Amplification-Free Nucleic Acid Testing with a Fluorescence One-Step-Branched DNA-Based Lateral Flow Assay (FOB-LFA).

The emergence of the global pandemic and the discovery of nucleic acid biomarkers in cancer diagnosis have fostered the development of more accurate and adaptive molecular diagnosis technologies. Current nucleic acid testing (NAT) methods either lack sensitivity or require tedious amplification operations, which could not meet the need for point-of-care (POC) NAT for on-site and community-based diagnosis. Here, we present a fluorescence one-step-bDNA-based lateral flow assay (FOB-LFA) method for amplification-free NAT to realize point-of-care pathogen detection and disease diagnosis. Take COVID-19 as an example, the developed FOB-LFA demonstrated a high sensitivity of 300 copies/mL for the RNA of the SARS-CoV-2 pseudovirus and exhibited high specificity among various homologous pseudoviruses. Further, the result of oropharyngeal swab sample detection suggested the great potential of FOB-LFA in clinical examination. The outstanding performance of FOB-LFA, including high sensitivity, high specificity, low cost, excellent portability, and minimized risk of nucleic acid leakage and contamination, can meet the POC testing demand for the diagnosis of various infectious and genetic diseases.

DNA branched junctions induced the enhanced fluorescence recovery of FAM-labeled probes on rGO for detecting Pb2.

The reduced graphene oxide (rGO) could strongly adsorb and quench the fluorescence of dye-labeled single-stranded DNA (ssDNA); thus, it is widely applied in fluorescent sensors. However, these sensors may suffer from a limited sensitivity due to the low fluorescence recovery when adding the complementary DNA (cDNA) sequence. In this work, the powerful DNA branched junctions were constructed to improve the fluorescence recovery of FAM-labeled probe on rGO. In the presence of target Pb2+, the ribonucleotide (rA) in the substrate was cleaved specifically and the catalytic hairpin assembly of three metastable hairpins was further initiated, accompanied by the formation of DNA branched junctions. Then, the liberated Pb2+ could be recyclable. Impressively, the DNA branched junctions not only hybridize with the FAM-labeled probes with a high efficiency, but also are significantly undesirable for the rGO. Thus, a high fluorescence recovery of FAM-labeled probe on rGO was expected. The integration of the high fluorescence recovery and dual-cycle signal amplification endows the sensing strategy with a good performance for Pb2+ detection, including low detection limit (0.17 nM), good selectivity, and satisfactory practical applicability. The proposed DNA branched junctions offer a novel avenue to improve the fluorescence recovery of the dye-labeled probes on rGO for biological analysis.

A Novel Branched DNA-Based Flowcytometric Method for Single-Cell Characterization of Gene Therapy Products and Expression of Therapeutic Genes.

Many preclinical and clinical studies of hematopoietic stem cell-based gene therapy (GT) are based on the use of lentiviruses as the vector of choice. Assessment of the vector titer and transduction efficiency of the cell product is critical for these studies. Efficacy and safety of the modified cell product are commonly determined by assessing the vector copy number (VCN) using qPCR. However, this optimized and well-established method in the GT field is based on bulk population averages, which can lead to misinterpretation of the actual VCN per transduced cell. Therefore, we introduce here a single cell-based method that allows to unmask cellular heterogeneity in the GT product, even when antibodies are not available. We use Invitrogen's flow cytometry-based PrimeFlow™ RNA Assay with customized probes to determine transduction efficiency of transgenes of interest, promoter strength, and the cellular heterogeneity of murine and human stem cells. The assay has good specificity and sensitivity to detect the transgenes, as shown by the high correlations between PrimeFlow™-positive cells and the VCN. Differences in promoter strengths can readily be detected by differences in percentages and fluorescence intensity. Hence, we show a customizable method that allows to determine the number of transduced cells and the actual VCN per transduced cell in a GT product. The assay is suitable for all therapeutic genes for which antibodies are not available or too cumbersome for routine flow cytometry. The method also allows co-staining of surface markers to analyze differential transduction efficiencies in subpopulations of target cells.

Extensive Expression Analysis of Htt Transcripts in Brain Regions from the zQ175 HD Mouse Model Using a QuantiGene Multiplex Assay.

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by a CAG repeat expansion within exon 1 of the huntingtin (HTT) gene. HTT mRNA contains 67 exons and does not always splice between exon 1 and exon 2 leading to the production of a small polyadenylated HTTexon1 transcript, and the full-length HTT mRNA has three 3'UTR isoforms. We have developed a QuantiGene multiplex panel for the simultaneous detection of all of these mouse Htt transcripts directly from tissue lysates and demonstrate that this can replace the more work-intensive Taqman qPCR assays. We have applied this to the analysis of brain regions from the zQ175 HD mouse model and wild type littermates at two months of age. We show that the incomplete splicing of Htt occurs throughout the brain and confirm that this originates from the mutant and not endogenous Htt allele. Given that HTTexon1 encodes the highly pathogenic exon 1 HTT protein, it is essential that the levels of all Htt transcripts can be monitored when evaluating HTT lowering approaches. Our QuantiGene panel will allow the rapid comparative assessment of all Htt transcripts in cell lysates and mouse tissues without the need to first extract RNA.

Dual coding potential of a 2',5'-branched ribonucleotide in DNA.

Branchpoints in RNA templates are highly mutagenic, but it is not known yet whether this also applies to branchpoints in DNA templates. Here, we report how nucleic acid polymerases replicate a 2',5'-branched DNA (bDNA) molecule. We constructed long-chained bDNA templates containing a branch guanosine and T7 promoters at both arms by splinted ligation. Quantitative real-time PCR analysis was used to investigate whether a branchpoint blocks DNA synthesis from the two arms in the same manner. We find that the blocking effect of a branchpoint is arm-specific. DNA synthesis from the 2'-arm is more than 20,000-fold decreased, whereas from the 3'-arm only 15-fold. Our sequence analysis of full-length nucleic acid generated by Taq DNA polymerase, Moloney murine leukemia virus reverse transcriptase, and T7 RNA polymerase from the 2'-arm of bDNA shows that the branched guanine has a dual coding potential and can base-pair with cytosine and guanine. We find that branchpoint templating is influenced by the type of the surrounding nucleic acid and is probably modulated by polymerase and RNase H active sites. We show that the branchpoint bypass by the polymerases from the 3'-arm of bDNA is predominantly error-free, indicating that bDNA is not as highly mutagenic as 2',5'-branched RNA.

Understanding Signal and Background in a Thermally Resolved, Single-Branched DNA Assay Using Square Wave Voltammetry.

Electrochemical bioanalytical sensors with oligonucleotide transducer molecules have been recently extended for quantifying a wide range of biomolecules, from small drugs to large proteins. Short DNA or RNA strands have gained attention recently due to the existence of circulating oligonucleotides in human blood, yet challenges remain for adequately sensing these targets at electrode surfaces. In this work, we have developed a quantitative electrochemical method which uses target-induced proximity of a single-branched DNA structure to drive hybridization at an electrode surface, with readout by square-wave voltammetry (SWV). Using custom instrumentation, we first show that precise control of temperature can provide both electrochemical signal amplification and background signal depreciation in SWV readout of small oligonucleotides. Next, we thoroughly compared 25 different combinations of binding energies by their signal-to-background ratios and differences. These data served as a guide to select the optimal parameters of binding energy, SWV frequency, and assay temperature. Finally, the influence of experimental workflow on the sensitivity and limit of detection (LOD) of the sensor is demonstrated. This study highlights the importance of precisely controlling temperature and SWV frequency in DNA-driven assays on electrode surfaces while also presenting a novel instrumental design for fine-tuning of such systems.

Novel Assay to Detect RNA Polymerase I Activity In Vivo.

This report develops an analytically validated chromogenic in situ hybridization (CISH) assay using branched DNA signal amplification (RNAscope) for detecting the expression of the 5' external transcribed spacer (ETS) of the 45S ribosomal (r) RNA precursor in formalin-fixed and paraffin-embedded (FFPE) human tissues. 5'ETS/45S CISH was performed on standard clinical specimens and tissue microarrays (TMA) from untreated prostate carcinomas, high-grade prostatic intraepithelial neoplasia (PIN), and matched benign prostatic tissues. Signals were quantified using image analysis software. The 5'ETS rRNA signal was restricted to the nucleolus. The signal was markedly attenuated in cell lines and in prostate tissue slices after pharmacologic inhibition of RNA polymerase I (Pol I) using BMH-21 or actinomycin D, and by RNAi depletion of Pol I, demonstrating validity as a measure of Pol I activity. Clinical human prostate FFPE tissue sections and TMAs showed a marked increase in the signal in the presumptive precursor lesion (high-grade PIN) and invasive adenocarcinoma lesions (P = 0.0001 and P = 0.0001, respectively) compared with non-neoplastic luminal epithelium. The increase in 5'ETS rRNA signal was present throughout all Gleason scores and pathologic stages at radical prostatectomy, with no marked difference among these. This precursor rRNA assay has potential utility for detection of increased rRNA production in various tumor types and as a novel companion diagnostic for clinical trials involving Pol I inhibition.Implications: Increased rRNA production, a possible therapeutic target for multiple cancers, can be detected with a new, validated assay that also serves as a pharmacodynamic marker for Pol I inhibitors. Mol Cancer Res; 15(5); 577-84. ©2017 AACR.

Hormone Receptor Expression Analyses in Neoplastic and Non-Neoplastic Canine Mammary Tissue by a Bead Based Multiplex Branched DNA Assay: A Gene Expression Study in Fresh Frozen and Formalin-Fixed, Paraffin-Embedded Samples.

Immunohistochemistry (IHC) is currently considered the method of choice for steroid hormone receptor status evaluation in human breast cancer and, therefore, it is commonly utilized for assessing canine mammary tumors. In case of low hormone receptor expression, IHC is limited and thus is complemented by molecular analyses. In the present study, a multiplex bDNA assay was evaluated as a method for hormone receptor gene expression detection in canine mammary tissues. Estrogen receptor (ESR1), progesterone receptor (PGR), prolactin receptor (PRLR) and growth hormone receptor (GHR) gene expressions were evaluated in neoplastic and non-neoplastic canine mammary tissues. A set of 119 fresh frozen and 180 formalin-fixed, paraffin-embedded (FFPE) was comparatively analyzed and used for assay evaluation. Furthermore, a possible association between the hormone receptor expression in different histological subtypes of canine malignant mammary tumors and the castration status, breed and invasive growth of the tumor were analyzed. The multiplex bDNA assay proved to be more sensitive for fresh frozen specimens. Hormone receptor expression found was significantly decreased in malignant mammary tumors in comparison to non-neoplastic tissue and benign mammary tumors. Among the histological subtypes the lowest gene expression levels of ESR1, PGR and PRLR were found in solid, anaplastic and ductal carcinomas. In summary, the evaluation showed that the measurement of hormone receptors with the multiplex bDNA assay represents a practicable method for obtaining detailed quantitative information about gene expression in canine mammary tissue for future studies. Still, comparison with IHC or quantitative real-time PCR is needed for further validation of the present method.

Non-isotopic Method for In Situ LncRNA Visualization and Quantitation.

In mammals and other eukaryotes, most of the genome is transcribed in a developmentally regulated manner to produce large numbers of long noncoding RNAs (lncRNAs). Genome-wide studies have identified thousands of lncRNAs lacking protein-coding capacity. RNA in situ hybridization technique is especially beneficial for the visualization of RNA (mRNA and lncRNA) expression in a heterogeneous population of cells/tissues; however its utility has been hampered by complicated procedures typically developed and optimized for the detection of a specific gene and therefore not amenable to a wide variety of genes and tissues.Recently, bDNA has revolutionized RNA in situ detection with fully optimized, robust assays for the detection of any mRNA and lncRNA targets in formalin-fixed paraffin-embedded (FFPE) and fresh frozen tissue sections using manual processing.

Establishment of a new quality control and vaccine safety test for influenza vaccines and adjuvants using gene expression profiling.

We have previously identified 17 biomarker genes which were upregulated by whole virion influenza vaccines, and reported that gene expression profiles of these biomarker genes had a good correlation with conventional animal safety tests checking body weight and leukocyte counts. In this study, we have shown that conventional animal tests showed varied and no dose-dependent results in serially diluted bulk materials of influenza HA vaccines. In contrast, dose dependency was clearly shown in the expression profiles of biomarker genes, demonstrating higher sensitivity of gene expression analysis than the current animal safety tests of influenza vaccines. The introduction of branched DNA based-concurrent expression analysis could simplify the complexity of multiple gene expression approach, and could shorten the test period from 7 days to 3 days. Furthermore, upregulation of 10 genes, Zbp1, Mx2, Irf7, Lgals9, Ifi47, Tapbp, Timp1, Trafd1, Psmb9, and Tap2, was seen upon virosomal-adjuvanted vaccine treatment, indicating that these biomarkers could be useful for the safety control of virosomal-adjuvanted vaccines. In summary, profiling biomarker gene expression could be a useful, rapid, and highly sensitive method of animal safety testing compared with conventional methods, and could be used to evaluate the safety of various types of influenza vaccines, including adjuvanted vaccine.

Effect of nine diets on xenobiotic transporters in livers of mice.

1. Lifestyle diseases are often caused by inappropriate nutrition habits and attempted to be treated by polypharmacotherapy. Therefore, it is important to determine whether differences in diet affect the disposition of drugs. Xenobiotic transporters in the liver are essential in drug disposition. 2. In the current study, mice were fed one of nine diets for 3 weeks. The mRNAs of 23 known xenobiotic transporters in livers of mice were quantified by microarray analysis, and validated by branched DNA assay. The mRNAs of 15 transporters were altered by at least one diet. Diet-restriction (10) and the atherogenic diet (10) altered the expression of the most number of transporters, followed by western diet (8), high-fat diet (4), lab chow (2), high-fructose diet (2) and EFA-deficient diet (2), whereas the low n-3 FA diet had no effect on these transporters. Seven of the 11 xenobiotic transporters in the Slc family, three of four in the Abcb family, two of four in the Abcc family and all three in the Abcg family were changed significantly. 3. This first comprehensive study indicates that xenobiotic transporters are altered by diet, and suggests there are likely diet-drug interactions due to changes in the expression of drug transporters.

Real-time detection of H5N1 influenza virus through hyperbranched rolling circle amplification.

An isothermal amplification method was developed for the sensitive detection of the H5N1 influenza virus. The padlock probe specifically bound to the H5N1 target and circularized with T4 DNA ligase enzyme. Then this circular probe was amplified by hyperbranched rolling circle amplification (HRCA) using Phi29 DNA polymerase. The fluorescence intensity was recorded at different intervals by intercalation of SYBR green molecules into the double-stranded product of the HRCA reaction. At an optimum time of 88 min, a calibration plot with fine linearity was obtained. Using HRCA based on a padlock probe and Phi29 DNA polymerase, high selectivity and sensitivity were achieved. The biosensor response was linear toward H5N1 in the concentration range from 10 fM to 0.25 pM, with a detection limit of 9 fM at a signal/noise ratio of 3. By replacing the heat shock with pH shock, not only was the procedure for detection of H5N1 influenza simplified, but also the DNA molecules were protected from possible breaking at high temperature.

Intracranial meningiomas, the VEGF-A pathway, and peritumoral brain oedema.

Meningiomas are the second-most common intracranial tumours in adults. They are derived from the arachnoid cells, and although approximately 90% of meningiomas are benign, more than half of all meningiomas develop peritumoral brain oedema (PTBE), which increases morbidity. The PTBE can be treated with steroid therapy, but this treatment is not specific, is not always effective, and involves long-term side-effects. Meningiomas are treated with radiation therapy, stereotactic radio-surgery or surgical resection. At the moment surgical resection is the only definite treatment, and the removal of the tumour also removes the PTBE. Based on the localization of the meningioma, surgery can be complicated. Although PTBE around meningiomas is frequent, the mechanisms behind its development are not clearly understood. It is believed that due to tumour growth and local tissue hypoxia, angiogenesis is increased and leads to the formation of PTBE. The angiogenic protein vascular endothelial growth factor A (VEGF-A) is believed to be involved in the formation of PTBE around meningiomas, as several studies have found that it is increased in meningiomas with PTBE. VEGF-A is also known as vascular permeability factor due to its ability to increase the permeability of capillaries. Paper I examines the VEGF-A protein and mRNA levels in 101 intracranial meningiomas. The PTBE is quantified on MRI, and capillary length and tumour water content are measured and compared to control brain tissue. Possible co-factors to PTBE like meningioma localization and subtypes are also examined. Forty-three of the patients have primary, solitary, supratentorial meningiomas with PTBE. The correlation between PTBE or edema index with the VEGF-A protein and mRNA, capillary length, and tumour water content is investigated in these patients. A novel method is used for mRNA quantification. It involves direct amplification of the mRNA with probes and branched DNA in order to produce a chemiluminescence signal that can be measured using a luminometer. The paper shows that the oedema index is correlated to the VEGF-A protein and mRNA, and that capillary length is correlated to the PTBE. It also finds that VEGF-A protein and mRNA, capillary length and water content is increased in meningiomas compared to control tissue, suggesting that VEGF-A is produced in, and possibly secreted from the meningiomas. In addition, supratentorial meningiomas are shown to have larger PTBE compared to infratentorial meningiomas, suggesting that infratentorial meningiomas are diagnosed and removed earlier, due to earlier symptom development based on the anatomical features of the fossa posterior. Finally, a gender-specific difference in tumour water content and VEGF-A protein is revealed (higher and lower in females, respectively). Paper II is a method-comparison study pitting the chemiluminescence assay against the often used quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) assay. In RT-qPCR, RNA is isolated, measured, reverse transcribed, purified, amplified via real-time PCR, and analyzed. The method is robust and reliable, albeit laborious to some extent. The chemiluminescence assay detects RNA directly without the need for RNA purification, complement DNA synthesis or cyclic amplification. By comparing the output of the two protocols to a dilution series ranging from 1 to 128 times of the homogenized samples, the precision of the protocols is measured. Furthermore, VEGF-A/GAPDH ratios are quantified for 15 tissue samples and the results compared between the two protocols, showing significant correlation. The study finds that the chemiluminescence assay is competitive to RT-qPCR, and reflects a similar pattern in gene expression measurement with a similar precision. Whether one method or the other should be used depends on the variability of the samples, budget, and time. RT-qPCR has a much wider dynamic range, and is preferable in case of significant sample inter-variability. It is also less expensive, and gives the user more flexibility as homemade reagents can be used. On the other hand, the chemiluminescence assay is straight forward, requires less hands-on-time, and can be used on formalin-fixed and paraffin-embedded (FFPE) tissue. Paper III continues the investigations in paper I. The sample size is increased so that 22 angiomatous and secretory meningiomas are compared to 40 non-angiomatous meningiomas and 10 control brain tissue samples. Angiomatous and secretory meningiomas are chosen because they are known to have larger PTBE compared to other meningiomas. In addition to VEGF-A, capillary length, and PTBE, the VEGF-A tyrosine kinase receptor VEGFR-2 mRNA and protein levels are also examined. VEGFR-2 is a transmembrane receptor found on endothelial cells. It binds VEGF-A and thereby increases angiogenesis. VEGFR-2's co-receptor neuropilin-1 is also examined. Neuropilin-1 is an agonist of angiogenesis through complex-binding of VEGF-A, but it can also work as an inhibitor through competitive binding of semaphorin-3A. The complex binding of semaphorin-3A to neuropilin-1 can also induce endothelial cell apoptosis, thus working as an antagonist of angiogenesis. The study finds that VEGF-A mRNA, VEGF-A protein, and neuropilin-1 mRNA are higher in angiomatous and non-angiomatous meningiomas compared to controls. VEGFR-2 protein is higher, and neuropilin-1 protein lower in angiomatous meningiomas compared to controls. The mean capillary length is 3614 mm/mm3 in angiomatous, 605 mm/mm3 in non-angiomatous meningiomas, and 229 mm/mm3 in the controls. Non-angiomatous and angiomatous meningioma patients have equally sized tumours. The mean PTBE around the angiomatous meningiomas is 695 cm3, i.e. 477 cm3 larger than the non-angiomatous meningiomas (p = 0.0045), and the mean oedema index is twice the size compared to the non-angiomatous meningiomas. Further comparison between the two meningioma groups shows that mean VEGF-A mRNA, VEGFR-2 protein, and neuropilin-1 mRNA is significantly higher and neuropilin-1 protein is lower in the angiomatous meningiomas. We believe that the VEGF-A pathway participates in the formation of PTBE in meningiomas by inducing formation of "leaky" capillaries, resulting in secretion of VEGF-A and plasma to the peritumoural brain tissue. It may therefore be worth pursuing therapies targeted directly against VEGF-A and its receptors through drugs like bevacizumab, sorafenib, sunitifib, and cediranib.

Single-molecule fluorescence in situ hybridization: quantitative imaging of single RNA molecules.

In situ detection of RNAs is becoming increasingly important for analysis of gene expression within and between intact cells in tissues. International genomics efforts are now cataloging patterns of RNA transcription that play roles in cell function, differentiation, and disease formation, and they are demonstrating the importance of coding and noncoding RNA transcripts in these processes. However, these techniques typically provide ensemble averages of transcription across many cells. In situ hybridization-based analysis methods complement these studies by providing information about how expression levels change between cells within normal and diseased tissues, and they provide information about the localization of transcripts within cells, which is important in understanding mechanisms of gene regulation. Multi-color, single-molecule fluorescence in situ hybridization (smFISH) is particularly useful since it enables analysis of several different transcripts simultaneously. Combining smFISH with immunofluorescent protein detection provides additional information about the association between transcription level, cellular localization, and protein expression in individual cells.

Development and evaluation of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Actinobacillus pleuropneumoniae based the dsbE-like gene

This paper reports on the development and validation of a loop-mediated isothermal amplification assay (LAMP) for the rapid and specific detection of Actinobacillus pleuropneumoniae (A. pleuropneumoniae). A set of six primers were designed derived from the dsbE-like gene of A.pleuropneumoniae and validate the assay using 9 A. pleuropneumoniae reference/field strains, 132 clinical isolates and 9 other pathogens. The results indicated that positive reactions were confirmed for all A. pleuropneumoniae strains and specimens by LAMP at 63ºC for 60 min and no cross-reactivity were observed from other non-A.pleuropneumoniae including Haemophilus parasuis, Escherichia coli, Pasteurella multocida, Bordetella bronchiseptica, Streptococcus suis, Salmonella enterica, Staphylococcus, porcine reproductive and respiratory syndrome virus (PRRSV), and Pseudorabies virus. The detection limit of the conventional PCR was 10² CFU per PCR test tube, while that of the LAMP was 5 copies per tube. Therefore, the sensitivity of LAMP was higher than that of PCR. Moreover, the LAMP assay provided a rapid yet simple test of A. pleuropneumoniae suitable for laboratory diagnosis and pen-side detection due to ease of operation and the requirement of only a regular water bath or heat block for the reaction.

A facile, branched DNA assay to quantitatively measure glucocorticoid receptor auto-regulation in T-cell acute lymphoblastic leukemia.

Glucocorticoid (GC) steroid hormones are used to treat acute lymphoblastic leukemia (ALL) because of their pro-apoptotic effects in hematopoietic cells. However, not all leukemia cells are sensitive to GC, and no assay to stratify patients is available. In the GC-sensitive T-cell ALL cell line CEM-C7, auto-up-regulation of RNA transcripts for the glucocorticoid receptor (GR) correlates with increased apoptotic response. This study aimed to determine if a facile assay of GR transcript levels might be promising for stratifying ALL patients into hormone-sensitive and hormone-resistant populations. The GR transcript profiles of various lymphoid cell lines and 4 bone marrow samples from patients with T-cell ALL were analyzed using both an optimized branched DNA (bDNA) assay and a real-time quantitative reverse transcription-polymerase chain reaction assay. There were significant correlations between both assay platforms when measuring total GR (exon 5/6) transcripts in various cell lines and patient samples, but not for a probe set that detects a specific, low abundance GR transcript (exon 1A3). Our results suggest that the bDNA platform is reproducible and precise when measuring total GR transcripts and, with further development, may ultimately offer a simple clinical assay to aid in the prediction of GC-sensitivity in ALL patients.

A sensitive branched DNA HIV-1 signal amplification viral load assay with single day turnaround.

Branched DNA (bDNA) is a signal amplification technology used in clinical and research laboratories to quantitatively detect nucleic acids. An overnight incubation is a significant drawback of highly sensitive bDNA assays. The VERSANT® HIV-1 RNA 3.0 Assay (bDNA) ("Versant Assay") currently used in clinical laboratories was modified to allow shorter target incubation, enabling the viral load assay to be run in a single day. To dramatically reduce the target incubation from 16-18 h to 2.5 h, composition of only the "Lysis Diluent" solution was modified. Nucleic acid probes in the assay were unchanged. Performance of the modified assay (assay in development; not commercially available) was evaluated and compared to the Versant Assay. Dilution series replicates (>950 results) were used to demonstrate that analytical sensitivity, linearity, accuracy, and precision for the shorter modified assay are comparable to the Versant Assay. HIV RNA-positive clinical specimens (n = 135) showed no significant difference in quantification between the modified assay and the Versant Assay. Equivalent relative quantification of samples of eight genotypes was demonstrated for the two assays. Elevated levels of several potentially interfering endogenous substances had no effect on quantification or specificity of the modified assay. The modified assay with drastically improved turnaround time demonstrates the viability of signal-amplifying technology, such as bDNA, as an alternative to the PCR-based assays dominating viral load monitoring in clinical laboratories. Highly sensitive bDNA assays with a single day turnaround may be ideal for laboratories with especially stringent cost, contamination, or reliability requirements.

A facile, branched DNA assay to quantitatively measure glucocorticoid receptor auto-regulation in T-cell acute lymphoblastic leukemia / 癌症

Glucocorticoid (GC) steroid hormones are used to treat acute lymphoblastic leukemia (ALL) because of their pro-apoptotic effects in hematopoietic cells. However, not all leukemia cells are sensitive to GC, and no assay to stratify patients is available. In the GC-sensitive T-cell ALL cell line CEM-C7, auto-up-regulation of RNA transcripts for the glucocorticoid receptor (GR) correlates with increased apoptotic response. This study aimed to determine if a facile assay of GR transcript levels might be promising for stratifying ALL patients into hormone-sensitive and hormone-resistant populations. The GR transcript profiles of various lymphoid cell lines and 4 bone marrow samples from patients with T-cell ALL were analyzed using both an optimized branched DNA (bDNA) assay and a real-time quantitative reverse transcription-polymerase chain reaction assay. There were significant correlations between both assay platforms when measuring total GR (exon 5/6) transcripts in various cell lines and patient samples, but not for a probe set that detects a specific, low abundance GR transcript (exon 1A3). Our results suggest that the bDNA platform is reproducible and precise when measuring total GR transcripts and, with further development, may ultimately offer a simple clinical assay to aid in the prediction of GC-sensitivity in ALL patients.

[The application of xMAP technology in genetic typing of measles virus].

The genetic typing of measles virus in clinical samples using xMAP technology was applied. The study provided the calculation and application of specific oligonucleotide probes of genotypes D4, D6 and D7 of measles virus. The strain HobO96 genotype A of measles virus as a check sample was used. The technical approaches to the optimization of preparatory work organization and to the process of identification of measles virus genotypes are described. The presence of genotypes D4, D6 and D7 in clinical samples is proved by the sequence analysis. The genetic typing effectiveness of technique of DNA hybridization using xMAP technology on the instrumental base BioPlex (BioRad, USA) is demonstrated.