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PubMed1995

Molecular detection of pre-ATL state among healthy HTLV-1 carriers in an endemic area of Japan.

Adult T-cell leukemia/lymphoma (ATL) usually develops after age 40 in Japan, suggesting a long latency since HTLV-I is considered to be transmitted mainly from mothers to babies via breast milk. Our previous studies had suggested that HTLV-I carriers who have a monoclonal integration of HTLV-I proviral DNA in the peripheral blood cells, designated pre-ATL, account for about 2% of healthy carriers and are at high risk of developing ATL. Their ages ranged from 32 to 80 (median 57). Nevertheless, many cases of pre-ATL showed a long-lasting carrier state (10-year probability around 90%). In the present investigation we conducted a large-scale molecular detection of monoclonal integration in a population (481 cases) of healthy carriers with ages ranging from 16 to 82 (median 49) for the purpose of clarifying the earliest onset of this pre-ATL state. Southern-blot analysis of DNA extracted from peripheral blood mononuclear cells revealed 6 cases (1.2%) with a monoclonal band. All of these 6 were older than 40; no single positive case was found in 220 carriers under age 40. These results indicate that the molecularly detectable pre-ATL state also develops after a long latency. Thus the pre-ATL state seems to be a subtype of ATL showing an extremely indolent course of disease development, but not merely an early phase of all subtypes of ATL. We propose that, in order to reveal a promoter(s) responsible for the development of ATL from the pre-ATL state, intensive epidemiological investigation of life style, eating habits, occupation and exposure to carcinogens must be conducted on this unique group prone to ATL. It is also important to perform such an epidemiological study on the general population of carriers by paying special attention to the first 3-4 decades of each carrier's life in Japan and by comparing the data with those from carriers in different geographical areas of the world.

Adolescent↗
PubMed1999

Molecular detection of micrometastases and circulating tumor cells in solid tumors.

The detection of circulating tumor cells and micrometastases may have important prognostic and therapeutic implications. Because their numbers can be very small, these tumor cells are not easily detected using conventional methods. In the last decade, molecular techniques have been widely used for the detection of occult tumor cells. The objective of this report is the application of these molecular tools to solid tumors. A systematic review of all related English-language articles published in the last 32 years was performed. The molecular detection of occult tumor cells can be accomplished by PCR amplification of tumor-specific abnormalities present in the DNA or mRNA of malignant cells. The other main PCR strategy for the detection of CTC and micrometastases involves amplification of tissue-specific mRNA. This latter method was often applied to solid tumors, whereas the former was occasionally used. PCR was shown to be superior to conventional techniques in detecting occult tumor cells, allowing the identification of 1 malignant cell mixed with 1 to 10 million normal cells. In some reports, PCR is shown to be a strong predictor of outcome. The molecular detection of circulating tumor cells and micrometastases in solid tumors can be accomplished using highly sensitive PCR assays. The central question of whether PCR reliably predicts relapse and survival remains unanswered for many types of solid tumor. If PCR-based assays are found to be a reliable tool, they will likely have a major impact on the management of these malignancies.

False Negative Reactions↗
PubMed2003

The molecular detection of micrometastatic breast cancer.

BACKGROUND: The rapid evolution of molecular technology and novel markers provides the opportunity to establish a more effective means to detect micrometastatic breast cancer. Given the controversies concerning application and clinical relevance, this review critically evaluates the current status of these molecular staging technologies. DATA SOURCES: Breast cancer literature addressing (1). molecular detection methodologies (immunohistochemistry, reverse transcriptase polymerase chain reaction, and microarray analysis); (2). specific tissue applications such as lymph nodes, bone marrow aspirate, and peripheral blood; (3). expert commentary concerning the clinical applications and pitfalls of these technologies; and (4). recent data from our molecular diagnostics laboratory. CONCLUSIONS: Molecular detection technologies such as reverse transcriptase polymerase chain reaction and microarray analyses are being developed that will likely have future application as cancer diagnostics. Further work is needed to establish assays that are validated by prospective clinical studies. Early identification of clinically relevant disease could lead to new treatment or staging approaches for breast cancer.

Bone Marrow Neoplasms↗
PubMed2000

Statistical methods for detecting molecular adaptation.

The past few years have seen the development of powerful statistical methods for detecting adaptive molecular evolution. These methods compare synonymous and nonsynonymous substitution rates in protein-coding genes, and regard a nonsynonymous rate elevated above the synonymous rate as evidence for darwinian selection. Numerous cases of molecular adaptation are being identified in various systems from viruses to humans. Although previous analyses averaging rates over sites and time have little power, recent methods designed to detect positive selection at individual sites and lineages have been successful. Here, we summarize recent statistical methods for detecting molecular adaptation, and discuss their limitations and possible improvements.

Journal Article↗
PubMed2003

Molecular detection methods developed for a systemic rickettsia-like bacterium (RLB) in Penaeus monodon (Decapoda: Crustacea).

Molecular detection methods were developed to aid in the diagnosis of a rickettsia-like bacterium (RLB) which caused severe mortalities of farm-raised Penaeus monodon in Madagascar. Using primers derived from the 16S rRNA gene of bacteria, a PCR assay was optimized to amplify this region of the genome of the RLB, using extracted DNA from infected P. monodon tissue as the template. The resulting amplified PCR product was sequenced and 2 novel primers were selected from the variable region of the gene. These primers amplified a 532 bp fragment of DNA originating from the rickettsia-infected samples. The PCR assay was optimized and tested on DNA extracted from specific pathogen-free (SPF) P. vannamei tissue and several other strains of bacteria. The PCR assay with the rickettsia-specific primers was specific for this RLB and did not amplify the other DNA samples tested. The 532 bp PCR-amplified fragment was labeled with digoxigenin (DIG) for in situ hybridization assays. This probe was tested on SPF, RLB and bacteria-infected shrimp specimens preserved in Davidson's fixative. The probe was specific for both natural and experimental rickettsial infections. Hybridization with this probe required a stringent temperature of 65 degrees C, otherwise cross-reactivity was observed with other types of bacteria.

Animals↗
PubMed2002

Molecular detection of p16 promoter methylation in the serum of colorectal cancer patients.

Assays based on the molecular detection of genetic changes in serum have been shown as potential diagnostic tools for colorectal cancer. We examined the methylation status of p16 in colorectal cancers using methylation-specific PCR (MSP). Forty-four of 94 (47%) cancer DNA exhibited abnormal promoter methylation of p16 gene while no corresponding normal DNA exhibited such methylation. Subsequently, we examined whether aberrant methylation could be detected in corresponding serum DNA, and found that 13 of 44 (30%) patients with p16 promoter methylation in tumor DNA demonstrated abnormal methylation in their serum DNA. Moreover, abnormal methylation was found in the serum of patients in all clinical stages, suggesting that early colorectal cancer could be detected using the MSP method.

Adenocarcinoma↗
PubMed2001

Outcome prediction by molecular detection of minimal residual disease in bone marrow for advanced neuroblastoma.

BACKGROUND: We have determined whether sequential molecular detection of minimal residual disease (MRD) in bone marrow (BM) could predict the outcome of patients with advanced neuroblastoma (NB). PROCEDURE: Bone marrow samples from 19 patients over 12 months of age with stage 4 neuroblastoma were sequentially examined for tumor cell contamination by detecting tyrosine hydroxylase (TH) mRNA using reverse transcription-polymerase chain reaction (RT-PCR). All patients received repetitive multi-drug chemotherapy including cisplatin, cyclophosphamide or ifosphamide, adriamycin, and etoposide or vincristine. Seventeen patients received myeloablative therapy with hematopoietic stem cell transplantation after achieving complete remission. RESULTS: All but one patient were histologically positive for tumor cells in BM samples at diagnosis, and they became negative for tumor cells within 3 months histologically. By the RT-PCR analysis, all patients were positive for TH mRNA in BM samples at diagnosis, and they became negative for TH mRNA 1 to 13 months after the start of chemotherapy. Six patients whose BM samples became negative for TH mRNA within 4 months after the start of chemotherapy remained alive without evidence of disease (median 61 months, range 20-76). In contrast, 12 of 13 patients whose BM samples remained positive at that time developed relapse and 10 of them died of disease (median 24 months, range 13-43). There was a statistically significant difference in survival between the two groups (P < 0.05). No significant difference of clinical characteristics by the MRD positivity at 4 months after the start of chemotherapy. CONCLUSIONS: Persistence of MRD in BM at 4 months after the start of chemotherapy could predict poor prognosis in advanced neuroblastoma.

Adrenal Gland Neoplasms↗
PubMed2005

Imaging and detecting molecular interactions of single transmembrane proteins.

Single-molecule atomic force microscopy (AFM) provides novel ways to characterize structure-function relationships of native membrane proteins. High-resolution AFM-topographs allow observing substructures of single membrane proteins at sub-nanometer resolution as well as their conformational changes, oligomeric state, molecular dynamics and assembly. Complementary to AFM imaging, single-molecule force spectroscopy experiments allow detecting molecular interactions established within and between membrane proteins. The sensitivity of this method makes it possible to detect the interactions that stabilize secondary structures such as transmembrane alpha-helices, polypeptide loops and segments within. Changes in temperature or protein-protein assembly do not change the position of stable structural segments, but influence their stability established by collective molecular interactions. Such changes alter the probability of proteins to choose a certain unfolding pathway. Recent examples have elucidated unfolding and refolding pathways of membrane proteins as well as their energy landscapes. We review current and future potential of these approaches to reveal insights into membrane protein structure, function, and unfolding as we recognize that they could help answering key questions in the molecular basis of certain neuro-pathological dysfunctions.

Animals↗
PubMed2005

First molecular detection of Babesia vogeli in dogs from Brazil.

The present work describes the detection and first molecular characterization of Babesia vogeli in dogs, naturally infected in Brazil and even in South America. Microscopic examination of Giemsa-stained peripheral blood smears collected from dogs originating from four different locations in Brazil revealed the presence of large Babesia merozoites and trophozoites (>2.5 microm). DNA was extracted from infected blood samples and PCR amplifications of the 18S rDNA were carried out. As a reference, DNA from an isolate of B. vogeli originated from Egypt was used. PCR products were purified and sequenced. The DNA sequences demonstrated 100% identity among the Brazilian isolates. Comparisons with the 18S rDNA sequence of the B. vogeli isolate from Egypt and with other B. vogeli sequences from Spain, France, Japan, Australia and South Africa confirmed the affiliation of all Brazilian isolates to the species B. vogeli.

Animals↗
PubMed2004

Phenotypic and molecular detection of CTX-M-beta-lactamases produced by Escherichia coli and Klebsiella spp.

Organisms producing CTX-M-beta-lactamases are emerging around the world as a source of resistance to oxyiminocephalosporins such as cefotaxime (CTX). However, the laboratory detection of these strains is not well defined. In this study, a molecular detection assay for the identification of CTX-M-beta-lactamase genes was developed and used to investigate the prevalence of these enzymes among clinical isolates of Escherichia coli and Klebsiella species in the Calgary Health Region during 2000 to 2002. In addition, National Committee for Clinical Laboratory Standards (NCCLS) recommendations were evaluated for the ability to detect isolates with CTX-M extended-spectrum beta-lactamases (ESBLs). The PCR assay consisted of four primer sets and demonstrated 100% specificity and sensitivity for detecting different groups of CTX-M-beta-lactamases in control strains producing well-characterized ESBLs. Using these primer sets, 175 clinical strains producing ESBLs were examined for the presence of CTX-M enzymes; 24 (14%) were positive for bla(CTX-M-1-like) genes, 95 (54%) were positive for bla(CTX-M-14-like) genes, and the remaining 56 (32%) were negative for bla(CTX-M) genes. Following the NCCLS recommendations for ESBL testing, all of the control and clinical strains were detected when screened with cefpodoxime and when both cefotaxime and ceftazidime with clavulanate were used as confirmation tests.

Anti-Bacterial Agents↗
PubMed2001

The significance of bcr-abl molecular detection in chronic myeloid leukemia patients "late," 18 months or more after transplantation.

The bcr-abl chimeric messenger RNA is frequently detected in chronic myeloid leukemia (CML) patients after bone marrow transplantation. It was previously reported that the relapse risk of bcr-abl detection 6 to 12 months after transplantation was greater than 40%. This risk decreased as the time between transplantation and detection increased. To further define the relapse risk associated with bcr-abl molecular detection in "late" CML survivors, 379 consecutive CML patients alive at 18 months after transplantation or later were studied. Ninety of 379 patients (24%) had at least one positive bcr-abl test 18 months after transplantation or later; 13 of 90 bcr-abl-positive patients (14%) and 3 of 289 bcr-abl-negative patients (1.0%) relapsed. The median time from bcr-abl detection to relapse was 916 days (range, 251-2654 days). The hazard ratio of relapse associated with bcr-abl detection was 19.2 (P <.0001). The stage of disease, chronic graft-versus-host disease, and the donor type did not alter the association between bcr-abl and relapse. Quantification of bcr-abl was performed on 344 samples from 85 bcr-abl-positive patients by means of a real-time quantitative reverse transcriptase-polymerase chain reaction assay. The median bcr-abl change of patients who relapsed was significantly greater than those that remained in remission (P =.002). The median bcr-abl level at relapse was 40 443 bcr-abl copies per microg RNA (range, 960-299 552). Of 73 bcr-abl-positive patients who failed to relapse, 69% had only one positive test at a median of 24 copies bcr-abl per microg RNA. The detection of bcr-abl is common following transplantation. The prognostic significance of a qualitative bcr-abl can be refined by quantitative assays and thus may target patients who would benefit from early intervention.

Adolescent↗
PubMed2002

Molecular detection of XO - Turner syndrome.

Turner syndrome is caused by haploinsufficiency of the short arm of X-chromosome, and is usually diagnosed by karyotyping. This procedure is time-consuming, expensive and unfeasible for population screening. We propose molecular detection of 45XO Turner patients based on the ability of HpaII, a methylation sensitive endonuclease, to induce the cleavage of non-methylated DNA in the active X-allele. Genomic DNA was obtained from 22 patients with Turner syndrome confirmed by karyotype (45XO, N = 18; 45XO/46XX, N = 4). After digestion, DNA was amplified with primers directed to exon 1 of the androgen receptor (AR) gene and to the GAPDH control gene. Normal control females or mosaic patients, with a second methylated X-chromosome, escaped from HpaII digestion and produced a band corresponding to AR gene amplification. 45XO patients have just one active non-methylated X-chromosome, completely digested by HpaII, thus preventing the amplification of the AR gene. Three of the 45XO cases gave amplified bands, suggesting low-frequency mosaicisms that are not detected by karyotyping. Compared to classical karyotype studies for the detection of 45XO Turner patients, this new molecular method is simpler, faster and less expensive.

Base Sequence↗
PubMed2003

Molecular detection of ascomycetes associated with Fucus serratus.

The association of ascomycetes with Fucus serratus was investigated by comparing the broad-based molecular and cultural diversities of healthy and dead fronds. Four PCR primer pairs were used to amplify the 18S (primers NS1-FR1; NS1-EF3) or 28S rRNA (primers NL209-NL912; NL359-NL912) genes directly from the DNA of algal thalli. Two novel primer pairs, NL209-NL912 and NL359-NL912 giving product sizes of 700 and 559 bp respectively, were designed to amplify the 28S rDNA from ascomycetes specifically. All primer combinations amplified DNA from 33 reference taxa isolated from Fucus serratus, and the products generated by primers NS1-FR1GC and NL359-NL912GC were separated in 18-38% and 38-60% denaturant gradients respectively after DGGE. The 18S rDNA DGGE system resolved eight bands from algal DNA, but many of the sequences separated were not fungal, whereas the 28S rDNA system resolved seven bands that were all identified as ascomycetes. Phylogenetic analysis and BLAST search results of environmental sequences revealed the presence of four main ascomycete groups: (1) the Halosphaeriales, (2) the Hypocreales, (3) an unidentified Lulworthiales complex, and (4) the Pleosporales. Few fungal isolates were detected molecularly suggesting that fungal colonisation of fronds was limited, mainly to species in dead casts.

Ascomycota↗
PubMed2001

Disease outcome may be predicted by molecular detection of minimal residual disease in bone marrow in advanced neuroblastoma: a pilot study.

PURPOSE: This pilot study focussed on whether sequential molecular detection of minimal residual disease (MRD) in bone marrow (BM) could predict the outcome of patients with advanced neuroblastoma. PATIENTS AND METHODS: Bone marrow samples from 21 patients older than age 12 months with stage IV neuroblastoma were sequentially examined for tumor cell contamination by detecting tyrosine hydroxylase (TH) messenger ribonucleic acid (mRNA) using reverse transcription-polymerase chain reaction (RT-PCR). Twenty patients received myeloablative therapy with hematopoietic stem cell transplantation after achieving complete remission. RESULTS: All BM samples of patients except that of one patient was cytologically positive for neuroblastoma cells at diagnosis, and they became negative for neuroblastoma cells within 3 months by cytologic examination. By RT-PCR analysis, BM samples of all patients were positive for TH mRNA at diagnosis, and samples of 19 patients became negative for TH mRNA 1 to 13 months after the start of chemotherapy. Six patients whose BM samples became negative for TH mRNA within 4 months after the start of chemotherapy remained alive without evidence of disease (median 76 mos, range 36-91). In contrast, out of 15 patients whose BM samples remained positive, 10 patients had relapse develop and 9 patients died from disease (median 15 mos, range 10-25). There was a statistically significant difference in disease-free survival between the two groups (P < 0.05). CONCLUSION: Persistence of MRD in BM may predict poor prognosis in advanced neuroblastoma.

Adrenal Gland Neoplasms↗
PubMed2006

Freezing induces biased results in the molecular detection of Flavobacterium columnare.

Specific PCR detection and electron microscopy of Flavobacterium columnare revealed the risk of false-negative results in molecular detection of this fish pathogen. Freezing and thawing destroyed the cells so that DNA was for the most part undetectable by PCR. The detection of bacteria was also weakened after prolonged enrichment cultivation of samples from infected fish.

DNA, Bacterial↗
PubMed2006

Enzyme-linked immunosorbent assay of nucleic acid sequence-based amplification for molecular detection of M. tuberculosis.

An enzyme-linked immunosorbent assay of nucleic acid sequence-based amplification (NASBA-ELISA) was developed for molecular detection of Mycobacterium tuberculosis. The primers targeting 16S rRNA were used for the amplification of bacterial RNA by the isothermal digoxigenin (DIG)-labeling NASBA process, resulting in the accumulation of DIG-labeled RNA amplicons. The amplicons were hybridized with a specific biotinylated DNA probe which was non-covalently immobilized on streptavidin-coated microtiter plate. The RNA-DNA hybrids were colorimetrically detected by the addition of an anti-DIG antibody HRP conjugate and 2,2-azino-di-(3-ethylbenzthiazolinsulfonate) substrate. Using this method, as little as 1 x 10(2) CFU ml(-1) of M. tuberculosis was detected within less than 5h. Results obtained from the clinical specimens showed 85.7% and 96% sensitivity and specificity, respectively. No interference was encountered in the amplification and detection of M. tuberculosis in the presence of non-target bacteria, confirming the specificity of the method.

Base Sequence↗
PubMed2006

Molecular detection of tyrosinase transcripts in peripheral blood from patients with malignant melanoma: correlation of PCR sensitivity threshold with clinical and pathologic disease characteristics.

BACKGROUND: Positive molecular detection of tyrosinase transcripts (TYR mRNA) in RNA extracts of peripheral blood (PB) samples from patients with malignant melanoma provides evidence of disease dissemination. METHODS: Total RNA extracted from PB was quantified and subjected to RT-PCR under ultra-sensitive and reduced-sensitivity PCR conditions using SSRT-II. Positive TYR mRNA detection in 78 melanoma patients and 40 healthy volunteers was correlated with clinical stage, Breslow's evaluation of tumor thickness, Clark's assessment of tumor invasion, the location of the primary tumor site, and tumor histology. The assay sensitivity was evaluated by spiking PB with the melanoma cell line SK-MEL-28. RESULTS: Using ultra-sensitive PCR conditions, eight out of 40 RNA (20%) samples from healthy volunteers and 50 out of 78 RNA (64.1%) samples from melanoma patients tested positive. Using reduced-sensitivity PCR conditions, we found only two positives in 40 RNA samples from healthy subjects and 20 positives in 78 RNA samples (25.6%) from melanoma patients. Only positive PCR samples for the reduced-sensitivity PCR assay correlated significantly with stage IV (metastatic) disease (p=0.0395). There was no significant correlation between positive TYR mRNA samples for either PCR condition (ultra-sensitive and reduced-sensitivity) with Breslow's classification of tumor thickness, Clark's assessment of tumor invasion, location of the primary tumor site, and type of tumor histology. CONCLUSIONS: We conclude that reduced-sensitivity rather than ultra-sensitive PCR conditions correlate with clinical stage in melanoma patients.

Adult↗