The impact of genetic diversity in protozoa on molecular diagnostics

Detection of intestinal parasitic protists, commonly referred to as 'intestinal protozoa,' by PCR is increasingly used not only for identification or confirmation but also as a first-line diagnostic tool. Apart from the ability to sample correctly and extract parasite DNA directly from faeces, primer and probe specificity and sensitivity affect predictive values and hence the utility of diagnostic assays. Molecular characterization of intestinal protists is necessary to design primers and probes because this is the basic material for current and future improved diagnostic PCRs for either detecting all genetic variants or specifically differentiating among such variants. As an example, this paper highlights the existence of interspecific and intraspecific genetic diversity among intestinal, unicellular parasites and its implications for nucleic acid-based diagnostic assays.     

基于CRISPR的快速灵敏便捷分子检测

快速灵敏检测技术对疾病防控必不可少。特别是新冠疫情暴发以来,人们深刻认识到快速灵敏检测技术的重要性。近年来,以CRISPR/Cas为代表的基因编辑技术带来了生物技术革命性的进步。CRISPR的核酸检测技术因其快速、准确、灵敏、经济等特点,正在引发分子诊断革新,并已被成功应用于传染病、遗传病、肿瘤基因突变诊断,以及食品安全等领域。本文归纳了基于CRISPR的多种核酸检测体系及应用,并对未来CRISPR核酸检测发展趋势及结合人工智能的智能化检测进行了展望。     

Microarray for diagnostics of human pathogenic influenza A viruses subtypes

An oligonucleotide microchip was developed for diagnostics of human pathogenic Influenza A viruses subtypes. It contains discriminating probes for H1-, H2-, H3-, H5-, H7- and H9-subtypes of hemagglutinin and for N1-, N2-, and N7-subtypes of neuraminidase. The additional set of probes was used for M-gene of Influenza A viruses definition. Microchip was tested on samples pathogenic H5N1 avian influenza viruses, pandemic H1N1 swine influenza viruses and seasonal H1N1 and H3N2 influenza viruses. The microchip can be used for the analysis of both cultured strains and clinical specimens.     

Proteomics, nanotechnology and molecular diagnostics

Sequencing of the human genome opened the way to the exploration of the proteome and this has lead to the identification of large numbers of proteins in complex biological samples. The identification of diagnostic patterns in samples taken from patients to aid diagnosis is in the early stages of development. The solution to many of the technical challenges in proteomics and protein based molecular diagnostics will be found in new applications of nanomaterials. This review describes some of the physical and chemical principles underlying nanomaterials and devices and outlines how they can be used in proteomics; developments which are establishing nanoproteomics as a new field. Nanoproteomics will provide the platform for the discovery of next generation biomarkers. The field of molecular diagnostics will then come of age.     

Bacteriophages: The viruses for all seasons of molecular biology

Bacteriophage research continues to break new ground in our understanding of the basic molecular mechanisms of gene action and biological structure. The abundance of bacteriophages in nature and the diversity of their genomes are two reasons why phage research brims with excitement. The pages of Virology Journal will reflect the excitement of the "New Phage Biology."     

Modern molecular cytogenetic techniques in genetic diagnostics

During the past decade, fluorescence in situ hybridization (FISH) has become an important complementing application in genetic diagnostics. The use of variable FISH techniques enhances the thorough interpretation of numerical and complex chromosome aberrations, bridging the gap between conventional chromosome banding analysis and molecular genetic DNA studies. This review gives a brief overview of the different molecular cytogenetic FISH techniques and applications currently used in routine genetic diagnostics and focus on their advantages and limitations.     

Fungal molecular diagnostics: a mini review

Conventional methods to identify fungi have often relied on identification of disease symptoms, isolation and culturing of environmental organisms, and laboratory identification by morphology and biochemical tests. Although these methods are still fundamental there is an increasing move towards molecular diagnostics of fungi in all fields. In this review, some of the molecular approaches to fungal diagnostics based on polymerase chain reaction (PCR) and DNA/RNA probe technology are discussed. This includes several technological advances in PCR-based methods for the detection, identification and quantification of fungi including real-time PCR which has been successfully used to provide rapid, quantitative data on fungal species from environmental samples. PCR and probe based methods have provided new tools for the enumeration of fungal species, but it is still necessary to combine the new technology with more conventional methods to gain a fuller understanding of interactions occurring in the environment. Since its introduction in the mid 1980's PCR has provided many molecular diagnostic tools, some of which are discussed within this review, and with the advances in micro-array technology and real-time PCR methods the future is bright for the development of accurate, quantitative diagnostic tools that can provide information not only on individual fungal species but also on whole communities.     

The value of microarray techniques for quantitative gene profiling in molecular diagnostics

There has been an explosion of interest in microarray technologies that allow the quantification of whole-genome RNA expression data. The apparent correlation of expression profiles with clinically relevant parameters such as disease outcome has raised expectations with respect to the clinical usefulness of the data generated. Yet the accuracy and biological relevance of these data remain contentious, even in basic research applications. Therefore, numerous issues related to format, quality, validation and interpretation remain to be resolved before microarray profiling can become a diagnostic tool of clinical relevance for routine work.     

Toward molecular diagnostics of mood disorders in psychiatry

Mental disorders are highly prevalent and often difficult to diagnose. There is a significant gap between advances in their pharmacotherapy and the present lack of objective biologic tests for diagnosis. The special complexity of diagnosis in psychiatry is related to the absence of objective diagnostic "gold standards", co-morbidity, heterogeneity and equifinality, quantitative trait loci, and locus heterogeneity. Here, we review recent findings relating to diagnostic, pathophysiological, and linkage markers for mood disorders at the biochemical level involving monoamine neurotransmitters, hormones, and signal-transducing G proteins. Identification of biological diagnostic markers could enable segregating mood disorders to several biologically different subtypes. New-era methods and strategies involving genomics, proteomics, multi-marker approach and single nucleotide polymorphisms have the potential to revolutionize future diagnosis in psychiatry.     

Genetic counseling in the era of molecular diagnostics

Veterinarians with an interest in theriogenology will often be asked by small animal clients for advice concerning hereditary diseases in their breeds. Many new DNA-based tests for analysis of genetic diseases and traits (e.g. coat color) are now available for use by both breeders and veterinarians. With appropriate interpretation, these tests can be invaluable tools in a breeding program. For example, they can be used to produce animals free of specific diseases, to quickly eliminate a disease from an entire breed, or to select for specific traits in breeding stock. Selection strategies that do not take into account maintaining genetic diversity of the breed may be detrimental and reduce the potential for future improvement.     

High-throughput protein arrays: prospects for molecular diagnostics

High-throughput protein arrays allow the miniaturized and parallel analysis of large numbers of diagnostic markers in complex samples. Using automated colony picking and gridding, cDNA or antibody libraries can be expressed and screened as clone arrays. Protein microarrays are constructed from recombinantly expressed, purified, and yet functional proteins, entailing a range of optimized expression systems. Antibody microarrays are becoming a robust format for expression profiling of whole genomes. Alternative systems, such as aptamer, PROfusion, nano- and microfluidic arrays are all at proof-of-concept stage. Differential protein profiles have been used as molecular diagnostics for cancer and autoimmune diseases and might ultimately be applied to screening of high-risk and general populations.     

Improved molecular diagnostics for ornithine transcarbamylase deficiency.

Since the cloning of the cDNA for X-linked ornithine transcarbamylase (OTC) in 1984, diagnostic accuracy of OTC deficiency for prenatal and carrier detection has been greatly improved by the use of linkage analysis. However, the use of RFLP-based diagnosis is limited in this and in other new mutation diseases. Here we report both the use of direct mutation detection by new PCR-based techniques and our experience with linkage-based diagnosis in 18 families. We have previously reported the use of chemical mismatch cleavage to detect mutations first in amplified mRNA and then in genomic DNA of patients. This technique has now been utilized for prenatal diagnosis. Primers for specific amplification of OTC exons 1, 3, 5, 9, and 10 have been developed and been employed to map deletions of the OTC gene in two families. These primers also have been used to detect alterations in the TaqI sites found in exons 1, 3, 5, and 9. Four novel mutations of the OTC gene leading to abolition of a TaqI site in the OTC cDNA were discovered. One of these mutations is in exon 1; two lie in exon 3; and one is in exon 9. In addition, we have used the PCR products as probes to identify the exon-specific bands seen on Southern blots and to map the polymorphic BamHI and MspI sites, which are commonly used for linkage analysis. This information will facilitate the interpretation of altered band patterns seen in deletion cases and in cases of point mutations affecting restriction sites. Utilization of the appropriate combination of these molecular techniques permitted accurate diagnostic evaluations in 17 of 18 families.