Development of a method for the separation of HLA-A, -B and -C haploid using biotinylated probe and streptavidin magnetic beads / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To develop a method for separating the human leukocyte antigen (HLA)-A, -B and -C haploid using biotinylated probes and streptavidin magnetic beads in order to solve ambiguous HLA genotyping results.</p><p><b>METHODS</b>Based on sequence information of HLA alleles from the IMGT/HLA database, the 5-biotinylated probes were designed. The probe was mixed and extended with corresponding genomic DNA, and incubated with streptavidin magnetic beads, which could form a streptavidin magnetic beads-biotin-probe DNA complex. The unique DNA haploid binding to corresponding probe was isolated after washes and elution. The separated haploid genomic DNA was used as template for HLA-A, -B and -C loci amplification and sequencing analysis.</p><p><b>RESULTS</b>Among the 12 HLA-A probes, 19 HLA-B probes and 13 HLA-C probes, DNA sequencing has confirmed that 9 HLA-A probes, 9 HLA-B probes and 5 HLA-C probes could successfully separate the haploid from genomic DNA samples.</p><p><b>CONCLUSION</b>The developed method for HLA-A, -B and -C haploid separation is reliable, which can solve certain ambiguity and improve the accuracy of HLA genotyping.</p>

Multidrug-Resistant Tuberculosis Presenting as Miliary Tuberculosis without Immune Suppression: A Case Diagnosed Rapidly with the Genotypic Line Probe Assay Method / 결핵및호흡기질환

Miliary tuberculosis (TB) is a rare extrapulmonary form of TB, and there have been only two reports of miliary TB associated with infection with multidrug-resistant (MDR)-TB pathogen in an immunocompetent host. A 32-year-old woman was referred to our hospital because of abnormal findings on chest X-ray. The patient was diagnosed with MDR-TB by a line probe assay and was administered proper antituberculous drugs. After eight weeks, a solid-media drug sensitivity test revealed that the pathogen was resistant to ethambutol and streptomycin in addition to isoniazid and rifampicin. The patient was then treated with effective antituberculous drugs without delay after diagnosis of MDR-TB. To the best of our knowledge, this is the first case of miliary TB caused by MDR-TB pathogen in Korea.

Micrometástasis: estrategias para su detección / Micrometastases: strategies for detection

La micrometástasis o enfermedad mínima residual ha adquirido una importancia trascendental en oncología al representar un verdadero problema clínico que debe ser solucionado, ya que aún se desconoce la respuesta de estos focos tumorales a los diferentes tratamientos que se usan para el control del cáncer. Aun cuando este es un problema específico fundamental a ser solucionado, ya existen métodos de ensayo inmunohistoquímicos y de biología molecular, que han permitido la ubicación de microfocos de células tumorales en diferentes órganos y tejidos, existiendo diferentes técnicas para determinar y cuantificar estas lesiones. Dentro de estas técnicas destacan la citometría de flujo y diferentes técnicas moleculares que van desde las ya tradicionales hasta las más nuevas y sofisticadas. El objetivo de la presente revisión está dirigido evaluar los nuevos métodos de diagnóstico que permitan la identificación de esta enfermedad residual, lo cual serviría para establecer tratamientos individualizados que pudieran prevenir la recurrencia de la enfermedad en los pacientes de cáncer bajo tratamiento.

Micrometastasis or minimal residual disease has become critically important in oncology since it represents a true clinical problem that must be solved, as the response of these tumor foci to the different treatments that are used for the control of cancer, is still unknown. Even though this is a fundamental specific problem to be solved, there are already immunohistochemical and molecular biology diagnostic methods that have allowed microfoci location of tumor cells in various organs and tissues, and different techniques are available to determine and quantify these lesions. Within these techniques, flow cytometry and different molecular methods are included, and they range from the traditional to the newest and most sophisticated. The goal of this review was aimed to evaluate new diagnostic methods that permit the identification of this residual disease, which would serve to establish individualized treatments and prevent the recurrence of the disease in cancer patients under treatment.

The application of small molecule bioactive probes in the identification of cellular targets / 药学学报

Identification of the cellular targets of bioactive compounds is a major challenge and a key issue in chemical biology and drug discovery. As an important technology in functional proteomics, small molecule probes play a pivotal role in the identification of cellular targets of bioactive compounds. This review is intended to introduce the application principles and structural design philosophy of chemical probes for the purpose of mechanistic study. Recent cases of successful application were also discussed to further demonstrate the principles and significance ofbioactive small molecule-based probes.

Applications and perspectives of DNA stable-isotope probing in metagenomics: a review / 生物工程学报

DNA stable-isotope probing (DNA-SIP) is a recently developed method with which the incorporation of stable isotope from a labeled substrate is used to identify the function of microorganisms in the environment. The technique has now been used in conjunction with metagenomics to establish links between microbial identity and particular metabolic functions. The combination of DNA-SIP and metagenomics not only permits the detection of rare low-abundance species from metagenomic libraries but also facilitates the detection of novel enzymes and bioactive compounds. We summarize recent progress in SIP-metagenomic techniques and applications and discuss prospects for this combined approach in environmental microbiology and biotechnology.

Connection of magnetic antisense probe with SK-Br-3 oncocyte mRNA nucleotide detected by high resolution atomic force microscope / 生物医学工程学杂志

The present paper is aimed to detect superparamagnetic iron oxide labeled c-erbB2 oncogene antisense oligonucleotide probe (magnetic antisense probe) connected with SK-Br-3 oncocyte mRNA nucleotide by high resolution atomic force microscope (AFM). We transfected SK-Br-3 oncocyte with magnetic antisense probe, then observed the cells by AFM with high resolution and detected protein expression and magnetic resonance imagine (MRI). The high resolution AFM clearly showed the connection of the oligonucleotide remote end of magnetic antisense probe with the mRNA nucleotide of oncocyte. The expression of e-erbB2 protein in SK-Br3 cells were highly inhibited by using magnetic antisense probe. We then obtained the lowest signal to noise ratio (SNR) of SK-Br-3 oncocyte transfected with magnetic antisense probe by MRI (P<0.05). These experiments demonstrated that the high resolution AFM could be used to show the binding of magnetic antisense probe and SK-Br-3 mRNA of tumor cell nuclear.

Use of multiplex ligation-dependent probe amplification (MLPA) for Duchenne muscular dystrophy (DMD) gene mutation analysis.

Background & objectives: Duchenne (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive disorders, caused by mutations in the dystrophin gene. Genetic diagnosis of the proband becomes crucial, and forms the base for carrier analysis, genetic counselling, prediction of natural history and prognosis, and eligibility for therapeutic strategies. Traditional multiplex PCR assay is the common method used in India to detect DMD gene deletions, mainly in the hot-spot region. Deletions of exons outside the usual 18 or 21 exons in the hot-spot, duplications and carrier analysis are often left without precise genetic diagnosis and require efficient dosage/quantitative analysis. In this study we evaluated the efficacy of using multiplex PCR (mPCR) of 30 exons followed by multiplex ligation-dependent probe amplification (MLPA), to study deletions and duplications in the DMD gene in patients clinically diagnosed as BMD/DMD. Methods: Using an algorithm of mPCR and MLPA which was less invasive and cost-effective, we performed retrospective and prospective analysis on 150 male patients. Results: Multiplex PCR could pick up deletions in 103 of the 150 cases. MLPA was able to detect deletions and duplications including nine additional mutations. Further, the borders of the deletions and duplications were more accurately defined by this recent methodology, which enables one to determine the effect of the mutation on the reading frame. In all, including the single exon deletions, MLPA was efficient in accurately confirming mutations in 35 per cent of all cases. Ten novel mutations were identified in this study. Overall, this approach confirmed mutations in 75 per cent of the patients in our study. Interpretations & conclusions: The systematic approach/algorithm used in this study offers the best possible economical mutation analysis in the Indian scenario.

Modeling of diffusion transport through oral biofilms with the inverse problem method / 国际口腔科学杂志·英文版

<p><b>AIM</b>The purpose of this study was to develop a mathematical model to quantitatively describe the passive transport of macromolecules within dental biofilms.</p><p><b>METHODOLOGY</b>Fluorescently labeled dextrans with different molecular mass (3 kD, 10 kD, 40 kD, 70 kD, 2000 kD) were used as a series of diffusion probes. Streptococcus mutans, Streptococcus sanguinis, Actinomyces naeslundii and Fusobacterium nucleatum were used as inocula for biofilm formation. The diffusion processes of different probes through the in vitro biofilm were recorded with a confocal laser microscope.</p><p><b>RESULTS</b>Mathematical function of biofilm penetration was constructed on the basis of the inverse problem method. Based on this function, not only the relationship between average concentration of steady-state and molecule weights can be analyzed, but also that between penetrative time and molecule weights.</p><p><b>CONCLUSION</b>This can be used to predict the effective concentration and the penetrative time of anti-biofilm medicines that can diffuse through oral biofilm. Furthermore, an improved model for large molecule is proposed by considering the exchange time at the upper boundary of the dental biofilm.</p>

Applications of fluorescent molecular imaging in tumor detection / 生物医学工程学杂志

As a recently emerging molecular imaging technique, fluorescent molecular imaging has the advantage of fast imaging, simple operation, high sensitivity, low cost, nonionizing radiation and so on. Through the real-time, non-invasive, specific tracking and detection of tumorigenesis, metastases, angiogenesis, and the therapeutic response to antitumor drugs in vivo, we can study the physiological and pathological processes within tumors at cellular and molecular levels. Fluorescent molecular imaging provides effective methods for the early detection and targeted therapy of tumor, for the intraoperative visualization of tumor foci, as well as for the development of new antitumor drugs in the future clinical practice.

Progress of molecular imaging research on the use of RNA interference in malignant tumors / 生物医学工程学杂志

RNA interference (RNAi) is applied in the gene therapy of malignant tumors as an effective technique to degrade its homologous mRNA specifically. This technique has been developed quickly in broad prospects. Molecular imaging plays an important role both in gene imaging and in molecular signal transduction. It is used as a direct approach to study the onset and development of disease. The technique of RNAi can be used for preparing molecular probes, for making earlier and specific diagnosis, and for targeted gene therapy of malignant tumors.

Development in ligase-mediated techniques for bio-molecular analysis / 生物工程学报

Two oligonucleotide probes are permitted to anneal to the nucleic acid target of interest so that the ends of two probes immediately become adjacent to each other. The ligase can then efficiently join the two juxtaposed oligonucleotide probes by the formation of a phosphodiester bond if and only if perfectly matched base-pairs at the nick are present. During past 20 years, many ligase-mediated techniques have been developed for analyzing various bio-molecules, such as known/unknown point mutations, small-scale insertions and deletions, CpG islands methylation, large sets of single nucleotide polymorphisms (SNPs), specific proteins and DNA regions with which some other proteins can interact. Since the ligation reaction can be easily integrated into other techniques, certain advances have been already achieved. These novel approaches retain high accuracy through multiple hybridization and enzymatic processing events, and provide inherent quality control checking. In this article, we provide a comprehensive review of the ligase-mediated techniques for bio-molecular analysis.

Application study on multicolor combinational probe coding real-time PCR in detection of foodborne pathogens / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate the detection limit of multicolor combinational probe coding real-time PCR (MCPC-PCR) in detection of Salmonella and Staphylococcus aureus suspended in the food samples, and to apply MCPC-PCR to detect the samples of food poisoning.</p><p><b>METHODS</b>Series concentration of bacterium suspension (10(1) - 10(9) CFU/ml) was prepared by using 22 simulated samples including fresh meat and cakes and then MCPC-PCR was applied to detect Salmonella and Staphylococcus aureus in 22 samples. Enrichment broth of 101 frozen samples and 5 early patients' anal swabs in food poisoning cases were detected after the DNA samples were extracted.</p><p><b>RESULTS</b>The limits of MCPC-PCR assay in detecting Salmonella and Staphylococcus aureus were about 10(2) copies/test; 101 frozen enrichment broth of samples in food poisoning cases were detected by MCPC-PCR assay, of 23 positive samples, 18 were confirmed by bacteriology techniques; 96 samples detected by MCPC-PCR and bacteriology techniques had the same results, and the coincidence rate was 95.05%. Anal swabs, collected from 5 of early patients in a food poisoning case gave a clue to be Vibrio parahaemolyticus by MCPC-PCR assay and then were perfectly consistent with bacteriology assay.</p><p><b>CONCLUSION</b>As a method of high sensitivity and good specificity, MCPC-PCR assay can quickly and conveniently detect multiple pathogens existing in food samples, therefore we recommend it to be used in rapidly screening or simultaneous detection of food-borne diseases.</p>

Applications of chemical genetics in biomedical research / 生物医学工程学杂志

Chemical genetics is the science which takes the small molecular compounds as tools to solve the genetics problems or to disturb/adjust normal biological process so as to find out protein functions. Because the small molecules have the diverse chemical characters and the ability to identify the target proteins, they also can be filtrated on the basis of phenotype. So the methods of chemical genetics have been applied in almost all of the researches on biology and medicine. In this paper, the methods to acquire small molecular compounds are introduced. The enormous progress achieved in the field of combinatorial chemistry, which has allowed the rapid production of a large number of chemically diverse molecules, is an important prerequisite to make chemical libraries available to academic researchers. And the applications of the compounds in early embryo development, cell differentiation, on-set and course of disease are discussed, too. The application of small molecules has an enormous impact on our understanding of cell biology. There are many examples where small molecules, in combination with genetic screens, have facilitated the dissection of complex cellular processes.

Estudo comparativo entre um sistema de sonda genética e métodos clássicos na identificação das micobactérias / Gene probes versus classical methods in the identification of mycobacteria

OBJETIVO: O aparecimento da co-infecção tuberculose/HIV e o aumento de casos de doenças provocadas por micobactérias não-tuberculosas (MNT) exigem repostas laboratoriais rápidas tanto no isolamento como na identificação das micobactérias. O objetivo deste trabalho foi avaliar a identificação das micobactérias através de sonda genética em comparação com os métodos bioquímicos clássicos. MÉTODOS: Entre 2002 e 2004, foram analisadas 178 culturas de micobactérias, confirmadas como bacilos álcool-ácido resistentes e obtidas de isolados clínicos de pacientes sintomáticos respiratórios ou com suspeita clínica de tuberculose pulmonar e/ou micobacterioses, atendidos nas Unidades de Saúde da Baixada Santista. RESULTADOS: A sonda genética identificou 137 amostras (77%) como complexo Mycobacterium tuberculosis e 41 (23%) como MNT. A discordância observada de 3% entre os métodos ocorreu apenas no ano de implantação (2002). Ao comparar os métodos, a sensibilidade, especificidade, valor preditivo positivo e valor preditivo negativo da sonda genética foram 98%, 93%, 98% e 93%, respectivamente. CONCLUSÕES: Apesar do custo elevado, a identificação de micobactérias pela técnica molecular é mais rápida: máximo de 3 h vs. 28-30 dias para os métodos clássicos. A utilização de sondas genéticas é uma técnica molecular validada, simples e disponível no mercado, com elevada especificidade, sensibilidade e rapidez, o que justifica sua implantação e uso rotineiro em laboratórios de referência, facilitando o diagnóstico e permitindo uma intervenção clínica ágil.

OBJECTIVE: The emergence of tuberculosis/HIV co-infection and the increase in the number of cases of infection with nontuberculous mycobacteria (NTM) require rapid laboratory test results in the isolation and identification of mycobacteria. The objective of this study was to evaluate the identification of mycobacteria by means of gene probes in comparison with that obtained using classical biochemical methods. METHODS: Between 2002 and 2004, 178 mycobacterial cultures, all testing positive for acid-fast bacilli, were analyzed. Samples were obtained from clinical specimens of patients with respiratory symptoms or with clinical suspicion of pulmonary tuberculosis/mycobacteriosis who were treated in the greater metropolitan area of Santos. RESULTS: The gene probe identified 137 samples (77%) as Mycobacterium tuberculosis complex and 41 (23%) as NTM. Discordant results between the methods (3%) were obtained only in the year of implementation (2002). When comparing the methods, the sensitivity, specificity, positive predictive value and negative predictive value of the gene probe method were 98%, 93%, 98% and 93%, respectively. CONCLUSIONS: Despite the cost, the identification of mycobacteria using the molecular technique is faster: maximum 3 h vs. 28-30 days for classical methods. The use of gene probes is a validated molecular technique. It is fast, easy to use and readily available on the market. It has high specificity and sensitivity, which justifies its implementation and routine use in referral laboratories, since it facilitates the diagnosis providing agile clinical interventions.

Detection of enterotoxigenic Escherichia coli by real-time polymerase chain reaction by using MGB-TaqMan probe technique / 中华预防医学杂志

<p><b>OBJECTIVE</b>To develop a real-time polymerase chain reaction(PCR) based on TaqMan technology by using a new MGB probe for detecting enterotoxigenic Escherichia coli (ETEC) in paper.</p><p><b>METHODS</b>Primers and MGB probe were designed in the ecoding region of heat-stable toxin of ETEC. Real-time PCR detected ETEC by using the exterior standard method with protracting standard curves. The specificity, sensitivity, accuracy, stability of real-time PCR system was evaluated. An internal negative antithesis was added to the real-time PCR system in order to get rid of the false positive of system. Using UNG enzyme expelled the contamination of PCR reaction.</p><p><b>RESULTS</b>Primers and MGB probe were suited to the Real-time PCR. The assay showed that the method was quick, special, sensitive and stable. The real-time PCR system could detect ETEC in a large scale. The assay might be finished in two hour.</p><p><b>CONCLUSION</b>These observations suggested that real-time PCR based on MGB probe should be an excellent candidate for a standard ETEC detection method.</p>

Use of the AMPLIFIED [TM] Mycobacterium tuberculosis direct test for the diagnosis of tuberculosis

A comparative analysis was made of 3 conventional tests for tuberculosis [TB] versus a DNA probe technique among suspected TB patients at a reference centre in Greece. During 2004, we tested 2961 biological specimens from 2234 patients with the following methods: Ziehl-Neelsen staining, LowensteinoJensen culture, BACTEC mycobacteria growth indicator tubes [MGIT] and the Gen-Probe AMPLIFIED[TM] Mycobacterium tuberculosis direct test [MTD]. Of a total of 136 TB patients diagnosed and under anti-TB treatment, 133 of them [98%] were positive by amplified MTD. There were 112 TB [82%] detected by the MGIT method, 102 [75%] by Lowenstein-Jensen culture and 75 [55%] by Ziehl-Neelsen staining. Using MTD the positive result is ready within hours compared with days or weeks

Application of superparamagnetic iron oxide labeled antisense oligodeoxynucleotide probe in cellular magnetic resonance imaging / 中国医学科学院学报

<p><b>OBJECTIVE</b>To prepare the superparamagnetic iron oxide (SPIO)-labeled antisense oligodeoxynucleotide (ASODN) probe and evaluate the application of this probe in cellular magnetic resonance imaging (MRI).</p><p><b>METHODS</b>We prepared the SPIO-labeled ASODN probe using chemical cross linking method to conjugate SPIO to ASODN, detected its configuration by atomic force microscopy, determined the conjugating rate and biology activation by high performance liquid chromatography, and detected the stability by polyacrylamide gel electrophoresis. After that, we transfected the SK-Br3 oncocytes which had over-expression of the c-erbB2 oncogene by this probes, observed the intracellular iron distribution by optical microscope, measured iron content by atomic absorption spectroscopy, and observed the signal change by MRI.</p><p><b>RESULTS</b>Atomic force microscope showed that the SPIO-labeled ASODN probe was mostly spherical and well-distributed, with a diameter of 25-40 nm and a conjugating rate of 100%. This probe had inhered biological activity and stability. In addition, light microscopy revealed an intracellular uptake of iron oxides in the transfected SK-Br3 oncocyte, and the iron content of the group of transfected SK-Br3 oncocytes was significantly higher than those of other contrast groups (all P < 0.01). MRI showed that transfected SK-Br3 oncocyte had the lowest signal among all other cells (all P < 0.05).</p><p><b>CONCLUSIONS</b>We prepared the SPIO-labeled ASODN probe successfully. It can effectively transfect SK-Br3 oncocyte and enter SK-Br3 oncocyte, and thus reduce the signal intension in MRI.</p>

Avaliação do crescimento em cordas na identificação presuntiva do complexo Mycobacterium tuberculosis / Detection of cord factor for the presumptive identification of Mycobacterium tuberculosis complex

OBJETIVO: O Mycobacterium tuberculosis, sob certas condições apropriadas, cresce em cordões de serpentinas, denominados de fator corda, ou crescimento em cordas. O objetivo deste estudo é avaliar a detecção do fator corda como método de identificação presuntiva do complexo M. tuberculosis, comparando-o aos testes de tipificação (TIP) convencionais. MÉTODO: Foram analisadas 743 cepas, de janeiro de 2002 a dezembro de 2005, na Área de Micobactérias do Instituto Adolfo Lutz - Santos, obtidas de isolados clínicos coletados de pacientes sintomáticos respiratórios ou com suspeita clínica de tuberculose pulmonar e/ou micobacterioses, atendidos nas Unidades Básicas de Saúde da Baixada Santista. Foram feitos esfregaços das cepas de micobactérias isoladas em meio líquido MB/BacT e meio sólido, Lowenstein-Jensen ou Ogawa-Kudoh, sendo 301 (40,5 por cento) cepas em meio líquido e 442 (59,5 por cento) em meio sólido. RESULTADOS: Os resultados de sensibilidade, especificidade e valores preditivos positivos e negativos, obtidos com a comparação do desempenho do método em ambos os meios de isolamento e TIP convencionais, foram respectivamente 98,5, 88, 97 e 93 por cento. Observou-se maior sensibilidade do método em meio sólido (100 por cento), com uma diferença de sensibilidade entre os meios analisados de apenas 2,7 por cento. CONCLUSÕES: Conclui-se, pelos resultados obtidos, que o fator corda é um critério real e rápido na identificação do complexo M. tuberculosis; além disso, em laboratórios com alta prevalência do complexo M. tuberculosis e que não dispõem de técnicas que permitam a precocidade de sua identificação, o fator corda possibilita o direcionamento aos testes conclusivos de identificação e adicionais de sensibilidade que se façam necessários.

OBJECTIVE: Virulent strains of the Mycobacterium tuberculosis complex, under certain appropriate conditions, grow as characteristic ropes, bundles or serpentine cords known as cord factor or growth in cords. The objective of the present study was to evaluate cord factor detection as a method of achieving presumptive identification of the M. tuberculosis complex, comparing it to conventional typing tests. METHODS: A total of 743 strains were analyzed from January of 2002 to December of 2005 in the Mycobacteria Sector of the Adolfo Lutz Institute, located in the city of Santos, Brazil. Samples were obtained from clinical specimens collected from patients with respiratory symptoms treated at basic health clinics in the greater metropolitan area of Santos. Ziehl-Neelsen-stained smears were prepared, 301 (40.5 percent) in MB/BacT broth and 442 (59.5 percent) on solid media, either Lowenstein-Jensen or Ogawa-Kudoh. RESULTS: The sensitivity, specificity, positive predictive value and negative predictive value obtained during the performance comparison of the two methods (cord factor detection and conventional typing) using both isolation media were, respectively, 98.5, 88, 97 and 93 percent. The method was more sensitive on solid medium (100 percent), and the difference in sensitivity between the two media types was only 2.7 percent. CONCLUSIONS: Taking into consideration the results obtained, we conclude that, in laboratories with a high incidence of M. tuberculosis complex isolation and limited economic resources, cord factor detection is a fast and valid criterion for identifying these mycobacteria using liquid or solid medium. It also enables subsequent conclusive identification tests, as well as additional sensitivity tests when necessary.

Development and field application of a molecular probe for the primary pathogen of the coral disease white plague type II

Abstract: One of the current problems in the field of coral disease research is that of tracking coral pathogens in the natural environment.A promising method to do this is by use of pathogen-specific molecular probes. However,this approach has been little used to date.We constructed,and validated in the laboratory,a fluoro-chrome-labeled molecular probe specific to Aurantimonas coralicida ,the bacterial pathogen of the Caribbean coral disease white plague type II (WPII).We then used the probe to test field samples of diseased coral tissue for the presence of this pathogen.Probe design was based on a unique subset (25 nucleotides)of the complete16S rRNA gene sequence derived from a pure culture of the pathogen.The pathogen-specific probe was labeled with the fluorochrome GreenStar*™FITC (fluorescein isothiocyanate,GeneDetect Ltd,New Zealand).As a control, we used the universal eubacterial probe EUB 338,labeled with a different fluorochrome (TRITC,tetra-methyl-rhodamine isothiocyanate).Both probes were applied to laboratory samples of pure cultures of bacteria, and field samples collected from the surface of the disease line of corals exhibiting signs of white plague (types I and II),healthy controls,and corals with an uncharacterized disease ("patchy necrosis ").All samples were analyzed using fluorescence in situ hybridization (FISH).We have determined that the probe is specific to our laboratory culture of the coral pathogen,and does not react with other bacterial species (the eubacterial probe does).The WPII pathogen was detected in association with diseased coral samples collected from coral colonies on reefs of the Bahamas (n=9 samples)exhibiting signs of both WPI and WPII.Diseased (and healthy)tissue samples (n=4)from corals exhibiting signs of "patchy necrosis "were also assayed.In this case the results were negative, indicating that the same pathogen is not involved in the two diseases.Incorporation and use of pathogen-specific probes can...

Micro machining of micro-cantilever probes for efficient deposition for biochips / 生物工程学报

Biochip technology will bring a tremendous revolution to life science and medical research in 21 century. Microarray assays represent an essential technical advance in biomedical research. Recently, the demand for microarray assay technology has spring up. Therefore, low cost and flexible techniques are needed to meet specific requirements for increasingly integrated biochips. Also performance must be improved in terms of speed and sensitivity. To this end, promising approaches, mainly based on micro and nanotechnologies, have been developed. In this paper, the design and microfabrication of a novel type of micro-cantilever probe are introduced. These probes were fabricated using silicon dioxide by Micro-electromechanical System (MEMS) techniques, and they featured one micron split gap, microchannels and self-replenishing reservoirs. All fabricated micro-cantilever probe were tested on Nanoarrayer instrumentation. Cy3-streptavidin was loaded as biological sample and patterned on DSU gold surface. Results showed these probes were capable of generating high quality biological arrays with routine spot sizes of 2 - 3 microns and could deposit at least three thousand spots without reloading. The spot size could potentially achieve sub-micron when probe size was further shrunk down by the high-resolution lithography technique or more precise microfabrication technologies, such as E-beam lithography. To further improve sample loading efficiency, it is needed to modify the cantilever surface in order to better confine sample inside the microchannel and reservoir, which will be researched in the future.