Molecular Detection Systems Research Articles

Distinctive Spectra of Cytogenetic Alterations in Over Five Hundred East Asian Patients with Acute Leukemia

Abstract 4408 Background:Racial difference of spectra of chromosomal aberrations in various hematological malignancies was previously reported between Asian and western countries. Therefore, characterization of the cytogenetic profile is still a matter of interests for identifying cytogenetic risk factors in the current risk-adapted chemotherapy. The aim of this study was to investigate the spectrum of chromosomal aberrations in East Asian (Korean) acute leukemia patients, and to propose a panel of leukemic fusion genes suitable for the development of new molecular detection systems. Patients and Methods:We prospectively analyzed bone marrow samples from 502 patients with acute leukemia referred for screening leukemic fusion genes by simultaneous analysis of conventional cytogenetics, FISH and multiplex RT-PCR system in Chonnam National University Hwasun Hospital (Hwasun, Korea) for last 5 years. Results:A total of 334 patients were diagnosed with AML, 155 with ALL and 13 with mixed phenotype acute leukemia. Twenty types in 28 fusion genes were detected in 42.4% of the total patients by multiplex RT-PCR system and in 33.9% by conventional cytogenetics including FISH. In the group of AML, the common fusion genes were 57 PML/RARa, 38 AML1/MGT8, 12 CBFb/MYH11, 11 MLL1, 5 BCR/ABL1, SET/CAN, PLZF/RARA and TLS/ERG in 2 cases each, and one of each AML1/MDS1, TEL/ABL and TEL/MN1. In the group of ALL, the following fusion genes were detected: 33 BCR/ABL1, 22 TEL/AML1, 10 MLL1, 7 E2A/PBX, 2 SET/CAN and SIL/TAL, one TEL/ABL. In addition, cytogenetically cryptic translocations were detected in 14.8% patients with normal karyotype or numerical aberrations by conventional cytogenetics. Among 288 patients with negative results by multiplex RT-PCR system, one child with early pre-B-ALL harbored MLL1/AF4, and 8 patients had 3 types of clinically significant chromosomal aberrations such as t(3;3)(q21;q26.2), t(8;14)(p24.1;q32) and i(17)(q10), which were all confirmed by conventional cytogenetics. Conclusions:The current study demonstrated the spectrum and frequency of chromosomal aberrations in Korean patients with acute leukemia, which are different from Caucasian data. Also these results may offer important implications for the development of new multiplex molecular detection system. Disclosures:No relevant conflicts of interest to declare.

Detection of viral sequence fragments of HIV-1 subfamilies yet unknown

BackgroundMethods of determining whether or not any particular HIV-1 sequence stems - completely or in part - from some unknown HIV-1 subtype are important for the design of vaccines and molecular detection systems, as well as for epidemiological monitoring. Nevertheless, a single algorithm only, the Branching Index (BI), has been developed for this task so far. Moving along the genome of a query sequence in a sliding window, the BI computes a ratio quantifying how closely the query sequence clusters with a subtype clade. In its current version, however, the BI does not provide predicted boundaries of unknown fragments.ResultsWe have developed Unknown Subtype Finder (USF), an algorithm based on a probabilistic model, which automatically determines which parts of an input sequence originate from a subtype yet unknown. The underlying model is based on a simple profile hidden Markov model (pHMM) for each known subtype and an additional pHMM for an unknown subtype. The emission probabilities of the latter are estimated using the emission frequencies of the known subtypes by means of a (position-wise) probabilistic model for the emergence of new subtypes. We have applied USF to SIV and HIV-1 sequences formerly classified as having emerged from an unknown subtype. Moreover, we have evaluated its performance on artificial HIV-1 recombinants and non-recombinant HIV-1 sequences. The results have been compared with the corresponding results of the BI.ConclusionsOur results demonstrate that USF is suitable for detecting segments in HIV-1 sequences stemming from yet unknown subtypes. Comparing USF with the BI shows that our algorithm performs as good as the BI or better.

Spectra of Chromosomal Aberrations in 325 Leukemia Patients and Implications for the Development of New Molecular Detection Systems

This study investigated the spectrum of chromosomal abnormalities in 325 leukemia patients and developed optimal profiles of leukemic fusion genes for multiplex RT-PCR. We prospectively analyzed blood and bone marrow specimens of patients with acute leukemia. Twenty types of chromosomal abnormalities were detected in 42% from all patients by commercially available multiplex RT-PCR for detecting 28 fusion genes and in 35% by cytogenetic analysis including FISH analysis. The most common cytogenetic aberrations in acute myeloid leukemia patients was PML/PARA, followed by AML1/MGT8 and MLL1, and in acute lymphoid leukemia patients was BCR/ABL, followed by TEL/AML1 and MLL1 gene rearrangement. Among the negative results for multiplex RT-PCR, clinically significant t(3;3)(q21;q26.2), t(8;14)(q24;q32) and i(17)(q10) were detected by conventional cytogenetics. The spectrum and frequency of chromosomal abnormalities in our leukemia patients are differed from previous studies, and may offer optimal profiles of leukemic fusion genes for the development of new molecular detection systems.

Detection and differentiation of Vibrio spp. in seafood and fish samples with cultural and molecular methods

Vibrio spp. as natural inhabitants of sea- and brackwater of both tropical and temperate regions of the world are commonly found in different kinds of seafood. Even among the three main human pathogenic species Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus most of the isolates from seafood do not carry the different virulence factors responsible for foodborne infections. Therefore, the risk assessment of Vibrio spp. in seafood is currently based mainly on the knowledge of the genetic setting of foodborne strains. For the detection and differentiation of Vibrio spp. ( V. parahaemolyticus, V. cholerae and V. vulnificus) three probe-based multiplex real-time PCR systems were developed and validated. One real-time PCR system simultaneously detects V. parahaemolyticus, V. cholerae and V. vulnificus on genus level combined with an Internal Amplification Control. The detection limit for the system was between 1 cfu/mL and 10 cfu/mL in pure culture and in different artificially contaminated sample material, e. g. prawns or Alaska Pollock. The other two PCR systems were implemented for the detection of different virulence genes of V. parahaemolyticus and V. cholerae isolates. The molecular detection systems were applied for the investigation of 338 raw and cooked seafood and fish samples for the presence of the different Vibrio spp. The collected data indicate that the PCR systems can be useful for rapid detection and differentiation of Vibrio spp. in different food matrices as basis for a preventive consumer protection policy.

Analysis of volatile arsenic compounds formed by intestinal microorganisms: rapid identification of new metabolic products by use of simultaneous EI-MS and ICP-MS detection after gas chromatographic separation

For the investigation of biotransformation processes of heteroelements in the environment, both molecular and element-sensitive detection systems are used in hyphenation to chromatographic separation. In this work, we studied the potential of our recently developed gas chromatographic system with parallel electron impact mass spectrometry and inductively coupled plasma mass spectrometry (GC/EI-MS/ICP-MS) for non-target screening and subsequent identification of volatile arsenic compounds formed by fecal microorganisms. By combining the strengths of element-sensitive detection by ICP-MS and molecular identification by EI-MS, it was possible to identify five mixed arsenic/sulfur species (Me2AsSH, dimethyl-mercapto-arsine, Me2AsSMe, dimethyl-methylthio-arsine, Me2AsSSMe, dimethyl-methyldithio-arsine, (Me2As)2S, thio-bis(dimethylarsine) and MeAs(SMe)(SEt), methyl-methylthio-ethylthio-arsine) as well as one mixed arsenic/selenium compound (Me2AsSeMe, dimethyl-methylseleno-arsine) in the headspace of fecal incubations. Identification of all compounds was verified by synthesis experiments. Three of these species, namely Me2AsSeMe, (Me2As)2S and MeAs(SMe)(SEt), have not been described in environmental or human matrices before. Finally, the advantages of elemental and molecular detection after gas chromatographic separation are discussed.

A hospital-wide study of the impact of introducing a personal data assistant-augmented blood culture round

Blood culture is the cornerstone of an established aetiological diagnosis of septicaemia. The automated blood culture systems used for this purpose have changed little in the last decade, and the clinical value of results depends on a variety of factors, including pre- and post-analytical variables. Growing scepticism over the value of blood culture results and pressure for the introduction of molecular detection systems have prompted a critical path analysis of pre-, peri- and post-analytical stages in the generation of positive blood culture results. The impact of a positive blood culture was studied in a teaching hospital for 12 months before and 12 months after the introduction of a microbiologist's blood culture round. Active culture reporting via a blood culture ward round was supported by a personal data assistant database of contemporaneous laboratory and clinical data. Hospital occupancy and death register records were subsequently obtained through the State Government data linkage project. There was no evidence that faster laboratory generation of positive blood culture results, faster reporting of results or direct clinical interaction with the patient's primary medical team reduced the risk of death in hospital. However, there was a threefold increase in the rate of death in hospital following a 1 day delay in collection of blood cultures after hospital admission (P=0.0010). The overall duration of hospital stay for patients with a positive blood culture fell by 2.5 days compared with the previous 12 month period (P=0.0003). The interval between the initial positive culture result and patient discharge fell by 2 days (P=0.0010). This difference was attributed to shorter overall admissions and shorter intervals between positive cultures containing Gram-positive cocci and subsequent patient discharge (P=0.0018). An increased mortality rate from community-acquired bacteraemic infections was associated with delayed culture collection, but not with a prolonged laboratory processing interval. Thus, the speed of conventional blood culture analysis and the form of clinical reporting have little direct effect on the clinical outcome of bacteraemia, but may contribute to a reduction in the length of hospital admission. Introduction of molecular identification tests, such as multiplex PCR methods, at the Gram-stain stage of blood culture is unlikely to affect the rate of death in hospital, but may reduce the length of hospital admission.

The Use of Magnetic Nanoparticles in the Development of New Molecular Detection Systems

Magnetic nanoparticles have been widely used in biomolecular separation and discrimination which coincidentally also represents the basis for most current day molecular diagnostic procedures. The specificity, affinity, and binding capacity of magnetic nanoparticles depends on their size, form, dispersion, and surface chemistry. In this review, we will briefly analyze how these factors affect biomolecular separations and focus on the use of magnetic nanoparticles in monitoring the microbial biodiversity in the environment. We found that magnetic nanoparticles are especially effective for biomolecular separations in environmental samples collected and preserved with fixatives. This feature, together with the high sample throughput capability and the generic low cost, makes magnetic nanoparticles particularly suitable for environmental microbial monitoring. Furthermore, key features that permit the optimization of magnetic nanoparticles-based separations and that can be useful in the development of new analytical procedures are also discussed.

Getting the most out of fluorescent amplified fragment length polymorphism

Amplified fragment length polymorphism (AFLP™) is one of the most widely applied molecular marker detection systems used today. Among the reasons for its popularity are its reproducibility, capacity to generate large numbers of data points in a single assay, and “off-the-shelf” universal applicability. The original AFLP protocol was developed using radioactive detection. The transfer of this technique to fluorescent detection on automated DNA fragment analysers not only removed the undesirable requirement for radioactivity but also provided the possibility for increased effectiveness and detection throughput. Unfortunately, a number of problems are frequently encountered with fluorescent AFLPs, particularly failure to amplify high molecular-weight fragments and generation of nonuniform peak distributions. Here, we describe an improved generic protocol for fluorescent AFLPs achieved mainly thorough optimization of the multiplexed selective amplification reaction. This improved protocol gives increased production of valuable high molecular-weight markers and uniform peak intensities, facilitating unambiguous scoring. The protocol has been successfully applied, without further optimization, to species of Salix and Populus (Salicaceae), Melampsora (Melampsoraceae, rust fungi) and Heracleum (Apiaceae), as well as sugar beet ( Beta vulgaris L. subsp. vulgaris , Amaranthaceae), the endangered species Ranunculus kadzunensis Makino (Ranunculaceae), and to Aphidius ervi Haliday (Braconidae), a parasitoid wasp.

Development of molecular approaches for the identification of transmission sites of schistosomiasis

Primers targeting the gene encoding the small subunit rRNA were designed to amplify DNA from Schistosoma mansoni with high specificity. Three PCR systems were developed: conventional PCR, two-step nested PCR (NPCR) and single-tube nested PCR (STNPCR). The limits of detection of parasite DNA for the conventional PCR, NPCR and STNPCR were 10 pg, 0.1 fg and 1 fg, respectively. The assays were highly specific for S. mansoni and did not recognise DNA from closely related non-schistosome trematodes. Using pools of Biomphalaria molluscs, PCR, NPCR and STNPCR were positive in 6/16 (37.5%), 15/16 (93.8%) and 13/16 (81.3%) of the tested samples, respectively, whereas the observation of cercariae shedding after exposure to light was able to detect S. mansoni infection in 6/16 (37.5%) of the pools. Thus, the molecular detection systems had a higher level of sensitivity than standard screening of intermediate hosts by cercarial shedding when DNA was purified from pools of snails collected from endemic areas. These PCR protocols have potential to be used as tools for monitoring of schistosome transmission.

Controlling the Sensing Volume of Metal Nanosphere Molecular Sensors

Noble metal nanoparticles and nanoshells support surface plasmons at optical frequencies. These resonances, known as localized surface plasmons (LSPs), are sensitive to the dielectric properties of the environment and, in particular, to the refractive index of the material close to the surface of the particle. This sensitivity can be exploited in molecular detection systems that use nanoparticles functionalized with receptors to (a) bind target molecules and (b) optically transduce the resulting change in the dielectric environment. Optimization of an optical nanoparticle sensor involves tailoring the particle to the target so as to maximize the sensitivity of spectroscopic features to the dielectric variation associated with binding of target molecules to the particle surface.

Dispersive one-dimensional (Mo/Si) nanocrystals for single molecular detection systems using x rays

To directly observe the behavior of single molecules in real time and space, the x-ray methodology, diffracted x-ray tracking, has been proposed. This system monitors the spots of a diffracted x ray from individual nanocrystals, which are labeled with the individual single molecules. To operate single molecular detection systems with x rays, it is necessary to fabricate dispersive nanocrystals and observe the diffracted spots from the nanocrystals. We succeeded in fabrications of dispersive one-dimensional (Mo/Si) nanocrystals with diameters of approximately 25 nm using reactive ion etching. Furthermore, we monitored well-defined diffracted x-ray spots from Mo/Si nanocrystals (2.5 bilayers: Mo/Si/Mo/Si/Mo, d-space=5.0 nm) in real time (2.0 ms).

Numeric simulation of plant signaling networks.

Plants have evolved an intricate signaling apparatus that integrates relevant information and allows an optimal response to environmental conditions. For instance, the coordination of defense responses against pathogens involves sophisticated molecular detection and communication systems. Multiple protection strategies may be deployed differentially by the plant according to the nature of the invading organism. These responses are also influenced by the environment, metabolism, and developmental stage of the plant. Though the cellular signaling processes traditionally have been described as linear sequences of events, it is now evident that they may be represented more accurately as network-like structures. The emerging paradigm can be represented readily with the use of Boolean language. This digital (numeric) formalism allows an accurate qualitative description of the signal transduction processes, and a dynamic representation through computer simulation. Moreover, it provides the required power to process the increasing amount of information emerging from the fields of genomics and proteomics, and from the use of new technologies such as microarray analysis. In this review, we have used the Boolean language to represent and analyze part of the signaling network of disease resistance in Arabidopsis.

The rapid diagnosis of isoniazid and rifampicin resistance in Mycobacterium tuberculosis--a molecular story.

Isoniazid and rifampicin resistance are assayed phenotypically by the resistance ratio, absolute concentration or proportion methods. Assay methods are often difficult to standardise and the World Health Organization (WHO) Global Programme on Drug Resistance is attempting to produce standardised drug resistance data worldwide. Broth-based methods are faster than solid media systems, and a commercial radiometric system, the Bactec 460, is arguably the fastest method and permits testing to be completed within 7-14 days; however, this method is expensive and requires disposal of radioactive material. Novel phenotypic methods that utilise mycobacteriophages have shown promise. Other molecular detection systems require knowledge of the genes encoding the drug target (the inhA/mabA, katG, oxyR and ahpC genes for isoniazid; rpoB for rifampicin) and the mutations producing resistance. These genotypic methods are limited in that not all resistance mechanisms are known, but advanced assays for rifampicin resistance that use gene sequencing, heteroduplex analysis, solid-phase hybridisation or single-strand conformation polymorphism analysis are becoming available.

Evaluation of surface kinetic data by the transform analysis of modulated molecular beam measurements

The merits of different modulated molecular beam detection systems are discussed and the application of modern data handling methods described. It is shown that by using time domain analysis together with fourier transform techniques, information on the kinetics of surface processes is obtained. Data at a number of excitation frequencies is obtained in a single experiment. A means of detecting non-linear surface processes, and an accurate method for the determination of reaction orders is given. The origin of spurious coherent signals is considered, and finally the application of the techniques is illustrated with data obtained from an investigation of the interaction of modulated As4 beams with GaAs surfaces.