Gene Amplification Techniques Research Articles

Gene libraries open up

By combining gene cloning and amplification techniques, a new one-pot, parallel synthesis method for the generation of long, repetitive genes is realized. The method promises to open up the discovery of protein polymer biomaterials.

Tuberculous Otitis Media with Facial Paralysis: A Clinical and Microbiological Diagnosis—A Case Report

The tuberculosis of the ear is rare, and in most cases the clinical picture resembles that of a chronic otitis media. The diagnosis is often delayed, and this can lead to irreversible complications such as hearing loss and/or facial paralysis. In view of its rare occurrence, we report a case of primary tuberculous otitis media in a 87-year-old female patient. The diagnosis was made on the basis of both histological and microbiological findings. In particular, gene amplification techniques such as real-time polymerase chain reaction are useful method for rapid diagnosis and detecting tuberculous bacilli usually present at very low number. Early diagnosis is essential for the prompt institution of antituberculous therapy.

Leptotrichia amnionii: certain pathogen in pyosalpinx

Leptotrichia amnionii has had a recent taxonomic definition (2002) and definitely belongs to fastidious Gram negative anaerobes group thanks to difficulties to culture and preserve. Few PubMed reports of microbiological detections are documented to date and this is the first isolation described in Italy. Frequently only DNA analysis in direct samples succeeds in finding it out. Our study describes a case of Leptotrichia amnionii successfully cultivated from aspirated pus, but only 16S rRNA gene amplification and subsequent sequencing technique could afford to identify.The work remarks the added value of DNA techniques in routine analysis of anaerobes.

Analysis of the Composition and Characteristics of Culturable Endophytic Bacteria Within Subnival Plants of the Tianshan Mountains, Northwestern China

This study first described the composition and characteristics of culturable endophytic bacteria isolated from wild alpine-subnival plant species growing under extreme environmental conditions (i.e., on the border of a glacier with frequently fluctuating and freezing temperatures, strong wind, and high ultraviolet radiation). Using a cultivation-dependent approach and 16S rRNA gene amplification techniques, 93 bacterial isolates showing different phenotypic properties were obtained from 20 different subnival plant species, of which gram-positive bacteria (61.5%), psychrotolerant bacteria (67.3%), and pigmented isolates (70.9%) accounted for a large proportion. All these characteristics of endophytes were closely related to the survival environment of their host plants and were in good agreement with microbes occurring in other cold environments. Phylogenetic analysis indicated that the endophytic isolates consisted of five phylogenetic groups comprising α-proteobacteria, γ-proteobacteria, the high G+C content gram-positive bacteria, the low G+C content gram-positive bacteria, and Flavobacterium-Bacteroides-Cytophaga. The largest generic diversity was found in the HGC group, while Clavibacter, Agreia, Rhodococcus, Sphingomonas, and Pseudomonas were the most prevalent genera. Of all isolates, 46.4% showed a high sequence similarity (98-100%) to strains discovered from other cold environments such as glaciers, tundra, and polar seas. Furthermore, 36.4% of the isolates produced Indole-3-acetic acid and 76.3% were able to solubilize mineral phosphate, which revealed that endophytic bacteria with multiple physiological functions were abundant and widespread in subnival plants. These results are essential for understanding the ecological roles of endophytes and as a foundation for further studying the interactions with plants and environment.

Real-time loop-mediated isothermal amplification assay for rapid and sensitive detection of anthrax spores in spiked soil and talcum powder.

Loop-mediated isothermal amplification (LAMP) assay is a powerful and innovative gene amplification technique that specifically amplifies the target gene under isothermal conditions with a high degree of sensitivity, rapidity and specificity. The major advantage of the LAMP assay is monitoring of amplified products without the requirement of any sophisticated equipment. In the present study a real time LAMP assay was employed for rapid and real time detection of Bacillus anthracis spores spiked in 0.1g of soil and talcum powder ranging from 2 to 10(7) spores. DNA was isolated from spiked soil and talcum powder using PBS containing 1% Triton X-100, and heat treatment. Isolated DNA was used as template for LAMP and PCR. LAMP amplification was obtained in 60min under isothermal condition at 63°C by employing a set of six primers targeting the pag gene of B. anthracis. The detection limit of LAMP assay in soil and talcum powder was found to be as low as 5 spores, compared to 10(3) spores and 10(4) spores by PCR in talcum powder and soil, respectively. The findings suggest that LAMP is a more rapid and sensitive assay than PCR for detecting anthrax spores, additionally the methodology to prepare DNA from spiked samples is simple, rapid and cost effective.

Pancreatic Tuberculosis: Role of Multidetector Computed Tomography

Pancreatic tuberculosis (TB) is an extremely rare clinical entity even in countries with high endemism [1]. Men and women are affected equally, with a mean age of approximately 40 years [2]. It usually occurs as a complication of miliary TB and immunodeficiency, with isolated involvement of the pancreas being extremely rare. It is frequently misdiagnosed as pancreatic neoplasm. Lately, the increased incidence of this entity is related to the frequency of abdominal TB in immunocompromised patients and the improvement in imaging methods of the pancreas. Because the clinical and radiographic presentation mimics pancreatic cancer, its diagnosis is a clinical challenge, and most cases are diagnosed after surgical exploration. The tuberculin skin test is useful in the diagnosis of pancreatic TB, but it has a false-negative rate of 10%e25% [3]. The definitive diagnosis rests on histologic and bacteriologic evidence of TB. Pancreatic TB should be kept in mind among the differential diagnosis of pancreatic solitary masses, especially in young people in developing countries [4]. Detecting Mycobacterium tuberculosis DNA by a polymerase chain reaction in the tissue obtained from the lesion allows sameday results, with up to 77% sensitivities, although sample contamination and poor assay design can result in false negatives. Advancement in gene amplification techniques can now identify genes associated with antibiotic resistance but cannot give information regarding drug sensitivities, and,

Effects of palindrome structure on Dhfr amplification in Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells are widely used in producing therapeutic proteins. Gene amplification techniques are frequently used in improving protein production, and the dihydrofolate reductase (DHFR) gene amplification system is most widely used for the CHO cell line. We previously constructed a CHO genomic bacterial artificial chromosome (BAC) library from a mouse Dhfr-amplified CHO DR1000L-4N cell line and found one BAC clone (Cg0031N14) containing a CHO genomic DNA sequence adjacent to Dhfr. The BAC clone contained a large palindrome structure with a small inverted repeat in the junction region. To investigate the effect of the palindrome structure derived from the BAC clone Cg0031N14 on Dhfr amplification in CHO cells, we constructed plasmids that contain part or the whole junction region of the palindrome structure. The transfected CHO DG44 cells containing part or the whole junction region of the palindrome structure could adapt quickly to high methotrexate (MTX) concentrations. Moreover, the cells containing the whole junction region of the palindrome structure showed a high ratio of GFP-positive cells during gene amplification. On the basis of these results, we estimated that the junction region plays an important role in gene amplification in CHO cells.

Identification and analysis of specific chromosomal region adjacent to exogenous Dhfr-amplified region in Chinese hamster ovary cell genome

Chinese hamster ovary (CHO) cells are widely used for the stable production of recombinant proteins. Gene amplification techniques are frequently used to improve of protein production, and the dihydrofolate reductase (DHFR) gene amplification system is most widely used in the CHO cell line. We previously constructed a CHO genomic bacterial artificial chromosome (BAC) library from a mouse Dhfr-amplified CHO DR1000L-4N cell line and one BAC clone (Cg0031N14) containing the CHO genomic DNA sequence adjacent to Dhfr was selected. To identify the specific chromosomal region adjacent to the exogenous Dhfr-amplified region in the CHO cell genome, we performed further screening of BAC clones to obtain other Dhfr-amplified regions in the CHO genome. From the screening by high-density replica filter hybridization using a digoxigenin-labeled pSV2-dhfr/hGM-CSF probe, we obtained 8 new BAC clones containing a Dhfr-amplified region. To define the structures of the 8 BAC clones, Southern blot analysis, BAC end sequencing and fluorescence in situ hybridization (FISH) were performed. These results revealed that all the selected BAC clones contained a large palindrome structure with a small inverted repeat in the junction region. This suggests that the obtained amplicon structure in the Dhfr-amplified region in the CHO genome plays an important role in exogenous gene amplification.

Characterization of Imjin Virus, a Newly Isolated Hantavirus from the Ussuri White-Toothed Shrew ( Crocidura lasiura )

Until recently, the single known exception to the rodent-hantavirus association was Thottapalayam virus (TPMV), a long-unclassified virus isolated from the Asian house shrew (Suncus murinus). Robust gene amplification techniques have now uncovered several genetically distinct hantaviruses from shrews in widely separated geographic regions. Here, we report the characterization of a newly identified hantavirus, designated Imjin virus (MJNV), isolated from the lung tissues of Ussuri white-toothed shrews of the species Crocidura lasiura (order Soricomorpha, family Soricidae, subfamily Crocidurinae) captured near the demilitarized zone in the Republic of Korea during 2004 and 2005. Seasonal trapping revealed the highest prevalence of MJNV infection during the autumn, with evidence of infected shrews' clustering in distinct foci. Also, marked male predominance among anti-MJNV immunoglobulin G antibody-positive Ussuri shrews was found, whereas the male-to-female ratio among seronegative Ussuri shrews was near 1. Plaque reduction neutralization tests showed no cross neutralization for MJNV and rodent-borne hantaviruses but one-way cross neutralization for MJNV and TPMV. The nucleotide and deduced amino acid sequences for the different MJNV genomic segments revealed nearly the same calculated distances from hantaviruses harbored by rodents in the subfamilies Murinae, Arvicolinae, Neotominae, and Sigmodontinae. Phylogenetic analyses of full-length S, M, and L segment sequences demonstrated that MJNV shared a common ancestry with TPMV and remained in a distinct out-group, suggesting early evolutionary divergence. Studies are in progress to determine if MJNV is pathogenic for humans.

Loop mediated isothermal amplification (LAMP): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases

Loop mediated isothermal amplification (LAMP) is a powerful innovative gene amplification technique emerging as a simple rapid diagnostic tool for early detection and identification of microbial diseases. The whole procedure is very simple and rapid wherein the amplification can be completed in less than 1 h under isothermal conditions employing a set of six specially designed primers spanning eight distinct sequences of a target gene, by incubating all the reagents in a single tube. Gene amplification products can be detected by agarose gel electrophoresis as well as by real‐time monitoring in an inexpensive turbidimeter. Gene copy number can also be quantified with the help of a standard curve generated from different concentrations of gene copy number plotted against time of positivity with the help of a real‐time turbidimeter. Alternatively, gene amplification can be visualised by the naked eye either as turbidity or in the form of a colour change when SYBR Green I, a fluorescent dsDNA intercalating dye, is employed. LAMP does not require a thermal cycler and can be performed simply with a heating block and/or water bath. Considering the advantages of rapid amplification, simple operation and easy detection, LAMP has potential applications for clinical diagnosis as well as surveillance of infectious diseases in developing countries without requiring sophisticated equipment or skilled personnel. Copyright © 2008 John Wiley & Sons, Ltd.

Les mycoplasmoses porcines

Swine mycoplasmoses Mycoplasmal diseases have a considerable impact on pig production. Pneumonia caused by Mycoplasma hyopneumoniae, whether alone or in combination with other pathogens, is a major concern worldwide. Culture and isolation of M. hyopneumoniae are difficult and often unsuccessful. However, gene amplification techniques (PCR) and serological analyses produce very interesting results. Antigenic diversity and differences in virulence between isolates have been demonstrated. Vaccines are effective to reduce symptoms and lung lesions, but they do not prevent the multiplication of M. hyopneumoniae in pigs. M. hyorhinis and M. hyosynoviae cause polyserositis and arthritis, generally in growing animals. The other species of mycoplasma appear to be non-pathogenic in swine.

Evaluation of Conventional Microbiological Procedures and Molecular Genetic Techniques for Diagnosis of Infections in Patients with Implanted Orthopedic Devices

Evaluation of Conventional Microbiological Procedures and Molecular Genetic Techniques for Diagnosis of Infections in Patients with Implanted Orthopedic Devices

Quantitative determination of free-DNA uptake in river bacteria at the single-cell level by in situ rolling-circle amplification.

Detection of plasmid DNA uptake in river bacteria at the single-cell level was carried out by rolling-circle amplification (RCA). Uptake of a plasmid containing the green fluorescent protein gene (gfp) by indigenous bacteria from two rivers in Osaka, Japan, was monitored for 506 h using this in situ gene amplification technique with optimized cell permeabilization conditions. Plasmid uptake determined by in situ RCA was compared to direct counts of cells expressing gfp under fluorescence microscopy to examine differences in detection sensitivities between the two methods. Detection of DNA uptake as monitored by in situ RCA was 20 times higher at maximum than that by direct counting of gfp-expressing cells. In situ RCA could detect bacteria taking up the plasmid in several samples in which no gfp-expressing cells were apparent, indicating that in situ gene amplification techniques can be used to determine accurate rates of extracellular DNA uptake by indigenous bacteria in aquatic environments.

Analysis of gene probes and gene amplification techniques for diagnosis and monitoring of treatment in childhood leprosy

Nucleic acid sequences of Mycobacterium leprae were detected using gene probes hybridizing with targeting ribosomal RNA (16S rRNA), ribosomal DNA (16S rDNA) and gene amplification techniques (PCR) in skin lesion of paediatric leprosy patients and the effect of treatment on the by these methods. Eighty paediatric leprosy patients were included in the study. Most cases (79%) were between 9 and 16 years of age. Cases were divided into three groups according to treatment status, viz. untreated (30), undergoing treatment (30), and at the end of treatment (20). Clinical and slit smear examination for acid fast bacilli (AFB) was performed and nucleic acids were extracted and fractionated from skin biopsies. M. leprae specific 16S rRNA and 16S rDNA was detected by hybridization with gene probes whereas the 36 kDa gene sequence was detected by a gene amplification assay (PCR). The cases were classified as paucibacillary (PB) and multibacillary (MB) by the standard criteria of WHO (1988). Positivity of 16S rRNA in PB cases decreased from 60% in untreated to 10.5% after 4-8 months of treatment whereas for 16S r DNA, it decreased from 50% to 21%, for PCR from 70% to 36.8% for the same specimen, and all became negative at 1 year. Similar trends were seen in MB cases where positivity in smear positive untreated cases decreased from 100% to 56.2% with 16S rRNA and 42.8% with 16S rDNA and PCR, respectively, after 9-12 months of treatment and all became negative at 2 years, except one case which remained positive with PCR. Similar results were observed in smear negative MB cases, 100% positivity detected by 16S r RNA and PCR, 75% detected by 16S rDNA decreased to zero after 9-12 months of therapy. This study suggests the potential usefulness of gene probes targeting 16S rRNA and 16S rDNA and PCR as supportive molecular tools for diagnosis of smear negative evolving MB disease and also monitoring the response to treatment, these observations however, needs to be validated in prospective follow up studies.

Norovirus genotypes causing gastroenteritis outbreaks in Finland 1998–2002

Background: Outbreak investigation methods for enteric viruses were improved in 1990s when gene amplification techniques were established in viral laboratories. Objectives: The objective of the study was to determine the causative agents for Finnish viral gastroenteritis outbreaks. Our aim was also to further characterise the norovirus strains, reveal the temporal occurrence of norovirus (NV) genotypes and to study some epidemiological aspects concerning the outbreaks. Study design: A total of 416 Finnish viral gastroenteritis outbreaks that occurred during 5 years (1998–2002), excluding those among hospitalised children, were investigated for enteric viruses. Stool samples were screened by electron microscopy as well as analyzed by specific noro- and astrovirus RT-PCR tests. Amplicon sequence analysis was used to find out norovirus genotypes. Results: Noroviruses caused 252 (60.6%) of the outbreaks; other viruses, astro- or rotavirus, caused four epidemics. Norovirus epidemics occurred in all kinds of settings, most often in hospitals (30.6%) and in restaurants and canteens (14.3%). Both NV genogroups were found every year, but NV GGII outbreaks always outnumbered those of GGI. All but one outbreak at hospitals and nursing homes were of genotype GII. Polymerase sequence analysis revealed a variety of NV genotypes; six GI and at least eight GII genotypes. The GI.3 Birmingham-like and GII.4 Bristol-like genotype appeared every year, whereas the other types were circulating for shorter periods or sporadically. During the study period the genotypes GII.4 (Bristol), GII.1 (Hawaii), an emerging genotype GIIb, and a new variant of GII.4 predominated in that order. Indication for rapid genetic changes in the genotype GII.4 was also noticed. Conclusions: Noroviruses were the most prevalent causative agents in the outbreaks. Many NV genotypes were circulating, and a shift in the predominant genotypes was evident between epidemic seasons.

Blood evidence: how DNA is revolutionizing the way we solve crimes

Blood evidence: how DNA is revolutionizing the way we solve crimes

Identification of novel eubacteria from spent mushroom compost (SMC) waste by DNA sequence typing: ecological considerations of disposal on agricultural land

A small study was undertaken to examine the microbiological characteristics of spent mushroom compost (SMC), which is the major waste by-product of the mushroom industry and which is regularly disposed off by application to agricultural land. The primary aim of this study was to examine SMC for the presence of faecal bacterial pathogens, including Campylobacter spp., Salmonella spp. and Listeria monocytogenes. Secondly it was desirable to quantify bacterial and fungal populations within SMC, and also qualitatively identify the diversity of bacterial populations within SMC, through employment of rDNA PCR and direct sequencing techniques on the culturable microflora. Conventional microbiological analyses of SMC material ( n=30) from six commercial operations in both Northern Ireland and the Republic of Ireland, failed to detect Salmonella spp , Listeria spp. or Campylobacter spp. in any of the SMC material examined. Total aerobic plate counts gave a mean count of log 10 7.01 colony forming units (cfu) per gram SMC material (range: log 10 6.53–7.52 cfu/g). Fungal counts gave a mean count of log 10 4.57 cfu per gram SMC material (range: log 10 3.93–4.98 cfu/g). From a total of greater than 50 colony picks, a total of 12 bacterial morphotypes were identified and were further examined by employment of partial 16S rRNA gene amplification and sequencing techniques, yielding several genera and species, including Bacillus licheniformis, Bacillus subtilis, Klebsiella/Enterobacter sp. Microbacterium sp. Paenibacillus lentimorbus, Pseudomonas mevalonii, Sphingobacterium multivorum and Stenotrophomonas sp. This is the first preliminary report on the microbial diversity of SMC waste and demonstrates the presence of several species that have not been previously described in SMC, in addition to two potentially novel species within the genera Microbacterium and Stenotrophomonas. It is thereby important to examine the ecological microbe–microbe and plant–microbe interactions that are occurring between the native bacterial soil flora and those added annually (theoretically estimated at approximately 10 18 cells) through the application of SMC. Such studies would be beneficial in helping to ascertain the ecological consequences involved in the disposal of SMC waste on agricultural land.

Analysis of the association between apolipoprotein E polymorphism and cardiovascular risk factors in an elderly population with longevity.

To establish the allelic and genotypic frequencies related to apolipoprotein E (ApoE) polymorphism and association of the genotypes with risk factors and cardiovascular morbidity in an elderly population with longevity. We analyzed 70 elderly patients aged 80 years or more who were part of the Projeto Veranópolis. We used the gene amplification technique through the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and cleavage with the restriction enzyme Hha I to identify the ApoE genotypes. The most frequent genotypes were compared considering biological variables and cardiovascular risks and morbidity. The frequencies of the E2, E3, and E4 alleles were 0.05, 0.84, and 0.11, respectively, and of the genotypes were as follows: E3E3 (0.70), E3E4 (0.22), E2E3 (0.06), and E2E2 (0.02). Individuals with the E3E4 had a mean age greater than those with the E3E3. No association was observed between the genotypes and the variables analyzed, except for obesity, which was associated with the E3E3 genotype. Individuals with the E3E4 genotype had high levels of LDL-cholesterol and fibrinogen as compared with those with the E3E3 genotype. The results suggest that the E4E4 genotype may be associated with early mortality. A balance between the protective or neutral factors and the cardiovascular risk factors may occur among the individuals with different genotypes, attenuating the negative effects of the E4 allele.

Cloning and sequencing of an alkaline protease gene from Bacillus lentus and amplification of the gene on the B. lentus chromosome by an improved technique.

A gene encoding an alkaline protease was cloned from an alkalophilic bacillus, and its nucleotide sequence was determined. The cloned gene was used to increase the copy number of the protease gene on the chromosome by an improved gene amplification technique.

Introduction of the gene amplification technique to decrease the risk of hepatitis C virus transmission by plasma products

The viral safety of plasma-derived products with respect to hepatitis C virus (HCV) is assured by selection of donors, screening of individual donations for antibodies to HCV and the incorporation of effective viral inactivation-removal steps into manufacturing processes. As antibody screening of single donations is not sufficient to completely eliminate HCV RNA positive plasmas from plasma pools, testing for HCV RNA by gene amplification techniques may be necessary to identify positive donations. Using modern molecular biology techniques, we developed a specific, sensitive and reproducible method for routine PCR screening for HCV RNA in plasma pools.