Polymerase Chain Reaction

A fraction of cats exposed to feline leukemia virus (FeLV) effectively contain virus and resist persistent antigenemia/viremia. Using real-time PCR (qPCR) to quantitate circulating viral DNA levels, previously we detected persistent FeLV DNA in blood cells of non-antigenemic cats considered to have resisted FeLV challenge. In addition, previously we used RNA qPCR to quantitate circulating viral RNA levels and determined that the vast majority of viral DNA is transcriptionally active, even in the absence of antigenemia.

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Solid-phase techniques have facilitated the handling of biochemical analytes. This has stimulated the development of systems by which large sample panels can be analyzed with high levels of security and quality. We describe a sample transfer device based on the principle of vacuum filtration, which enables parallel handling of 96 samples of analytes bound to Sepharose beads. The tool was employed for strand separation of DNA samples, by attracting the beads to filter probes while passing them between the reagent solutions. The samples were analyzed using Pyrosequencing technology and proved to yield genotyping results of high quality. The presented sample preparation procedure provides an important link in the development of integrated systems for rapid genetic analysis at a low cost. In addition, the same filter could be reused extensively with very low risk for detectable cross-contamination between assays and without any reduction in processing capacity, thus further reducing the cost per analyzed sample.

There is a general acceptance that cellular material will transfer from one person to another person's fingernails through everyday contact. However, the level or degree of contact required to transfer sufficient cellular material in order to obtain a DNA profile is not known. This study examined swabs from the fingernails of 40 volunteers and compared the DNA profiles obtained to the daily activities of that individual. The majority (78%) of high level profiles obtained were associated with recent intimate contact. However, high level profiles were also obtained from the fingernails of individuals who shared accommodation with their partner, flatmates and/or children. Low level profiles and single profiles were associated with all levels of contact.

Techniques of molecular biology have improved diagnostic sensitivity, accuracy and validity in forensic medicine very much, especially in the field of identification (paternity testing, stain analysis). Since more than 10 years these techniques -meanwhile well established in clinical disciplines -are used also for other applications in forensic medicine: determination of cause and manner of death, tissue identification by mRNA and microRNA, examination of gene expression levels (survival time, time since death, cause of death), toxicogenetics.

The host-feeding patterns of Anopheles quadrimaculatus Say and Anopheles punctipennis (Say) were examined in order to evaluate their potential contributions to the transmission of eastern equine encephalitis virus (EEEv) and other arboviruses in the northeastern U.S.A. Engorged mosquitoes of the two species were collected from EEEv foci in central New York (NY) and throughout New Jersey (NJ), and their bloodmeals were identified using a polymerase chain reaction (PCR)-based assay and sequencing portions of the mitochondrial cytochrome b gene. Analysis of 131 An. quadrimaculatus and 107 An. punctipennis from NY revealed that 97.7% and 97.2%, respectively, had acquired blood solely from mammalian hosts. Similarly, examination of 288 An. quadrimaculatus and 127 An. punctipennis from NJ showed 100% and 96.0%, respectively, contained mammalian-derived bloodmeals. Mosquitoes containing mixed bloodmeals from both avian and mammalian hosts were detected in 1.6% of An. quadrimaculatus from NY, and 2.8% and 4.0% of An. punctipennis from NY and NJ, respectively. White-tailed deer (Odocoileus virginianus) constituted the most common vertebrate host for these anopheline mosquitoes, accounting for 85.8-97.7% of all bloodmeals identified. The predominance of white-tailed deer as a source of bloodmeals supports enzootic amplification of deer-associated arboviruses in this region, including Jamestown Canyon, Cache Valley and Potosi viruses. One horse-and two human-derived bloodmeals were also detected in An. quadrimaculatus collected in NJ. Limited avian-derived bloodmeals were detected from mourning dove (Zenaida macroura), sharp-shinned hawk (Accipiter striatus) and house finch (Carpodacus mexicanus), mostly in mixed bloodmeals. Occasional feeding on avian hosts suggests that these mosquitoes may participate as epizootic-epidemic bridge vectors of EEEv from viraemic birds to mammalian hosts of concern, including horses and humans. An isolate of EEEv was recovered from the head and thorax of an An. punctipennis mosquito collected in NY.

Ehrlichia are obligate intracellular bacteria that belong to the family Rickettsiaceae. Human monocytic ehrlichiosis (HME) and human granulocytic ehrlichiosis (HGE) are the 2 ehrlichial diseases that are of greatest health concern in the United States. The agents causing HME and HGE are zoonotic pathogens requiring a mammalian reservoir and an arthropod vector. Differences in the geographic distribution of their tick vectors account for the concentration of HME in the South and southeastern United States and HGE in the Northeast and northern Midwest. Both infections have been reported in coastal regions from Rhode Island to Florida. HME and HGE are flulike illnesses that usually are self-limited but may be fatal. Diagnosis is confirmed by identification of the microorganism on blood smear or polymerase chain reaction or by detection of anti-ehrlichial antibody. Doxycycline is the antibiotic of choice for treatment of ehrlichiosis.

The gut bacteria exert phenotypic traits to the host but the factors which determine the gut bacterial profile (GBP) is poorly understood. This study aimed to understand the effect of ethnicity and geography on GBP of Mongoloid and Proto-Australoid tribes of India. Fecal bacterial diversity was studied in fifteen tribal populations representing four geographic regions (Assam, Telangana, Manipur and Sikkim) by DGGE followed by NGS analysis on Illumina MiSeq platform. Geography and diet had significant effect on GBP of the Indian tribes which was dominated by Prevotella. The effects were more prominent with lower taxonomic levels, indicating probable functional redundancy of the core GBP. A comparison with the worldwide data revealed that GBP of the Indian population was similar to the Mongolian population (Mongolia). The bacterial genera Faecalibacterium, Eubacterium, Clostridium, Blautia, Ruminococcus and Roseburia were found to be core genera in the representative populations of th...

Transforming growth factor alpha (TGFα) belongs to the epidermal growth factor (EGF) family and is known to play an important role during eyelid morphogenesis. In this study, we showed that ectopic expression of TGFα in the stroma of Kera-rtTA/tet-O-TGFα bitransgenic mice results in precocious eye opening, abnormal morphogenesis of the meibomian gland, tendon and tarsal plate malformation and epithelium hyperplasia. TGFα did not change proliferation and differentiation of meibocytes, but promoted proliferation and inhibited differentiation of the tarsal plate tenocytes. These results suggest that proper formation of the tendon and tarsal plate in the mouse eyelid is required for normal morphogenesis of the meibomian gland.

To determine the prevalence, molecular characteristics and hematological study of thalassemia in Tha Kradarn Subdistrict Chachoengsao Province. The present study population consisted of266 participants from Moo 19 Baan Na-Ngam, Chachoengsao Province, Thailand. After blood collection, all samples were screened for thalassemia by initial screening with the OF and DCIP tests and additional testing by CBC, RBC indices, hemoglobin typing and determination of Hb A2 and Hb E. All common alpha-thalassemia mutations were determined using the PCR with allele specific primers and Gap PCR for common deletions. The prevalence of alpha-thal 1, alpha-thal 2 and beta-thal were found as 2.72%, 11.26% and 0.97%, respectively. Regarding the abnormal hemoglobins, the prevalence of Hb E, Hb Constant Spring and Hb Pakse was 38.45%, 3.69% and 0.78%, respectively. MCV and MCH were significantly different between P-thalassemia as well as a-thal 1 carriers and normal subjects. In all alpha-thal 1 traits, it ...

We describe two newly discovered fungi living on the adaxial leaf surface of plants belonging to the Convolvulaceae, Ipomoea asarifolia and Turbina corymbosa. The fungi apparently are epibionts because hyphae were never observed to penetrate epidermal cells or stomata of their respective host plants, and most remarkably are intimately associated with secretory glands on the leaf surface. Hyphae and structures resembling chlamydospores and synnemata (but lacking conidia), formed by both fungal species are phenotypically nearly indistinguishable after in vitro growth or when examined in vivo on the leaf surface. Phylogenetic trees based on aligned DNA sequences from nuclear genes for b-tubulin (tubB) and RNA Polymerase II subunit 1 (rpbA), and the mitochondrial gene for ATP synthase F0 subunit A (atp6), grouped the fungal species in a clade within the Clavicipitaceae. Clavicipitaceous fungi isolated from the two different plant species could be distinguished by their atp6 and rpbA sequences, and nuclear genes for c-actin (actG), translation elongation factor 1-a (tefA), and 4-(c,c-dimethylallyl)tryptophan synthase (dmaW), the determinant step in ergoline (i.e. ergot) alkaloid biosynthesis. Based on these findings we propose a new fungal genus, Periglandula, gen. nov., and describe two new species, Periglandula ipomoeae sp. nov., from host plant I. asarifolia, and Periglandula turbinae sp. nov., from T. corymbosa.

Mutations in the DJ-1 gene have recently been shown to cause autosomal recessive Parkinson's disease. To estimate the prevalence of this mutation, an analysis was undertaken of 39 index cases of Parkinson's disease in whom a family history suggested autosomal recessive inheritance. No DJ-1 mutations were found in these patients, indicating that this gene is unlikely to be of numerical significance in clinical practice. The hypothesis was also tested that young onset Parkinson's disease patients in whom, despite extensive analysis, only a single heterozygous parkin mutation was found, might harbour a second mutation in the DJ-1 gene--that is, digenic inheritance. No patient was found with a single mutation in both DJ-1 and parkin genes, making this mode of inheritance unlikely. Finally it was confirmed that PARK6 and PARK7 (DJ-1), despite being phenotypically similar and mapping to the same small chromosomal region of 1p36, are caused by mutations in separate genes.

Identification of Candida cultured from various clinical specimens to the species level is increasingly necessary for clinical laboratories. Although sn PCR identifies the species within hours but its cost-effectiveness is to be considered. So there is always a need for media which help in the isolation and identification at the species level. The study aimed to evaluate the performance of different chromogenic media and to compare the effectiveness of the traditional phenotypic methods vs. seminested polymerase chain reaction (sn PCR) for identification of Candida species. One hundred and twenty seven Candida strains isolated from various clinical specimens were identified by conventional methods, four different chromogenic media and sn PCR. HiCrome Candida Differential and CHROMagar Candida media showed comparably high sensitivities and specificities in the identification of C. albicans, C. tropicalis, C. glabrata and C. krusei. CHROMagar Candida had an extra advantage of identify...

The pathogenic oomycete Aphanomyces invadans is the primary etiological agent in ulcerative mycosis, an ulcerative skin disease caused by a fungus-like agent of wild and cultured fish. We developed sensitive PCR and fluorescent peptide nucleic acid in situ hybridization (FISH) assays to detect A. invadans. Laboratory-challenged killifish (Fundulus heteroclitus) were first tested to optimize and validate the assays. Skin ulcers of Atlantic menhaden (Brevoortia tyrannus) from populations found in the Pamlico and Neuse River estuaries in North Carolina were then surveyed. Results from both assays indicated that all of the lesioned menhaden (n = 50) collected in September 2004 were positive for A. invadans. Neither the FISH assay nor the PCR assay cross-reacted with other closely related oomycetes. These results provided strong evidence that A. invadans is the primary oomycete pathogen in ulcerative mycosis and demonstrated the utility of the assays. The FISH assay is the first molecula...

Although the extraction and analysis of nucleic acids from formalin-fixed paraffin-embedded tissues is a routine and growing part of pathology practice, no generally accepted recommendations exist to guide laboratories in their selection of tissue fixation, processing and DNA/RNA extraction techniques. The aim of this study was to determine how fixation method and length, paraffin embedding, processing conditions and nucleic acid extraction methods affect quality and quantity of DNA and RNA, and their performance in downstream applications. Nine tissue samples were subjected to freezing, fixation in formalin for b24 h and 7 days followed by conventional processing, and fixation in molecular fixative for b 24 h and 7 days followed by rapid processing. DNA and RNA were isolated using in-house extraction and commercial kits, and assessed by PCR reactions for amplicons with varying sizes ranging from 268 to 1327 bp and one-step RT-PCR for 621 bp and 816 bp amplicons of housekeeping genes. Molecular fixative (MF) appeared to perform well under nearly all circumstances (extraction methods, fixation lengths and longer amplicons), often performing as well as frozen samples. Formalin fixation generally performed well only for shorter length amplicons and short fixation (b 24 h). WaxFree kit showed consistently higher success rates for DNA and poorer rates for RNA. RecoverAll kit generally performed suboptimally in combination with prolonged formalin fixation. In conclusion, the Molecular Fixative regardless of fixation length, and the rapid tissue processing system were able to preserve large DNA and RNA fragments in paraffin blocks, making these techniques preferable for use in downstream molecular diagnostic assays.

With the widespread use of protein electrophoresis in verte- brate systematics in the 1960s and 70s, several museums and universities established extensive collections of frozen tissues. Use of these special collections expanded exponentially with the development of the polymerase chain reaction (PCR) and improvements in obtaining nucle- otide sequences. Moreover, using PCR, DNA sequences can now be routinely obtained directly

Using a new strategy, capture-RT-PCR, bcrablfusion mRNA sequences were specifically and sensitively detected in samples of whole blood from leukemia patients with the Philadelphia chromosome. Sample processing required only mixing blood with the chaotropic salt, GuSCN, and mRNA was captured during a short incubation of prepared blood with an affinity membrane. Immobilized mRNA sequences were amplified without elution. No radioisotopes or Southern transfer were needed.

Trypanosome infections in their natural hosts are frequently di.cult to detect by microscopy\ and culture methods are unreliable and not suitable for all species of Trypanosoma[ A nested PCR strategy for detecting and identifying Trypanosoma species\ suitable for detecting both known and unknown trypanosomes\ is presented[ Thirty!two blood samples from 12 species of Australian birds and mammals were screened by a nested PCR for the presence of Trypanosoma sp[ ssrRNA[ Three infections were detected\ one in an eastern grey kangaroo "Macropus giganteus#\ one in a common wombat "Vombatus ursinus# and one in a platypus "Ornithorhynchus anatinus#[ The kangaroo and wombat are new host records for Trypanosoma sp[^the platypus parasite was Trypanosoma binneyi[ The three parasites could be distinguished by restriction fragment length polymorphisms of the ampli_ed fragment of the ssrRNA gene[ The kangaroo and wombat parasites were also isolated in a semi!solid blood agar medium[ The culture forms of the kangaroo trypanosome had an expanded~agellar sheath in which structures similar to hemidesmosomes were detected by EM[ The nested PCR was at least as sensitive as culture\ and analysis of the PCR products gave parasite!speci_c _ngerprints[ Therefore this method could be suitable for rapidly screening host animals for the presence of trypanosomes and identifying the infecting strain[ Þ 0888 Australian Society for Parasitology[ Published by Elsevier Science Ltd[ All rights reserved[ Keywords] Macropus giganteus^Ornithorhynchus anatinus^Ortholfersia macleayi^Trypanosoma binneyi^Trypanosoma cruzi^Vombatus ursinus^Guanidine thiocyanate

Genosensor technology relying on the use of carbon and gold electrodes is reviewed. The key steps of each analytical procedure, namely DNA-probe immobilisation, hybridisation, labelling and electrochemical investigation of the surface, are discussed in detail with separate sections devoted to labelfree and newly emerging magnetic assays. Special emphasis has been given to protocols that have been used with real DNA samples.

Echinococcosis is a frequent hepatic parasitic disease in several countries but it is practically absent in Mexico. A cattle strain of Echinococcus granulosus was identified by RAPD, PCR-RFLP and mitochondrial CO1 gene analysis in an autochthonous case. The parasite was obtained after a laparoscopic excision of a liver cyst from a patient that was symptomatic for 6 years but mis-diagnosed before hospitalization.

N-acetyl-β-D-hexosaminidase was purified from wheat bran and characterized. The purified enzyme showed two protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with molecular mass of 75 and 78 kDa. The enzyme exhibited optimum pH and temperature at 5.0 and 50 o C, respectively. The enzyme was active on the substrates of p-nitrophenyl-Nacetyl-β-D-glucosaminide (pNP-GlcNAc) and p-nitrophenyl-Nacetyl-β-D-galactosaminide (pNP-GalNAc), whereas inactive on pNP-β-D-glucopyranoside, pNP-β-D-galactopyranoside, swollen chitin, and colloidal chitin, suggesting high substrate specificity. The enzyme activity for pNP-GlcNAc was stable at pH 3-6 and under 50 o C. The K m , V max and K cat for pNP-GlcNAc were 0.014 mM, 0.05 µmol/min, and 3.01×10 6 min −1 , respectively. The enzyme could be completely inhibited at 1-10 mM HgCl 2 and AgNO 3, suggesting that the intact thiol group is essential for activity. β-N-Acetylhexosaminidase from wheat bran could inhibit the conidial germination and digest the hyphae of Fusarium solani.

Here we describe a non-HeLa-derived rep/cap cell line called K209, generated by stable transfection of A549 cells with a plasmid construct containing the P5 rep/cap cassette from AAV2. Infection of K209 cells with adenovirus leads to a 1000-fold amplification of the rep/cap gene with high-yield production of AAV vectors. The multiplicity of infection (MOI) of adenovirus that led to maximum amplification of the rep/cap gene and high-level production of AAV is 10 times higher in the HeLa-based cell line than that required in K209 cells. Our data suggest that papilloma-derived gene products present in HeLa cells are not required for high-yield production of AAV vectors.

The coat protein (CP) gene of 75 South African Potato virus Y (PVY) isolates was amplified using reversetranscriptase polymerase chain reaction (RT-PCR). The resulting cDNA products were cloned and sequenced. These sequences were used to identify the strains to which the isolates belonged. Some, when compared to reference sequences, belonged to the PVY N and PVY O strains. A number of isolates were found to demonstrate significant homology to PVY N strains from China. A large number of South African isolates possessed CP sequences showing evidence of recombination between PVY N and PVY O strains, similar to those of PVY NTN isolates. Multiplex RT-PCR analysis allowed further differentiation of PVY O isolates and revealed that the majority were of the PVY N -Wilga strain. It was deduced that the most likely way in which these isolates reached South Africa was via the importation of infected material.

code VAL-0008 versie 5.0 pagina 1 / 16 verantw GD revisie 9-3-2012 De inhoud van dit afgedrukte document is enkel geldig indien het overeenstemt met de meest recente elektronische versie © nadruk verboden I) Scope Implementatie van een moleculaire test: Parameter: Neisseria gonorrhoeae kwalitatief Matrix: Urine (M) en genitale wissers (V) in eSwab transportmedium Toestel: Rotorgene 6000 (Qiagen) Techniek: Real-time PCR Kit: Diagenode Neisseria gonorrhoeae real-time PCR kit + Qiagen Quantifast Probe PCR Master Mix Een nieuwe moleculaire test is nodig ter vervanging van de vroegere in-house NG NASBA assay, die specificiteitsproblemen vertoonde (commensale Neisseria species werden opgepikt). De kit van Diagenode is CE-IVD approved en is gevalideerd in combinatie met de Nuclisens EasyMAG en de Rotorgene 6000. De Master Mix wordt apart aangekocht bij Qiagen (Quantifast Probe PCR Master Mix). Onze start-en elutievolumes zijn verschillend met wat in de bijsluiter wordt aangeraden, dus zullen we de LOD opnieuw moeten bepalen. De kit is gebaseerd op het artikel "A duplex Neisseria gonorrhoeae real-time polymerase chain reaction assay targeting the gonococcal porA pseudogene and multicopy opa genes. Namray Goire, Michael D. Nissen, Genevera M. LeCornec, Theo P.Sloots, David M. Whiley. Diagnostic Microbiology and infectious disease 61 (2008) 6-12" en gebruikt dezelfde primers en probes voor de detectie van Neisseria gonorrhoeae. Hierdoor kunnen we de data uit dit artikel en gerelateerde artikels gebruiken in het validatiedossier.

The cacti family is a morphologically heterogeneous group comprising 100 genera and about 1500 species (Hernandez and Barcenas, 1996). With the exception of one genus, all members of this family are native to America (Hernandez and Barcenas, 1996). There are three subfamilies, Opuntioideae, Cactoideae, and Pereskioideae (Gibson and Nobel, 1986). DNA isolation from cacti is notoriously difficult because they contain high amounts of polysaccharides and secondary metabolites which form insoluble complexes with nucleic acids during extraction (Guillemaut and Marechal-Drouard, 1992). Like in other groups of plants, the secondary metabolites and polysaccharides in cacti inhibit enzyme action (Porebski et al., 1997). The polysaccharides are visually evident by their viscous, glue-like texture and they make the DNA unmanageable when pipeting and hard to amplify by the polymerase chain reaction (PCR) (Poresbski et al., 1997). We report an easy and inexpensive protocol to isolate DNA from cacti. We used this method to isolate DNA from 85 species (170 individuals) of 39 genera of the subfamilies Pereskioideae, Opuntioidea, and Cactoideae. This procedure is a modification of a protocol described by De la Cruz et al. (1995) for the Cacti family. It requires only a few grams of tissue and does not require destruction of the whole plant to produce high molecular weight genomic DNA. The DNA from this procedure can be amplified consistently by PCR and used for RAPD analysis.

A novel real-time quantitative PCR (QPCR) assay is described for monitoring CMV DNA load in clinical specimens using the LightCycler™. The assay is rapid ( B 40 min), easy to carry out, robust, reliable and is capable of detecting from 10 to over 2 ×10 5 CMV DNA copies with a wide linear range. Amplification and detection occur simultaneously, avoiding the need for post-PCR analysis and thereby minimising the risk of carryover contamination. The assay proved to be accurate, specific and reproducible when evaluated in three different laboratories. In addition, LightCycler™ results were comparable with those of TaqMan™, an independent real-time QPCR assay.

We evaluated the relationship between the intestinal microbiota composition and clinical outcome in a group of 15 high-risk patients admitted for acute infection and/or surgical/accidental trauma who were treated with systemic antibiotics according to standard intensive care unit (ICU) protocols. There was a high mortality rate amongst these patients, each of whom had a considerable organ failure score at admission, respiratory assistance during the most of their ICU stay and a long length of stay. All of these individuals received sedation and enteral nutrition, and the majority also received insulin, vasoactive drugs and some stress-ulcer prophylaxis agents. The intestinal microbiota composition was assessed using denaturing gradient gel electrophoresis (DGGE), a molecular biology tool used to characterize bacterial ecosystems. As all of the patient subjects were in good health prior to their acute illness and admission to the ICU, the first faecal samples obtained from this group showed a DGGE banding pattern that was similar to that of healthy subjects. After 1 week of critical illness, coupled with intensive care treatment, including antibiotics, a very definite alteration in the overall microbiota composition was evident, as revealed by a reduction in the number of DGGE bands. Further pronounced changes to the DGGE banding profiles could be observed in patients remaining in the ICU for 2 weeks. Moreover, a dominant band, identified by sequencing as highly related to Enterococcus, was detected in the DGGE profile of some of our patient subjects. We also performed real-time PCR and obtained results that were in agreement with our qualitative evaluations using DGGE. The degree of organ failure and ICU mortality was significantly higher in patients for whom a high reduction in microbiota biodiversity was coupled with a massive presence of enterococci. A statistically significant link between these two ecological traits and the use of clindamycin was also found.

Pine nuts are a part of traditional cooking in many parts of the world and have seen a significant increase in availability/use in the United States over the past 10 years. The U.S. Food and Drug Administration (US FDA) field offices received 411 complaints from U.S. consumers over the past three years regarding taste disturbances following the consumption of pine nuts. Using analysis of fatty acids by gas chromatography with flame ionization detection, previous reports have implicated nuts from Pinus armandii (Armand Pine) as the causative species for similar taste disturbances. This method was found to provide insufficient species resolution to link FDA consumer complaint samples to a single species of pine, particularly when samples contained species mixtures of pine nuts. Here we describe a DNA based method for differentiating pine nut samples using the ycf1 chloroplast gene. Although the exact cause of pine nut associated dysgeusia is still not known, we found that 15 of 15 samples from consumer complaints contained at least some Pinus armandii, confirming the apparent association of this species with taste disturbances.

Genetic manipulation of the oil-yielding crop plants for better oil quality through biotechnological methods is an important aspect of crop improvement. Due to the inherent absence of the Δ6-desaturase (d6D) function, Brassica juncea, an oil-yielding crop plant, is unable to synthesize γ-linolenic acid (GLA), a nutritionally important fatty acid although the crop plant synthesizes the precursor fatty acids required for GLA production. Cyanobacterial d6D introduces carbon–carbon double bond onto linoleic acid (C18:2) and α-linolenic acid (C18:3) by desaturation processes for production of GLA and octadecatetraenoic acid (OTA) respectively. In the present investigation, d6D coding sequence from Synechocystis sp. PCC6803 was cloned by polymerase chain reaction and introduced into B. juncea through Agrobacterium-mediated transformation technique. Both cytosolic as well as seed-specific expression of d6D were attempted. The transformed plants show production of GLA and OTA in contrast to their absence in the untransformed control plants adducing evidence for introgression and functional expression of the cyanobacterial d6D gene in B. juncea.

We compared PCR to conventional culture for the detection of vancomycin-resistant enterococci (VRE) in 30,835 rectal samples over a 3-year period. The positive and negative predictive values of vanB PCR were 1.42% and 99.9%, respectively. A positive vanB result by PCR is poorly predictive and necessitates culture for differentiation of VRE-positive and -negative individuals.

An efficient cell-free protein synthesis system has been developed using a novel energy-regenerating source. Using the new energy source, 3-phosphoglycerate (3-PGA), protein synthesis continues beyond 2 h. In contrast, the reaction rate slowed down considerably within 30-45 min using a conventional energy source, phosphoenol pyruvate (PEP) under identical reaction conditions. This improvement results in the production of twice the amount of protein obtained with PEP as an energy source. We have also shown that Gam protein of phage lambda, an inhibitor of RecBCD (ExoV), protects linear PCR DNA templates from degradation in vitro. Furthermore, addition of purified Gam protein in extracts of Escherichia coli BL21 improves protein synthesis from PCR templates to a level comparable to plasmid DNA template. Therefore, combination of these improvements should be amenable to rapid expression of proteins in a high-throughput manner for proteomics and structural genomics applications.

O 6 -methylguanine DNA methyltransferase (MGMT) is a DNA repair protein that restores mutagenic O 6 -methylguanine to guanine. MGMT methylation is frequently observed in sporadic colorectal cancer and was recently correlated with the C4T allele at SNP rs16906252, within the transcriptional enhancer element of the promoter. MGMT methylation has also been associated with KRAS mutations, particularly G4A transitions. We studied 1123 colorectal carcinoma to define the molecular and clinicopathological profiles associated with MGMT methylation. Furthermore, we assessed factors contributing to MGMT methylation in the development of colorectal cancer by studying the allelic pattern of MGMT methylation using SNP rs16906252, and the methylation status of neighbouring genes within 10q26 in selected tumours and matched normal colonic mucosa. MGMT methylation was detected by combined bisulphite restriction analysis in 28% of tumours and was associated with a number of characteristics, including CDKN2A methylation, absent lymphovascular space invasion and KRAS mutations (but not specifically with KRAS G4A transitions). In a multivariate analysis adjusted for age and sex, MGMT methylation was associated with the T allele of SNP rs16906252 (Po0.0001, OR 5.5, 95% CI 3.8-7.9). Low-level methylation was detected by quantitative methylation-specific PCR in the normal colonic mucosa of cases, particularly those with a correspondingly methylated tumour, as well as controls without neoplasia, and this was also associated with the C4T SNP. We show that the T allele at SNP rs16906252 is a key determinant in the onset of MGMT methylation in colorectal cancer, whereas the association of methylation at MGMT and CDKN2A suggests that these loci may be targets of a common mechanism of epigenetic dysregulation.

D De ee er r M Ma an na ag ge em me en nt t a an nd d R Re es se ea ar rc ch h Wildlife Society Bulletin 2005, 33(1):1-xxx Peer refereed Abstract Red deer (Cervus elaphus) and roe deer (Capreolus capreolus) are 2 of the most numerous wild ungulate species in Europe. They are sympatric over a large part of their total geographic range. There are no reliable techniques for differentiating between them when studies are based on hair or fecal sampling in the field where the 2 species' ranges overlap.

A procedure based on the polymerase chain reaction (PCR) has been developed to classify cucumber mosaic cucumovirus (CMV) isolates accurately into two subgroups. Two CMV-specific primers that flank the CMV capsid protein gene were used to amplify a DNA fragment of approximately 870 bp. Restriction enzyme analysis of this fragment produces distinct restriction patterns that assign the CMV isolate into one of two subgroups. These two restriction groups correlate with the previously established CMV subgroupings; this PCR-based method may provide a simple alternative to the serological assays used for typing CMV isolates.

To start determining the nature of meiotic incompetence in goat oocytes, we have examined the expression of one of the potential pre-MPF subunits: the cyclin B 1 . We have been isolating a small DNA probe encoding the goat cyclin B 1 box to analyze the expression of the cyclin B 1 gene in competent and incompetent goat oocytes. This probe was easily obtained by polymerase-chain-reaction (PCR) on reversetranscribed mRNA from granulosa cells, using cyclin B specific primers derived from a bovine cDNA. The transcript corresponding to cyclin B 1 in goat granulosa cells is 1.8 kb. In situ hybridization analysis indicated that competent and incompetent oocytes contained cyclin B 1 mRNA, but also that active cyclin B 1 mRNA synthesis occured at the end of the growth phase, e.g., when oocytes progressed in the acquisition of meoitic competence. Western blot analysis, performed with a monoclonal anticyclin B 1 antibody, revealed in competent and incompetent oocytes a polypeptide of 65kDa corresponding to the goat cyclin B 1 protein. This pattern of cyclin B 1 expression further suggested that meiotic incompetence in goat oocytes could not be primarily correlated with a lack of cyclin B 1 protein as potential pre-MPF subunit, but to a limiting amount of this protein. Mol. Reprod. Dev. 47:222-228, 1997. r 1997 Wiley-Liss, Inc.

A PCR-based method was applied to Campylobacter detection in poultry samples at the retail level. In total, 73 retail poultry samples purchased from supermarkets in the Basque Country area in the north of Spain were examined using both culture and molecular (alternative) methods. In our routine method, the worldwide ISO 10272:1995 standard of Preston broth incubated at 42 • C for conventional Campylobacter detection was adopted. The molecular method was comprised of a DNA extraction kit consisting of a single polypropylene spin column and PCR amplification of the Campylobacter 16S rRNA gene. A total of 54 raw samples were positive by either PCR or culture; among these, 50 were found to be positive by conventional plating and 54 by PCR. Concordant results, i.e., positive and negative in both methods, were found in 64 samples (94.1%). All positive samples by culture were also positive by PCR, resulting in 100% of positive concordance. Two samples (2.9%) positive after retesting by PCR were considered to be false-negatives. The detection limit of the PCR method was 5 CFUs that corresponded to 0.2 CFUs per 5 µl in the PCR mixture. The percentages of samples that required enrichment to prove Campylobacter presence were moderate, 18% by culture and 13% by PCR. Total analysis time was reduced to a few hours (within the working day) or 24 h when enrichment was required. Therefore, this PCR method proved to be useful as a routine diagnostic test for Campylobacter detection and confirmation of C. jejuni and C. coli in naturally contaminated poultry samples.