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PCR Amplification Products (pcr + amplification_products)
Selected AbstractsIdentification of cyanobacteria and their toxigenicity in environmental samples by rapid molecular analysisENVIRONMENTAL TOXICOLOGY, Issue 6 2001Judith A. Baker Abstract We report molecular analyses which identify cyanobacterial strains present in environmental samples. These analyses do not require the isolation and culture of strains. Identification of cyanobacteria used the polymerase chain reaction (PCR), based on the phycocyanin operon. Differentiation was either by restriction endonuclease digestion (restriction fragment length polymorphisms) or sequencing of the PCR products. Identification was based on sequence homology of the intergenic spacer region (IGS) between the ,- and ,-phycocyanin subunits (PC-IGS) with database records. We have found that the length and sequence of the PC-IGS is capable of predicting the genus accurately, but not the species. Toxigenicity was determined with oligonucleotide probes for key steps in the microcystin toxin synthesis pathway. We have shown that it is possible to easily and routinely obtain PCR amplification products and differentiate the strains in bloom samples. The methods can detect even minor components in bloom samples, which may not be apparent on microscopic examination. Genetic probes for microcystin toxigenicity are effective on environmental samples, eliminating the need for isolation and culture of the organisms. The use of a suite of tests described here will allow water managers to determine the presence and the type of cyanobacteria and their microcystin toxigenicity. © 2001 John Wiley & Sons, Inc. Environ Toxicol 16: 472,482, 2001 [source] Isolation of a cyprinid herpesvirus 2 from goldfish, Carassius auratus (L.), in the UKJOURNAL OF FISH DISEASES, Issue 11 2007K R Jeffery Abstract Haematopoietic necrosis virus [cyprinid herpesvirus 2 (CyHV-2)] was isolated during disease outbreaks in goldfish, Carassius auratus, at an ornamental fish retail site in southern England in 2004. Signs of disease included lethargy and inappetence and were first seen after water temperatures increased from 14,15 to 19,21 °C. External gross pathology included pale patches on the gills and skin and internally the spleen was enlarged, often with distinctive white nodules. The most prominent histopathological changes observed were necrotic lesions in the spleen and kidney and focal patches of necrosis in the gill lamellae. Necrotic cells often contained nuclei with marginated chromatin and pale intranuclear inclusions. Ultrastructural examination of the spleen tissue revealed typical herpesvirus-like particles measuring 100 nm in diameter. The virus was isolated from extracts of gill tissue in KF-1 cells at 20 °C and oligonucleotide primer sets were designed based on conserved gene sequences and used to amplify viral DNA by polymerase chain reaction (PCR). The PCR assays were then used to detect the virus in DNA extracted from tissues sampled during earlier disease investigations at the retail site owner's holding facility in 2002 and 2003 and stored at ,70 °C since then. Polymerase gene-specific PCR amplification products obtained from tissue samples and from the virus isolated in cell culture shared 100% nucleotide sequence identity with the published sequence for CyHV-2. [source] Diversity of streptomycetes in water-damaged building materials based on 16S rDNA sequencesLETTERS IN APPLIED MICROBIOLOGY, Issue 6 2002H. Rintala Aims:,The diversity of streptomycetes in two different types of water-damaged building materials was investigated. Methods and Results:,Direct PCR amplification of 16S rDNA from DNA isolated from building materials, cloning of the fragments and sequence analysis were used. In the phylogenetic analysis of the variable , region of the PCR amplification products, the sequences affiliated with five groups. Conclusions:,Several different sequences were found in both materials, suggesting the presence of several species. Also, previously unknown sequences were detected, although all the sequences clustered together with sequences of known species. Significance and Impact of the Study:,Streptomycetes are known as indicators for moisture and mould damage in buildings and potential health risk, but their diversity in indoor environments is still unknown. [source] Evaluation of infant methylenetetrahydrofolate reductase genotype, maternal vitamin use, and risk of high versus low level spina bifida defects,BIRTH DEFECTS RESEARCH, Issue 3 2003Kelly A. Volcik BACKGROUND Several studies have suggested that homozygosity for the C677T 5,10-methylenetetrahydrofolate reductase (MTHFR) variant is a potential risk factor for neural tube defects (NTDs), as individuals homozygous for the C677T allele have slightly elevated homocysteine concentrations under conditions of low folic acid intake. It has been hypothesized that maternal folic acid supplementation prevents NTDs by partially correcting reduced MTHFR activity associated with the variant form of the enzyme. METHODS Genomic DNA was extracted from newborn screening blood spots obtained from 145 infants with spina bifida (SB) and 260 nonmalformed control infants. The MTHFR C677T genotype was determined by restriction enzyme digestion of PCR amplification products with Hinf1. We investigated whether infant MTHFR genotype influenced the risk for the anatomic level of the SB lesion (high vs. low); we also explored whether maternal vitamin use influenced this risk. RESULTS Compared to controls, the frequency of SB infants with the homozygous 677 TT genotype was greatest in those infants with high level SB defects (26%; odds ratio [OR] = 2.9; 95% confidence interval [CI] = 0.9,10.1) than for those with low level SB defects (22%; OR = 1.8; 95% CI = 0.9,3.2). Furthermore, homozygous 677TT infants whose mothers did not use vitamins containing folic acid had a modestly increased risk of SB (OR = 1.8; 95% CI = 0.8,3.9), with this risk increasing more than three-fold (OR = 5.5; 95% CI = 0.8,28.1) for those infants with high level SB defects whose mothers did not use vitamins. CONCLUSIONS Based upon our observations, it is suggested that the association between the infant MTHFR homozygous variant genotype and spina bifida risk may be conditional upon both lesion level and maternal vitamin use. Birth Defects Research (Part A) 67:154,157, 2003. © 2003 Wiley-Liss, Inc. [source] Apolipoprotein E and apolipoprotein B genotypes and risk for spina bifidaBIRTH DEFECTS RESEARCH, Issue 5 2002Kelly A. Volcik Background Altered cholesterol metabolism and defects in cholesterol biosynthesis may influence abnormal central nervous system (CNS) development. During early stages of embryonic development, high levels of cholesterol are needed by rapidly proliferating cells that utilize cholesterol as a key cell membrane component. Alterations in cholesterol levels are influenced by variations in the apolipoprotein E (apoE) and apolipoprotein B (apoB) genes. The purpose of our study was to explore the possible association between infant genetic variations in the apoE and apoB genes and spina bifida (SB) risk. Methods Genomic DNA was extracted from newborn screening blood spots obtained from 26 infants with SB and 73 non-malformed control infants. ApoE and apoB genotypes were determined by restriction enzyme digestion of PCR amplification products. Results Genotype frequencies for the apoE and apoB polymorphisms were not statistically different between case and control infants. For each apoB polymorphism, however, the frequency of the wild-type allele was higher in SB infants as compared to controls. Additionally, the apoE genotype E2/E3 was observed more frequently in the controls than in SB infants [15% in controls compared to 4% in cases; OR = 0.2 (0,1.6)]. Conclusions Results from this study suggest that genetic variations in the apoE and apoB genes, known to regulate cholesterol metabolism, do not substantially contribute to the risk of SB in infants. Teratology 66:257,259, 2002. © 2002 Wiley-Liss, Inc. [source] Comparison of two different PCR amplification products (the 18-kDa protein gene vs.CLINICAL & EXPERIMENTAL DERMATOLOGY, Issue 4 2003RLEP repetitive sequence) in the diagnosis of Mycobacterium leprae Summary To determine the best molecular method for diagnosing leprosy, two sets of Mycobacterium leprae -specific primers were compared. Fresh biopsies and slit skin smear samples were obtained from 67 leprosy patients and examined by touchdown (TD) PCR using primers amplifying either a 129-bp fragment of the RLEP repetitive sequence or a 360-bp fragment of the 18-kDa protein gene of M. leprae. Seventeen of 30 (56.7%) biopsy specimens and four of 37 (10.8%) slit skin smear specimens were positive using the primer for the 18-kDa protein gene, whereas 24 of 30 (80%) biopsy and 27 of 37 (73%) slit skin smear samples showed detectable PCR products in the RLEP repetitive sequence. Twenty-one of 31 cases (67.7%) with a bacterial index of zero were PCR positive for the primer RLEP repetitive sequence. These results demonstrate that detection of M. leprae using PCR with primers to a RLEP sequence is more sensitive and specific than PCR with the 18-kDa protein gene primers and also slit smears with acid fast staining. PCR of RLEP repetitive sequences is therefore a useful means of detecting M. leprae DNA even when it is present at very low levels. [source] | |||||||||||||