Molecular probing of TNF: From identification of therapeutic target to guidance of therapy in inflammatory diseases.

Therapy by blocking tumor necrosis factor (TNF) activity is highly efficacious and profoundly changed the paradigm of several inflammatory diseases. However, a significant proportion of patients with inflammatory diseases do not respond to TNF inhibitors (TNFi). Prediction of therapeutic response is required for TNFi therapy. Isotope labeled anti-TNF antibodies or TNF receptor have been investigated to localize TNF production at inflammatory tissue in animal models and in patients with inflammatory diseases. The in vivo detection of TNF has been associated with treatment response. Recently, fluorophore labeled anti-TNF antibody in combination with confocal laser endomicroscopy in patients with Crohn's disease yielded more accurate and quantitative in vivo detection of TNF in the diseased mucosa. More importantly, this method demonstrated high therapeutic predication value. Fluorophore labeled TNF binding aptamers in combination with modern imaging technology offers additional tools for in vivo TNF probing.

Informative or noninformative calls for gene expression: a latent variable approach.

The strength and weakness of microarray technology can be attributed to the enormous amount of information it is generating. To fully enhance the benefit of microarray technology for testing differentially expressed genes and classification, there is a need to minimize the amount of irrelevant genes present in microarray data. A major interest is to use probe-level data to call genes informative or noninformative based on the trade-off between the array-to-array variability and the measurement error. Existing works in this direction include filtering likely uninformative sets of hybridization (FLUSH; Calza et al., 2007) and I/NI calls for the exclusion of noninformative genes using FARMS (I/NI calls; Talloen et al., 2007; Hochreiter et al., 2006). In this paper, we propose a linear mixed model as a more flexible method that performs equally good as I/NI calls and outperforms FLUSH. We also introduce other criteria for gene filtering, such as, R2 and intra-cluster correlation. Additionally, we include some objective criteria based on likelihood ratio testing, the Akaike information criteria (AIC; Akaike, 1973) and the Bayesian information criterion (BIC; Schwarz, 1978 ). Based on the HGU-133A Spiked-in data set, it is shown that the linear mixed model approach outperforms FLUSH, a method that filters genes based on a quantile regression. The linear model is equivalent to a factor analysis model when either the factor loadings are set to a constant with the variance of the latent factor equal to one, or if the factor loadings are set to one together with unconstrained variance of the latent factor. Filtering based on conditional variance calls a probe set informative when the intensity of one or more probes is consistent across the arrays, while filtering using R2 or intra-cluster correlation calls a probe set informative only when average intensity of a probe set is consistent across the arrays. Filtering based on likelihood ratio test AIC and BIC are less stringent compared to the other criteria.

Analytical sensitivity and efficiency comparisons of SARS-CoV-2 RT-qPCR primer-probe sets.

The recent spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exemplifies the critical need for accurate and rapid diagnostic assays to prompt clinical and public health interventions. Currently, several quantitative reverse transcription-PCR (RT-qPCR) assays are being used by clinical, research and public health laboratories. However, it is currently unclear whether results from different tests are comparable. Our goal was to make independent evaluations of primer-probe sets used in four common SARS-CoV-2 diagnostic assays. From our comparisons of RT-qPCR analytical efficiency and sensitivity, we show that all primer-probe sets can be used to detect SARS-CoV-2 at 500 viral RNA copies per reaction. The exception for this is the RdRp-SARSr (Charité) confirmatory primer-probe set which has low sensitivity, probably due to a mismatch to circulating SARS-CoV-2 in the reverse primer. We did not find evidence for background amplification with pre-COVID-19 samples or recent SARS-CoV-2 evolution decreasing sensitivity. Our recommendation for SARS-CoV-2 diagnostic testing is to select an assay with high sensitivity and that is regionally used, to ease comparability between outcomes.

Probe-specific mixed-model approach to detect copy number differences using multiplex ligation-dependent probe amplification (MLPA).

BACKGROUND: MLPA method is a potentially useful semi-quantitative method to detect copy number alterations in targeted regions. In this paper, we propose a method for the normalization procedure based on a non-linear mixed-model, as well as a new approach for determining the statistical significance of altered probes based on linear mixed-model. This method establishes a threshold by using different tolerance intervals that accommodates the specific random error variability observed in each test sample. RESULTS: Through simulation studies we have shown that our proposed method outperforms two existing methods that are based on simple threshold rules or iterative regression. We have illustrated the method using a controlled MLPA assay in which targeted regions are variable in copy number in individuals suffering from different disorders such as Prader-Willi, DiGeorge or Autism showing the best performace. CONCLUSION: Using the proposed mixed-model, we are able to determine thresholds to decide whether a region is altered. These threholds are specific for each individual, incorporating experimental variability, resulting in improved sensitivity and specificity as the examples with real data have revealed.

A statistical framework for consolidating "sibling" probe sets for Affymetrix GeneChip data.

BACKGROUND: Affymetrix GeneChip typically contains multiple probe sets per gene, defined as sibling probe sets in this study. These probe sets may or may not behave similar across treatments. The most appropriate way of consolidating sibling probe sets suitable for analysis is an open problem. We propose the Analysis of Variance (ANOVA) framework to decide which sibling probe sets can be consolidated. RESULTS: The ANOVA model allows us to separate the sibling probe sets into two types: those behave similarly across treatments and those behave differently across treatments. We found that consolidation of sibling probe sets of the former type results in large increase in the number of differentially expressed genes under various statistical criteria. The approach to selecting sibling probe sets suitable for consolidating is implemented in R language and freely available from http://research.stowers-institute.org/hul/affy/. CONCLUSION: Our ANOVA analysis of sibling probe sets provides a statistical framework for selecting sibling probe sets for consolidation. Consolidating sibling probe sets by pooling data from each greatly improves the estimates of a gene expression level and results in identification of more biologically relevant genes. Sibling probe sets that do not qualify for consolidation may represent annotation errors or other artifacts, or may correspond to differentially processed transcripts of the same gene that require further analysis.

Detection of Xanthomonas oryzae pv. oryzae in seeds using a specific TaqMan probe.

Xanthomonas oryzae pv. oryzae is the pathogen that causes bacterial leaf blight in rice. Bacterial leaf blight is the main cause for severe rice underproduction in many countries. However, with conventional methods it is difficult to quickly and reliably distinguish this pathogen from other closely related pathogenic bacteria, especially X. oryzae pv. oryzicola, the causal organism of bacterial leaf streak in rice. We have developed a novel and highly sensitive real-time method for the identification of this specific bacteria based on a TaqMan probe. This probe is designed to recognize the sequence of a putative siderophore receptor gene cds specific to X. oryzae pv. oryzae, and can be identified from either a bacterial culture or naturally infected rice seeds and leaves in only 2 h. The sensitivity of the method is 100 times higher than that of the current polymerase chain reaction (PCR) gel electrophoresis method for diagnosis.

Multiplex ligation-dependent probe amplification for the detection of chromosomal gains and losses in formalin-fixed tissue.

Molecular analysis on formalin-fixed paraffin-embedded tissue is of increasing importance in diagnostic histopathology and tumor research. Multiplex ligation-dependent probe amplification (MLPA) is a technique that can be used for detection of copy number alterations of up to 45 different DNA sequences in one experiment. It can be performed on partially degraded DNA, which makes this technique very suitable for analysis of formalin-fixed lesions. We tested the reliability of MLPA by analyzing DNA isolated from formalin-fixed melanomas that were previously characterized by comparative genomic hybridization (CGH), and additionally the applicability of MLPA was tested by analyzing 29 routinely processed melanocytic lesions. MLPA appears to be a reliable and efficient method to evaluate DNA copy number changes as 86% of the loci tested revealed concordant CGH results. Discordance mainly involved alterations that were detected by MLPA and not by CGH probably due to a combination of lower resolution of CGH and occasionally false positive MLPA results. For application of MLPA in a diagnostic setting, different probes on a specific region of interest should be used to prevent false positive MLPA results. In a research setting as well as in a diagnostic setting, MLPA is a fast technique to screen large numbers of formalin-fixed lesions for DNA gains and losses.

Exonuclease cycling assay: an amplified assay for the detection of specific DNA sequences.

An assay is described in which an oligonucleotide probe is specifically digested by lambda exonuclease only when it is annealed to its complementary sequence. In this assay, a cycling effect occurs whereby a small amount of target sequence acts as a specific co-factor in the enzymatic degradation of a larger number of molecules of an oligonucleotide probe. This amplification principle is demonstrated and the effect of the oligonucleotide probe sequence investigated. The necessary steps needed to convert this effect into a useful diagnostic tool are discussed.

Storage phosphor imaging technique improves the accuracy of RNA quantitation using 32P-labeled cDNA probes.

The use of the storage phosphor imaging technique to quantitate radioactivity on blots generated by hybridization with 32P-cDNA probes was evaluated and compared with screen-enhanced x-ray film autoradiography. Quantitation of RNA dot blots hybridized with a 28S ribosomal RNA-specific cDNA probe showed that storage phosphor imaging was more sensitive than screen-enhanced x-ray film autoradiography in identifying low amounts of total RNA (1-10 micrograms). Evaluation of Northern blots containing 30 micrograms of total RNA from human skin biopsies hybridized with a cDNA probe for the human acidic ribosomal phosphoprotein, PO, showed that both techniques detected random biological variability of this housekeeping gene in a similar manner. The two techniques exhibited a strong linear correlation in their ability to quantitate mRNA levels of a retinoic acid-inducible gene (RIS-1). This correlation was stronger at levels corresponding to 1-fold to 30-fold increases of signal and decreased beyond this range because of the insensitivity of the x-ray film. In conclusion, storage phosphor imaging is more accurate than screen-enhanced x-ray film autoradiography in identifying different RNA amounts (higher sensitivity) and in detecting increasing RNA signals at high levels of radioactivity (higher dynamic range).

Mixed chimerism after sex-mismatched allogeneic BMT: evaluation of two molecular techniques.

Two different molecular techniques were used to monitor chimerism following 17 non-T cell-depleted BMTs from female donors to male recipients: pHY10, a Y chromosome-specific probe (Southern or slot blots), and a set of primers for Y chromosome sequence-specific amplification by the polymerase chain reaction (PCR). On Southern blots, male DNA was detectable at a level less than 1% of 10 micrograms DNA while cross-reactivity with autosomal sequences was avoided. On slot blots, male DNA was reliably detectable at levels less than 0.5%, even in small sample (0.5 microgram DNA). With the PCR technique, male DNA was detectable at levels of 1:10(6) to 1:10(7) of 0.5 microgram DNA. Slot blot and PCR results were concordant in 19 of 23 samples. Both techniques demonstrated a constant small mixed chimerism during the first year after BMT and in four of nine patients, this chimerism persisted even longer (up to 29 months after BMT).

DNA copy number analysis by MAPH: molecular diagnostic applications.

DNA copy number variation is an important cause of genetic disease. There are several techniques available to detect copy number changes of various sizes, each with their limitations in resolution and cost. Here we outline the development of multiplex amplifiable probe hybridization (MAPH) into a high-throughput diagnostic technique for detecting copy number variation of almost any size. Its application in testing for genetic mutations causing diseases, such as familial breast cancer, Charcot-Marie-Tooth disease Type 1A, Duchenne/Becker muscular dystrophy and familial colorectal cancer is described, as well as its use in identifying chromosomal changes in some individuals with mental retardation. The analysis of the data produced by MAPH is also considered, along with its potential for automation and development of microarray-based MAPH.

Digoxigenin as an alternative probe labeling for in situ hybridization.

In situ hybridization (ISH) techniques have proven to be invaluable tools for diagnostic molecular laboratories in the detection of gene expression and viral infection in cell and tissue samples. Radioactively labeled probes are still widely used for ISH because of their high sensitivity in detection. Among the non-isotopic systems only biotinylated probes for viral DNA detection found broader acceptance. Recently we introduced the digoxigenin probe labeling and detection system for ISH in our diagnostic molecular laboratory. Our experiences with digoxigenin-labeled probes are summarized in this report in the form of technical recommendations for probe choice, labeling, quantification, hybridization and detection. Several ISH protocols using RNA, DNA and oligonucleotide probes for the detection of mRNA and viral DNA are reported. Advantages, disadvantages and pitfalls of the digoxigenin system as well as comparisons with radiolabeled and other non-isotopic systems are discussed. Digoxigenin-labeled probes provide an attractive alternative to radiolabeled probes and are superior to other nonradioactive probes for ISH. Probe labeling, quantification, hybridization and detection can be performed with low biohazard risk. Working efforts and costs applying digoxigenin-labeled probes or radiolabeled probes for ISH are similar. Digoxigenin-labeled probes are stable for several months, provide an equal sensitivity in detection and a higher degree of cellular resolution than radiolabeled probes. The turnaround time of procedures is short and results of diagnostic ISH can be obtained much quicker than using radiolabeled probes.

Identification of Staphylococcus epidermidis using a 16S rRNA-directed oligonucleotide probe.

Staphylococcus epidermidis is the most medically significant of the coagulase-negative staphylococci. An oligonucleotide probe (pSe) for identification of S. epidermidis was defined by comparing the sequences of the 16S rRNA variable region V6 from numerous coagulase-negative staphylococci. In order to increase the sensitivity of the detection, polymerase chain reaction amplification of the variable region with primers based on the conserved flanking sequences was applied. The detection limit of the polymerase chain reaction assay combined with pSe probe was shown to be 1 fg which corresponds to about one single bacterium. Additionally, a sensitive, non-radioisotopic system with chemiluminescence detection was tested.

An evaluation of the BD ProbeTec ET system for the direct detection of Mycobacterium tuberculosis in respiratory samples.

In controlling the spread of tuberculosis, early detection of disease caused by organisms of the Mycobacterium tuberculosis complex (MTBC) is vital. The BD ProbeTec ET system provides a method for the direct detection of MTBC by strand displacement amplification. Two hundred and five respiratory samples from patients with a high probability of tuberculosis were assessed by ProbeTec and by microscopy and culture for mycobacteria. ProbeTec positive results were obtained with 101 of 109 samples from which MTBC organisms were isolated. ProbeTec correctly signalled 78 of 81 samples that gave growths of mycobacteria other than tubercle bacilli (MOTT) as negative. Three samples gave false-positive results, corrected on repeat testing. Positive and negative predictive values (PPV, NPV) were 0.97 and 0.90 and the system showed a sensitivity and specificity of 92.7% and 96.0%, respectively. These values rose to PPV 0.97, NPV 0.96, sensitivity 97.1% and specificity 96.0% when data from the small number of gastric lavage samples tested were removed from the analysis. The BD ProbeTec ET system offers a robust and reliable molecular biological approach to the detection of MTBC organisms in respiratory samples in a semi-automated format.

Highly sensitive digoxigenin-labelled DNA probe for the detection of potato spindle tuber viroid.

A molecular probe pSPAv6.2(+), with concatameric insert representing 6.2-times repeated copy of potato spindle tuber viroid (PSTV) RNA, was labelled with digoxigenin and used to detect PSTV by dot-blot hybridization assay. The probe was highly sensitive and specific, detecting as little as 2.5 pg of PSTV RNA. Both severe and mild PSTV strains were detectable in 64-512-times diluted crude extracts from infected tomato leaves, and potato leaves, sprouts, and seeds. For extraction of plant tissue three buffers were compared to determine the lowest non-specific background and the highest sensitivity. The results showed that the digoxigenin-labelled probe is as sensitive as the 32P-labelled probe and can replace radioactive techniques in PSTV detection. With such high sensitivity, the probe is also potentially useful for detecting the viroid in composite samples of mass-indexing programs.

Evaluation of strand-specific RNA probes visualized by colorimetric and chemiluminescent reactions for the detection of B19 parvovirus DNA.

A dot-blot hybridization assay was developed to detect B19 DNA using strand-specific RNA probes labelled with digoxigenin. The sensitivity of the assays was evaluated either using 'plus' and 'minus' sense RNA probes in two different hybridization assays, or in two successive reactions of the same assay. The hybridized probes were revealed immunoenzymatically using anti-digoxigenin Fab fragments conjugated with alkaline phosphatase. The enzyme was visualized by colorimetric reaction. Since 'minus' sense RNA probe gave the best results in the dot-blot procedures, we increased the sensitivity of the hybridization assay visualizing the 'minus' sense digoxigenin-labelled RNA probe by chemiluminescent reaction. In these experimental conditions up to 20 fg of target B19 DNA could be visualized. In the search for B19 DNA, 4656 serum samples were analyzed by chemiluminescent reaction of 'minus' sense digoxigenin-labelled RNA probe and for comparison with the digoxigenin-labelled DNA probe. Positive results were confirmed by Southern blotting. Out of 4656 serum samples analyzed, 4648 gave negative results, 1 resulted positive to all the hybridization assays, 6 only using RNA probe and 1 only by DNA probe.

Effect of off-site transportation on detection of Neisseria gonorrhoeae in endocervical specimens.

OBJECTIVES: To evaluate both the effect of off-site transportation on detection of Neisseria gonorrhoeae in cultured endocervical specimens and the impact of transportation on viability of N. gonorrhoeae by comparison of culture with a nucleic acid probe assay. DESIGN: Three endocervical swabs were randomly collected; one was tested on-site using a nucleic acid-based assay (PACE 2NG System, Gen-Probe, Inc, San Diego, Calif), one was tested off-site following inoculation to modified Thayer-Martin agar (Remel, Lenexa, Kan), and a third swab was tested on-site by culture isolation. A nucleic acid amplification assay of the original swab for PACE 2NG testing was used to resolve discrepancies. SETTING: The emergency department of a university medical center. PATIENTS: Four hundred two patients were evaluated. The test population consisted of both asymptomatic and symptomatic patients. MAIN OUTCOME MEASURE: Positivity for N. gonorrhoeae by one or more of the test procedures, with discrepancy analysis when warranted. RESULTS: Of 402 specimens evaluated, the sensitivities for on-site and off-site testing using culture isolation for N. gonorrhoeae were 88.9% and 77.8%, respectively, in a population prevalence of 6.7%. However, the sensitivity for on-site PACE 2NG testing for N. gonorrhoeae was 96.3%. CONCLUSIONS: A decrease in sensitivity between on-site and off-site culture was found, which suggested transportation may have an adverse effect on the detection of N gonorrhoeae. However, with the limited population and prevalence, the difference was not found to be statistically significant. Further studies indicated that the nucleic acid probe assay was significantly more sensitive (P = .05) when compared with off-site testing using a culture isolation method, demonstrating that viability is an important consideration. These results suggested that a molecular probe assay should be considered in testing specimens for N. gonorrhoeae, especially when the specimen is to be transported off-site.

Non-similarity combinatorial problems.

Similarity problems intensively investigated in computational molecular biology have the following two stringology models: find the longest string included in any string of a given finite language, and find the shortest string including every string of a given finite language. These two problems are exemplified by the two well-known pairs of problems, the longest common subsequence (or substring) problem and the shortest common supersequence (or superstring) problem, interpretations. In this paper we consider opposite problems connected with string non-inclusion relations: find the shortest string included in no string of a given finite language and find the longest string including no string of a given finite language. The predicate "string alpha is not included in string beta" is interpreted either as "alpha is not a subsequence of beta" or as "alpha is not a substring of beta". The main purpose is to determine the complexity status of the non-similarity problems. Using graph approaches, we present NP-hardness proofs for the first interpretation and polynomial-time algorithms for the second one. Special cases of the problems, and related issues are discussed.

Development and evaluation of a non-isotopically labeled DNA probe for the diagnosis of infectious laryngotracheitis.

A digoxigenin-labeled cloned infectious laryngotracheitis virus (ILTV) DNA fragment was evaluated as a nonradioactive alternative probe in the diagnosis of infectious laryngotracheitis. The dot-blot hybridization protocol was optimized and was capable of detecting 40 pg of purified ILTV DNA and as few as 50 ILTV-infected chicken embryo liver cells. The utility of this approach for diagnostic use was evaluated through four ILTV inoculation trials using a mild field isolate, a virulent challenge strain, a tissue-culture-origin vaccine, and an egg-origin vaccine. Birds were examined for clinical signs of ILT, and conjunctival and pharyngeal swabs from inoculated and sentinel birds were tested for ILTV by the digoxigenin-labeled probe and by virus isolation. In general, higher numbers of ILTV-positive samples were detected by both assays from conjunctival swabs. For the non-vaccine strains, detection by dot-blot hybridization was equivalent to that for virus isolation. However, for the two vaccine strains, there was some lack of correlation between the dot-blot results and the virus-isolation results. The kappa values between virus-isolation results and dot-blot results for the tissue-culture-origin vaccine, egg-origin vaccine, Ont 1598 field isolate, and virulent strain were 0.00, 0.16, 0.39, and 0.24, respectively, for pharyngeal samples and 0.19, 0.29, 0.58, and 0.48, respectively, for conjunctival samples.