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This survey will discuss current and emerging isothermal and rapid polymerase chain reaction (PCR) based nucleic acid amplification methods for patient near diagnostics. To assess the clinical need of rapid diagnostics for infectious diseases based on nucleic acid tests (NATs) we performed and analysed a questionnaire among laboratories participating in corresponding INSTAND ring trials for external quality assurance. The questions concerning new amplification technologies like isothermal nucleic acid amplification, potentially suited to significantly decrease turnaround times, were complemented by questions to evaluate the present status of NATs. Besides end-users, companies were also addressed by sending out a manufacturer specific questionnaire. Analysis of the answers from 48 laboratories in 14 European countries revealed that a much shorter turnaround time is requested for selected pathogens compared to about 2 h or longer when applying temperature cycling amplification, i.e. PCR. In this context, most frequently mentioned were MRSA, norovirus, influenza A and B viruses, cytomegalovirus (CMV) as well as hepatitis B virus (HBV) and hepatitis C virus (HCV). At present, 8% of the laboratories having participated in this survey apply isothermal amplification of nucleic acid to identify infectious pathogens.
Microbead-based technologies represent elegant and versatile approaches for highly parallelized quantitative multiparameter assays. They also form the basis of various techniques for detection and quantification of nucleic acids and proteins. Nucleic acid-based methods include hybridization assays, solid-phase PCR, sequencing, and trapping assays. Microbead assays have been improved in the past decades and are now important tools in routine and point-of-care diagnostics as well as in life science. Its advances include low costs, low workload, high speed and high-throughput automation. The potential of microbead-based assays therefore is apparent, and commercial applications can be found in the detection and discrimination of single nucleotide polymorphism, of pathogens, and in trapping assays. This review provides an overview on microbead-based platforms for biosensing with a main focus on nucleic acid detection (including amplification strategies and on selected probe systems using fluorescent labeling). Specific sections cover chemical properties of microbeads, the coupling of targets onto solid surfaces, microbead probe systems (mainly oligonucleotide probes), microbead detection schemes (with subsections on suspension arrays, microfluidic devices, and immobilized microbeads), quantification of nucleic acids, PCR in solution and the detection of amplicons, and methods for solid-phase amplification. We discuss selected trends such as microbead-coupled amplification, heterogeneous and homogenous DNA hybridization assays, real-time assays, melting curve analysis, and digital microbead assays. We finally discuss the relevance and trends of the methods in terms of high-level multiplexed analysis and their potential in diagnosis and personalized medicine. Contains 211 references.
In the field of animal species differentiation, work on standardizing methods has been intensified in Germany in recent years, not least due to the horsemeat scandal in 2013. Even though there are now hardly ever any positive findings anymore in examinations to detect horse adulterations in foods such as lasagne, animal species differentiation altogether ranks high in detecting adulteration of foods. This article, therefore, summarises the current status of analytical techniques used in Germany with standardization at German level. It has been established by the working group "Biochemical and Molecular Biological Analytics" of the Lebensmittelchemische Gesellschaft (Food Chemistry Society within the German Chemical Society) with support of experts in the working group “Molecular biology techniques for differentiating plant and animal species" (§64 of the German Food and Feed Code - LFGB) and the "Immunology and molecular biology" task force of the food hygiene and food of animal origin working group (ALTS), both from Germany.
Work on standardising methods in the field of animal species differentiation has been intensified in Germany in recent years, not least due to the horsemeat scandal in 2013. Even though there are now hardly ever any positive findings any more in examinations to detect horse adulterations in foods such as lasagne, animal species differentiation altogether ranks high in detecting adulteration of foods. This article therefore summarises the current status of analytical techniques used in Germany with standardisation at German level. It has been established by the working group “Biochemical and Molecular Biological Analytics” of the Lebensmittelchemische Gesellschaft (Food Chemistry Society within the German Chemical Society) with support of experts in the working group “Molecular biology techniques for differentiating plant and animal species” (§ 64 of the German Food and Feed Code – LFGB) and the “Immunology and molecular biology” task force of the food hygiene and Food of animal origin working group (ALTS), both from Germany.
Nicht zuletzt bedingt durch den Pferdefleischskandal im Jahr 2013 wurden in Deutschland die Aktivitäten bei der Methodenstandardisierung im Bereich der Tierarten-Differenzierung in den vergangenen Jahren intensiviert. Wenn auch positive Befunde bei dem Nachweis von Bestandteilen aus Pferd in Lebensmitteln wie Lasagne derzeit so gut wie nicht mehr anzutreffen sind, so hat die Tierartendifferenzierung insgesamt beim Nachweis von Verfälschungen in Lebensmitteln einen hohen Stellenwert.
Diese Arbeit fasst daher den aktuellen Stand der Analytik in Deutschland mit Schwerpunkt bei der Standardisierung zusammen. Sie wurde erstellt durch die Arbeitsgruppe „Biochemische und molekularbiologische Analytik“ der Lebensmittelchemischen Gesellschaft mit Unterstützung von Experten der Arbeitsgruppe „Molekularbiologische Methoden zur Pflanzen- und Tierartendifferenzierung“ (§ 64 LFGB) sowie der ALTS-Arbeitsgruppe „Immunologie und Molekularbiologie“ (jeweils D).