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Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns.

Doi: 10.3791/50405

Next generation sequencing (NGS), massively parallel or deep sequencing are related terms that describe a DNA sequencing technology which has revolutionised genomic research. Using NGS an entire human genome can be sequenced within a single day. In contrast, the previous Sanger sequencing technology, used to decipher the human genome, required over a decade to deliver the final draft. Although in genome research NGS has mostly superseded conventional Sanger sequencing, it has not yet translated into routine clinical practice. The aim of this article is to review the potential applications of NGS in paediatrics.

Doi: 10.1136/archdischild-2013-304340

A new method for determining nucleotide sequences in DNA is described. It is similar to the “plus and minus” method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448] but makes use of the 2′,3′-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage ϕX174 and is more rapid and more accurate than either the plus or the minus method.

DOI: 10.1073/pnas.74.12.546

The 5′—-3′ exonuclease activity of the thermostable enzyme Thermus aquaticus DNA polymerase may be employed in a polymerase chain reaction product detection system to generate a specific detectable signal concomitantly with amplification. An oligonucleotide probe, nonextendable at the 3′ end, labeled at the 5′ end, and designed to hybridize within the target sequence, is introduced into the polymerase chain reaction assay. Annealing of probe to one of the polymerase chain reaction product strands during the course of amplification generates a substrate suitable for exonuclease activity. During amplification, the 5′—-3′ exonuclease activity of T. aquaticus DNA polymerase degrades the probe into smaller fragments that can be differentiated from undegraded probe. The assay is sensitive and specific and is a significant improvement over more cumbersome detection methods.

DOI: 10.1073/pnas.88.16.7276

Immunoassays are a powerful diagnostic tool and are widely used for the quantification of proteins and biomolecules in medical diagnosis and research. Enzyme-linked immunosorbent assay (ELISA) is the most commonly used immunoassay format and allows the detection of biomarkers at a very low concentration. The diagnostic platforms such as enzyme immunoassay (EIA), chemiluminescence (CL) assay, polymerase chain reaction (PCR), flow cytometry (FC), and mass spectrometry (MS) have been used to identify molecular biomarkers. However, these diagnostic tools requiring expensive equipment, long testing time, and qualified personnel that is not always available in small local hospitals with limited resources. The lateral flow immunoassay (LFIA) platform was developed for rapidly obtaining laboratory results and to make urgent decisions in emergency medicine, as well as the recently introduced concept of testing at the site of care (point-of-care, POC). The simultaneous measurement of different substances from a single sample called multiplex assays have become increasingly significant for in vitro quantification of multiple analytes in a single sample, thereby minimising cost, time, and volume. In multiplex immunoassays, the ligands are immobilized either in planar format (flat surface) or on microspheres in suspension that binds to target analytes in sample. The multiplex technology has established itself in proteomic networks and pathways, validation of genomic discoveries, and in the development of clinical biomarkers. In the present review article, various types of monoplex/simplex and complex/multiplex immunoassays have been analysed that are increasingly being applied in laboratory medicine. Also, some advantages and disadvantages of these multiplex assays have also been included such as experimental animals, in vitro tests using cell lines and tissue samples, 3-dimensional modelling and bioprinting, in silico tests, organ-on-chip, and computer modelling.

DOI: 10.1007/s00580-021-03302-4

Next Generation Sequencing (NGS) is a powerful tool that depends on loading a precise amount of DNA onto a flowcell. NGS strategies have expanded our ability to investigate genomic phenomena by referencing mutations in cancer and diseases through large-scale genotyping, developing methods to map rare chromatin interactions (4C; 5C and Hi-C) and identifying chromatin features associated with regulatory elements (ChIP-seq, Bis-Seq, ChiA-PET). While many methods are available for DNA library quantification, there is no unambiguous gold standard. Most techniques use PCR to amplify DNA libraries to obtain sufficient quantities for optical density measurement. However, increased PCR cycles can distort the library’s heterogeneity and prevent the detection of rare variants. In this analysis, we compared new digital PCR technologies (droplet digital PCR; ddPCR, ddPCR-Tail) with standard methods for the titration of NGS libraries. DdPCR-Tail is comparable to qPCR and fluorometry (QuBit) and allows sensitive quantification by analysis of barcode repartition after sequencing of multiplexed samples. This study provides a direct comparison between quantification methods throughout a complete sequencing experiment and provides the impetus to use ddPCR-based quantification for improvement of NGS quality.

Nature Scientific reports