimental/metabolism , Mice , Mice, Inbred C57BL , Oxidoreductases/metabolism , Time Factors , Tumor Cells, Cultured
12.
Plant Mol Biol ; 16(4): 647-61, 1991 Apr.
Article in English | MEDLINE | ID: mdl-1651125

ABSTRACT

With a view to cloning the root-knot nematode resistance gene Mi in tomato by chromosome walking, we have developed a molecular probe for the tightly linked acid phosphatase-1 (Aps-1) locus. The acid phosphatase-1 allozyme (APS-1(1], encoded by the Aps-1(1) allele originating from Lycopersicon peruvianum, was purified to apparent homogeneity from tomato roots and suspension cells. Microsequencing of CNBr and tryptic peptides generated from APS-1(1) provided a partial amino acid sequence, which accounted for approximately 23% of the protein and revealed two stretches of homology with soybean proteins KSH3 and VSP27, comprising 22 matches within 26 amino acid residues. The partial amino acid sequence information enabled us to isolate a 2.4 kb genomic Aps-1(1) sequence by means of the polymerase chain reaction (PCR), primed by degenerate pools of oligodeoxyribonucleotides, synthesized on the basis of the amino acid sequences. Synthesis of the 2.4 kb PCR product was specific for genomic templates carrying the L. peruvianum Aps-1(1) allele. Crucial to the priming specificity and the synthesis of the 2.4 kb genomic sequence was the use of degenerate primer pools in which the number of different primer species was limited by incorporating deoxyinosine phosphate residues at three and four base ambiguities. In using cDNA as a template, a 490 bp sequence was obtained, indicating a high proportion of intron sequences in the 2.4 kb genomic Aps-1(1) sequence. The Aps-1(1) origin of the PCR product was confirmed by RFLP (restriction fragment length polymorphism) analysis, using both a chromosome 6 substitution line and a pair of nearly isogenic lines, differing for a small chromosomal region around the Aps-1/Mi loci.


Subject(s)
Acid Phosphatase/genetics , Isoenzymes/genetics , Plants/genetics , Acid Phosphatase/isolation & purification , Alleles , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Chromatography , Chromatography, Ion Exchange , Durapatite , Electrophoresis, Polyacrylamide Gel , Genetic Markers , Hydroxyapatites , Immunity, Innate/genetics , Inosine/analogs & derivatives , Isoenzymes/isolation & purification , Molecular Sequence Data , Nematoda , Oligonucleotide Probes , Plant Diseases , Plants/enzymology , Polymerase Chain Reaction/methods , Sequence Homology, Nucleic Acid , Ultrafiltration
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