Development of molecular inversion probes for soybean progeny genomic selection genotyping.

Increasing rate of genetic gain for key agronomic traits through genomic selection requires the development of new molecular methods to run genome-wide single-nucleotide polymorphisms (SNPs). The main limitation of current methods is the cost is too high to screen breeding populations. Molecular inversion probes (MIPs) are a targeted genotyping-by-sequencing (GBS) method that could be used for soybean [Glycine max (L.) Merr.] that is both cost-effective, high-throughput, and provides high data quality to screen breeder's germplasm for genomic selection. A 1K MIP SNP set was developed for soybean with uniformly distributed markers across the genome. The SNPs were selected to maximize the number of informative markers in germplasm being tested in soybean breeding programs located in the northern-central and middle-southern regions of the United States. The 1K SNP MIP set was tested on diverse germplasm and a recombinant inbred line (RIL) population. Targeted sequencing with MIPs obtained an 85% enrichment for the targeted SNPs. The MIP genotyping accuracy was 93% overall, whereas homozygous call accuracy was 98% with <10% missing data. The accuracy of MIPs combined with its low per-sample cost makes it a powerful tool to enable genomic selection within soybean breeding programs.

Cost-effectiveness of nucleic acid amplification testing to guide treatment for vaginitis: a decision-modeling analysis.

We evaluated the cost-effectiveness of test-and-treat scenarios for vaginitis, scenarios based on clinical and microscopic examination (CME), nucleic acid amplification testing (NAAT), or nonamplified nucleic acid probe (probe) testing. The symptom resolution outcome and the payer cost of diagnosis and treatment were estimated in decision analytical models in a hypothetical patient population. Compared with probe testing, NAAT resulted in symptom resolution in more patients (615 versus 475 per 1000 tested) at a cost of $210 per incremental symptom resolution, a cost lower than the willingness to pay for symptom resolution ($871) implied by payer coverage for probe testing. Following a negative CME, the NAAT scenario resulted in symptom resolution in more patients (650 per 1000 patients tested) than did either CME (525) or the CME probe testing-based scenario (602) at incremental cost-effectiveness ratios lower than the willingness to pay implied by coverage for CME. Therefore, NAAT is likely to cost-effectively improve health outcomes for patients with vaginitis.

Comparison of Sanger sequencing for hepatitis C virus genotyping with a commercial line probe assay in a tertiary hospital.

BACKGROUND: The technique most frequently used to genotype HCV is quantitative RT-PCR. This technique is unable to provide an accurate genotype/subtype for many samples; we decided to develop an in-house method with the goal of accurately identifying the genotype of all samples. As a Belgium National Centre of reference for hepatitis, we developed in-house sequencing not only for 5'UTR and core regions starting from VERSANT LiPA amplicons but also for NS5B regions. The sequencing of VERSANT LiPA amplicons might be useful for many laboratories worldwide using the VERSANT LiPA assay to overcome undetermined results. METHODS: 100 samples from Hepatitis C virus infected patients analysed by the VERSANT HCV Genotype 2.0 LiPA Assay covering frequent HCV types and subtypes were included in this study. NS5B, 5'UTR and Core home-made sequencing were then performed on these samples. The sequences obtained were compared with the HCV genomic BLAST bank. RESULTS: All the samples were characterised by the VERSANT LiPA assay (8 G1a, 17 G1b, 6 G2, 11 G3, 13 G4, and 10 G6). It was not possible to discriminate between G6 and G1 by the VERSANT LiPA assay for 8 samples and 27 had an undetermined genotype. Forty-one samples were sequenced for the three regions: NS5B, 5'UTR and Core. Twenty-three samples were sequenced for two regions: 5' UTR and Core and 36 samples were sequenced only for NS5B. Of the 100 samples included, 64 samples were analysed for 5'UTR and Core sequencing and 79 samples were analysed for NS5B sequencing. The global agreement between VERSANT LiPA assay and sequencing was greater than 95%. CONCLUSIONS: In this study, we describe a new, original method to confirm HCV genotypes of samples not discriminated by a commercial assay, using amplicons already obtained by the screening method, here the VERSANT LiPA assay. This method thus saves one step if a confirmation assay is needed and might be of usefulness for many laboratories worldwide performing VERSANT LiPA assay in particular.

Cost-Effectiveness Analysis of Diagnosis of Duchenne/Becker Muscular Dystrophy in Colombia.

OBJECTIVES: To determine the cost-effectiveness ratio of different courses of action for the diagnosis of Duchenne or Becker muscular dystrophy in Colombia. METHODS: The cost-effectiveness analysis was performed from the Colombian health system perspective. Decision trees were constructed, and different courses of action were compared considering the following tests: immunohistochemistry (IHC), Western blot (WB), multiplex polymerase chain reaction, multiplex ligation-dependent probe amplification (MLPA), and the complete sequencing of the dystrophin gene. The time horizon matched the duration of sample extraction and analysis. Transition probabilities were obtained from a systematic review. Costs were constructed with a type-case methodology using the consensus of experts and the valuation of resources from consulting laboratories and the 2001 Social Security Institute cost manual. Deterministic sensitivity and scenario analyses were performed with one or more unavailable alternatives. Costs were converted from Colombian pesos to US dollars using the 2014 exchange rate. RESULTS: In the base case, WB was the dominant strategy, with a cost of US $419.07 and a sensitivity of 100%. This approach remains the dominant strategy down to a 98.2% sensitivity and while costs do not exceed US $837.38. If WB was not available, IHC had the best cost-effectiveness ratio, followed by MLPA and sequencing. CONCLUSIONS: WB is a cost-effective alternative for the diagnosis of patients suspected of having Duchenne or Becker muscular dystrophy in the Colombian health system. The IHC test is rated as the second-best detection method. If these tests are not available, MLPA followed by sequencing would be the most cost-effective alternative.

Detection of Mycobacterium avium complex DNA directly in clinical respiratory specimens: opportunities for improved turn-around time and cost savings.

We developed, evaluated, and implemented a Taqman multiplex real-time polymerase chain reaction (PCR) assay for the detection of Mycobacterium avium complex (MAC), targeting the 16S-23S rRNA internal transcribed spacer, which we have combined with an existing Mycobacterium tuberculosis complex assay for use directly in clinical respiratory specimens. Evaluation of the performance of this assay for MAC detection included 464 clinical respiratory specimens tested prospectively. This real-time PCR assay was found overall to have a sensitivity of 71.1%, a specificity of 99.5%, a positive predictive value of 98.0%, and a negative predictive value of 90.2% for MAC. The assay provides results prior to the availability of cultured material and identification, most within 24 h of specimen receipt, and may reduce the need to culture MAC-PCR-positive specimens when susceptibility testing is not requested. Additionally, we have found significant cost savings of approximately $21.00 per specimen and staff time reductions of 3.75 h per specimen with implementation of this assay.

Perspectives for on-site monitoring of progesterone.

The steroid hormone progesterone is the primary biomarker of the reproductive status of female mammals. Current techniques of monitoring progesterone are based predominantly on (enzyme) immunoassays, but these are too expensive to be affordable in daily screening programmes because of their associated labour costs and the need for laboratory facilities and/or equipment. Here, we discuss existing methods as well as new perspectives for (automated) application at point of care/need, e.g. the milking parlour. These make it apparent that a low-cost, fully automated progesterone assay system to monitor the reproductive status is far from being realised at present. Timely ovulation prediction techniques for artificial insemination and reproductive cycling are thus urgently needed, and promising perspectives will be highlighted.

Recent advances in peptide probe-based biosensors for detection of infectious agents.

Recent biological terrorism threats and outbreaks of microbial pathogens clearly emphasize the need for biosensors that can quickly and accurately identify infectious agents. The majority of rapid biosensors generate detectable signals when a molecular probe in the detector interacts with an analyte of interest. Analytes may be whole bacterial or fungal cells, virus particles, or specific molecules, such as chemicals or protein toxins, produced by the infectious agent. Peptides and nucleic acids are most commonly used as probes in biosensors because of their versatility in forming various tertiary structures. The interaction between the probe and the analyte can be detected by various sensor platforms, including quartz crystal microbalances, surface acoustical waves, surface plasmon resonance, amperometrics, and magnetoelastics. The field of biosensors is constantly evolving to develop devices that have higher sensitivity and specificity, and are smaller, portable, and cost-effective. This mini review discusses recent advances in peptide-dependent rapid biosensors and their applications as well as relative advantages and disadvantages of each technology.

Development of a Scorpion probe-based real-time PCR for the sensitive quantification of Bacteroides sp. ribosomal DNA from human and cattle origin and evaluation in spring water matrices.

Spring water from alpine catchments are important water resources but they can be vulnerable against faecal contamination. Potential faecal contamination sources are wildlife populations, pasturing activities, or alpine tourism. Unfortunately, no faecal source tracking method is available to date which is sensitive enough for appropriate spring water monitoring and source allocation. Our purpose was to develop a Duplex Scorpion real-time PCR approach for the specific and sensitive quantification of Bacteroides sp. 16S rDNA fragments from human and cattle origin. By the developed approach, detection of plasmids, carrying the respective biomarker sequence, was possible over a range of more than seven orders of magnitudes down to six copy numbers per PCR assay. Furthermore, the Duplex Scorpion real-time PCR allowed the specific quantification down to 50 targets in plasmid spiked spring water matrices. Results indicate that microbial source tracking appears feasible in spring water habitats by probe-based real-time PCR technologies. However, preliminary testing of the established approach on faecal samples collected from a representative alpine habitat did not allow unambiguous source allocation in all cases. In the future, the available sequence database has thus to be widened to allow reliable source tracking in alpine spring watersheds and even expand this approach to other potential faecal sources.

Large scale blood group genotyping.

Determination of predicted blood group phenotype by determination of genotype has been performed since the 1990s. This evolved due to the rapid accrual of information surrounding the molecular basis of blood group antigen expression, which started in 1990 with ABO and RH systems and has now resulted in the molecular description of 28 of the 29 blood groups. Blood group genotyping is currently performed mostly for fetal blood group incompatibility and for assessment of multi-transfused patients. Both of these clinical scenarios are either dangerous or technically difficult, respectively to define serologically. With the simultaneous development of mass scale genotyping platforms it has now permitted the application of them to blood group genotype determination. In this paper, I describe some recently published work that has demonstrated that mass scale genotyping approaches are feasible. These approaches may lead to more effective management of blood stocks and patient cross-matching by reducing the dependence on serology during the time critical pre-transfusion phase. It is most probable that large scale studies, perhaps involving many European Union and North American based blood suppliers, may drive the introduction of this technology and convince red cell serologists that this approach may allow their work to be more focussed.

Multifunctional encoded particles for high-throughput biomolecule analysis.

High-throughput screening for genetic analysis, combinatorial chemistry, and clinical diagnostics benefits from multiplexing, which allows for the simultaneous assay of several analytes but necessitates an encoding scheme for molecular identification. Current approaches for multiplexed analysis involve complicated or expensive processes for encoding, functionalizing, or decoding active substrates (particles or surfaces) and often yield a very limited number of analyte-specific codes. We present a method based on continuous-flow lithography that combines particle synthesis and encoding and probe incorporation into a single process to generate multifunctional particles bearing over a million unique codes. By using such particles, we demonstrate a multiplexed, single-fluorescence detection of DNA oligomers with encoded particle libraries that can be scanned rapidly in a flow-through microfluidic channel. Furthermore, we demonstrate with high specificity the same multiplexed detection using individual multiprobe particles.

Towards fast and inexpensive molecular diagnostic: the case of TP53.

BACKGROUND: Much research suggests that TP53 mutations have prognostic importance and sometimes are a significant factor in clinical oncology. A considerable effort has been made to develop fast and inexpensive methods for TP53 mutations detection. METHODS: On the basis of describing the role of TP53 as tumor suppressor gene and TP53 mutation spectrum, the authors discuss conventional methods and new technologies for TP53 mutations detection. This discussion is supported by more recent publications in the field of both molecular genetics and analysis technologies. RESULTS: Biosensors and gene chips are of considerable recent interest, due to their tremendous promise for obtaining sequence-specific information in a faster, simpler and cheaper manner compared to traditional methods. CONCLUSIONS: New methods such as biosensors and gene chips appear promising as analytical methods of detecting mutations.

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.

Exploring the new world of the genome with DNA microarrays.

Thousands of genes are being discovered for the first time by sequencing the genomes of model organisms, an exhilarating reminder that much of the natural world remains to be explored at the molecular level. DNA microarrays provide a natural vehicle for this exploration. The model organisms are the first for which comprehensive genome-wide surveys of gene expression patterns or function are possible. The results can be viewed as maps that reflect the order and logic of the genetic program, rather than the physical order of genes on chromosomes. Exploration of the genome using DNA microarrays and other genome-scale technologies should narrow the gap in our knowledge of gene function and molecular biology between the currently-favoured model organisms and other species.

Methods and reagents. Eliminating ghost bands from plasmid preps.

Methods and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methds-reagnts, available on the internet. This month's column updates the discussion of a ghost DNA band that continues to haunt netters. For details on how to partake in the newsgroup, see the accompanying box.

Multiplex PCR provides a low-cost alternative to DNA probe methods for rapid identification of Mycobacterium avium and Mycobacterium intracellulare.

A multiplex PCR designed to differentiate Mycobacterium tuberculosis complex organisms from M. avium and M. intracellulare was used to test 105 isolates identified by DNA probe methods as M. avium, M. intracellulare, or M. avium complex type X. The multiple PCR correctly identified 33 of 34 isolates identified by commercial probe methods as M. avium and all 51 isolates identified as M. intracellulare. The 20 isolates identified as M. avium complex type X by probe were identified as Mycobacterium spp. by the multiplex method. These results confirm that the multiplex PCR, which is simple to perform and cheaper than commercial probe methods, is suitable for routine identification of M. avium and M. intracellulare.