Dengue virus: a review on advances in detection and trends - from conventional methods to novel biosensors.

Dengue virus is an important arbovirus infection which transmitted by the Aedes female mosquitoes. The attempt to control and early detection of this infection is a global public health issue at present. Because of the clinical importance of its detection, the main focus of this review is on all of the methods that can offer the new diagnosis strategies. The advantages and disadvantages of reported methods have been discussed comprehensively from different aspects like biomarkers type, sensitivity, accuracy, rate of detection, possibility of commercialization, availability, limit of detection, linear range, simplicity, mechanism of detection, and ability of usage for clinical applications. The optical, electrochemical, microfluidic, enzyme linked immunosorbent assay (ELISA), and smartphone-based biosensors are the main approaches which developed for detection of different biomarkers and serotypes of Dengue virus. Future efforts in miniaturization of these methods open the horizons for development of commercial biosensors for early-diagnosis of Dengue virus infection. Graphical abstract Transmission of Dengue virus by the biting of an Aedes aegypti mosquito, the symptoms of Dengue hemorrhagic fever and the structure of Dengue virus and application of biosensors for its detection.

Co-amplification at lower denaturation temperature-PCR: methodology and applications.

Co-amplification at lower denaturation temperature-polymerase chain reaction (COLD-PCR) is a novel form of PCR that selectively denatures and amplifies low-abundance mutations from mixtures of wild-type and mutation-containing sequences, enriching the mutation 10 to 100 folds. Due to the slightly altered melting temperature (Tm) of the double-stranded DNA and the formation of the mutation/wild-type heteroduplex DNA, COLD-PCR methods are sensitive, specific, accurate, cost-effective and easy to maneuver, and can enrich mutations of any type and at any position, even unknown mutations within amplicons. COLD-PCR and its improved methods are now applied in cancer, microorganisms, prenatal screening, animals and plants. They are extremely useful for early diagnosis, monitoring the prognosis of disease and the efficiency of the treatment, drug selection, prediction of prognosis, plant breeding and etc. In this review, we introduce the principles, key techniques, derived methods and applications of COLD-PCR.

Molecular DNA Analysis in Forensic Identification.

BACKGROUND: Serological and biochemical identification methods used in forensics have several major disadvantages, such as: long time in processing biological sample and lack of sensitivity and specificity. In the last 30 years, DNA molecular analysis has become an important tool in forensic investigations. DNA profiling is based on the short tandem repeats (STR) and aids in human identification from biological samples. METHODS: Forensic genetics, can provide information on the events which occurred at the crime scene or to supplement other methods of forensic identification. Currently, the methods used in identification are based on polymerase chain reaction (PCR) analyses. This method analyses the autosomal STRs, the Y-chromosome, and the mitochondrial DNA. RESULTS: Correlation of biological samples present at the crime scene with identification, selection, and the probative value factor is therefore the first aspect to be taken into consideration in the forensic genetic analysis. CONCLUSIONS: In the last decade, because of the advances in the field of molecular biology, new biomarkers such as: microRNAs (miR), messenger RNA (mRNA), and DNA methylation have been studied and proposed to be used in the forensic identifications of body fluids.

Is this the real time for genomics?

In the last decades, molecular biology has moved from gene-by-gene analysis to more complex studies using a genome-wide scale. Thanks to high-throughput genomic technologies, such as microarrays and next-generation sequencing, a huge amount of information has been generated, expanding our knowledge on the genetic basis of various diseases. Although some of this information could be transferred to clinical diagnostics, the technologies available are not suitable for this purpose. In this review, we will discuss the drawbacks associated with the use of traditional DNA microarrays in diagnostics, pointing out emerging platforms that could overcome these obstacles and offer a more reproducible, qualitative and quantitative multigenic analysis. New miniaturized and automated devices, called Lab-on-Chip, begin to integrate PCR and microarray on the same platform, offering integrated sample-to-result systems. The introduction of this kind of innovative devices may facilitate the transition of genome-based tests into clinical routine.

Next-generation sequencing can reveal in vitro-generated PCR crossover products: some artifactual sequences correspond to HLA alleles in the IMGT/HLA database.

The high-resolution human leukocyte antigen (HLA) genotyping assay that we developed using 454 sequencing and Conexio software uses generic polymerase chain reaction (PCR) primers for DRB exon 2. Occasionally, we observed low abundance DRB amplicon sequences that resulted from in vitro PCR 'crossing over' between DRB1 and DRB3/4/5. These hybrid sequences, revealed by the clonal sequencing property of the 454 system, were generally observed at a read depth of 5%-10% of the true alleles. They usually contained at least one mismatch with the IMGT/HLA database, and consequently, were easily recognizable and did not cause a problem for HLA genotyping. Sometimes, however, these artifactual sequences matched a rare allele and the automatic genotype assignment was incorrect. These observations raised two issues: (1) could PCR conditions be modified to reduce such artifacts? and (2) could some of the rare alleles listed in the IMGT/HLA database be artifacts rather than true alleles? Because PCR crossing over occurs during late cycles of PCR, we compared DRB genotypes resulting from 28 and (our standard) 35 cycles of PCR. For all 21 cell line DNAs amplified for 35 cycles, crossover products were detected. In 33% of the cases, these hybrid sequences corresponded to named alleles. With amplification for only 28 cycles, these artifactual sequences were not detectable. To investigate whether some rare alleles in the IMGT/HLA database might be due to PCR artifacts, we analyzed four samples obtained from the investigators who submitted the sequences. In three cases, the sequences were generated from true alleles. In one case, our 454 sequencing revealed an error in the previously submitted sequence.

Large-scale tag/PCR-based gene expression profiling.

An intriguing enigma in molecular biology is how genes within a single genome are differentially expressed in different cell types of a multicellular organism, or in response to different developmental or environmental queues in a single cell type. Quantification of transcript levels on a genome-wide scale, often termed transcript profiling, provides a powerful approach to identifying protein-coding and non-coding RNAs functionally relevant to a given biological process. Indeed, transcriptome analysis has been a key area of biological inquiry for decades and successfully produced discoveries in a multitude of processes and disease states, and in an increasingly large number of organisms. The evolution of technologies with increasing levels of informational content, ranging from hybridization-based technologies such as Northern blot analysis and microarrays to tag/polymerase chain reaction (PCR)- and sequence-based technologies including differential display and SAGE, along with the next-generation sequencing, has provided hope for revealing the molecular details of biological systems as they respond to change. This review is an overview of selected high throughput tag/PCR-based methods for genome-wide expression profiling amenable to high-throughput automated operation in any standard laboratory.

Target-enrichment strategies for next-generation sequencing.

We have not yet reached a point at which routine sequencing of large numbers of whole eukaryotic genomes is feasible, and so it is often necessary to select genomic regions of interest and to enrich these regions before sequencing. There are several enrichment approaches, each with unique advantages and disadvantages. Here we describe our experiences with the leading target-enrichment technologies, the optimizations that we have performed and typical results that can be obtained using each. We also provide detailed protocols for each technology so that end users can find the best compromise between sensitivity, specificity and uniformity for their particular project.

Recent advances in quantitative PCR (qPCR) applications in food microbiology.

Molecular methods are being increasingly applied to detect, quantify and study microbial populations in food or during food processes. Among these methods, PCR-based techniques have been the subject of considerable focus and ISO guidelines have been established for the detection of food-borne pathogens. More particularly, real-time quantitative PCR (qPCR) is considered as a method of choice for the detection and quantification of microorganisms. One of its major advantages is to be faster than conventional culture-based methods. It is also highly sensitive, specific and enables simultaneous detection of different microorganisms. Application of reverse-transcription-qPCR (RT-qPCR) to study population dynamics and activities through quantification of gene expression in food, by contrast with the use of qPCR, is just beginning. Provided that appropriate controls are included in the analyses, qPCR and RT-qPCR appear to be highly accurate and reliable for quantification of genes and gene expression. This review addresses some important technical aspects to be considered when using these techniques. Recent applications of qPCR and RT-qPCR in food microbiology are given. Some interesting applications such as risk analysis or studying the influence of industrial processes on gene expression and microbial activity are reported.

Clinical features and corresponding immune function status of recurrent viral polymerase chain reaction positivity in patients with COVID-19 : A meta- analysis and systematic review.

INTRODUCTION: Coronavirus disease 2019 (COVID-19) was declared a global pandemic in March 2020. Since then, several studies have found COVID-19 patients with recurrent viral polymerase chain reaction (PCR) positivity. METHODS: On May 6, 2021, an exhaustive literature search of the Web of Science, PubMed, Cochrane Library, Chinese National Knowledge Infrastructure databases, Embase, Wan Fang Data, VIP database, Sinomed database, BioRxiv, MedRxiv, and Research Square was conducted to find describing the laboratory indicators of recurrent and non-recurrent viral PCR positivity in patients with COVID-19. The data were statistically analyzed using STATA version 15.0. RESULTS: In total, 22 studies-comprising 5154 laboratory-confirmed COVID-19 cases-were included in the analyses. Patients with less severe COVID-19 illness (i.e. those clinically classified as mild or common-type) seemed to exhibit recurrent PCR positivity more commonly than patients with more severe illness (i.e. those classified as severe or critical). There were also significant differences between the two groups in terms of the rates of headaches and dizziness, in addition to the levels of aspartate aminotransferase, C reactive protein, interleukin-6, and lactate dehydrogenase. Further, there were variations in the ratio of CD4+ T cells/CD8+ T cells on admission to the hospital. CONCLUSION: In comparison to COVID-19 patients with non-recurrent viral PCR positivity, patients with recurrent virus PCR positivity seem to experience more severe immune function suppression upon hospital admission.

Accumulation of Mitochondrial DNA Microsatellite Instability in Malaysian Patients with Primary Central Nervous System Tumors.

AIM: To determine the mitochondrial microsatellite instability (mtMSI) status in a series of Malaysian patients with brain tumors. Furthermore, we analyzed whether the mtMSI status is associated with the clinicopathological features of the patients. MATERIAL AND METHODS: Forty fresh frozen tumor tissues along with blood samples of brain tumor patients were analyzed for mtMSI by PCR amplification of genomic DNAs, and the amplicons were directly sequenced in both directions using Sanger sequencing. RESULTS: Microsatellite analysis revealed that 20% (8 out of 40) of the tumors were mtMSI positive with a total of 8 mtMSI changes. All mtMSI markers were detected in D310 and D16184 of the D-loop region. Additionally, no significant association was observed between mtMSI status and clinicopathological features. CONCLUSION: The variations, specifically the mtMSI, suggest that the mitochondrial DNA (mtDNA) can be targeted for genomic alteration in brain tumors. Therefore, the specific role of mtDNA alteration in brain tumor development and prognosis requires further investigation.

Clinical validation of a novel quantitative assay for the detection of MGMT methylation in glioblastoma patients.

BACKGROUND: The promoter hypermethylation of the methylguanine-DNA methyltransferase gene is a frequently used biomarker in daily clinical practice as it is associated with a favorable prognosis in glioblastoma patients treated with temozolamide. Due to the absence of adequately standardized techniques, international harmonization of the MGMT methylation biomarker is still an unmet clinical need for the diagnosis and treatment of glioblastoma patients. RESULTS: In this study we carried out a clinical validation of a quantitative assay for MGMT methylation detection by comparing a novel quantitative MSP using double-probe (dp_qMSP) with the conventional MSP in 100 FFPE glioblastoma samples. We performed both technologies and established the best cutoff for the identification of positive-methylated samples using the quantitative data obtained from dp_qMSP. Kaplan-Meier curves and ROC time dependent curves were employed for the comparison of both methodologies. CONCLUSIONS: We obtained similar results using both assays in the same cohort of patients, in terms of progression free survival and overall survival according to Kaplan-Meier curves. In addition, the results of ROC(t) curves showed that dp_qMSP increases the area under curve time-dependent in comparison with MSP for predicting progression free survival and overall survival over time. We concluded that dp_qMSP is an alternative methodology compatible with the results obtained with the conventional MSP. Our assay will improve the therapeutic management of glioblastoma patients, being a more sensitive and competitive alternative methodology that ensures the standardization of the MGMT-biomarker making it reliable and suitable for clinical use.

Ultrafast Photonic PCR Based on Photothermal Nanomaterials.

Over the past few decades, PCR has been the gold standard for detecting nucleic acids (NAs) in various biomedical fields. However, there are several limitations associated with conventional PCR, such as complicated operation, need for bulky equipment, and, in particular, long thermocycling time. Emerging nanomaterials with photothermal effects have shown great potential for developing a new generation of PCR: ultrafast photonic PCR. Here, we review recent applications of photothermal nanomaterials in ultrafast photonic PCR. First, we introduce emerging photothermal nanomaterials and their light-to-heat energy conversion process in photonic PCR. We then review different photothermal nanomaterial-based photonic PCRs and compare their merits and drawbacks. Finally, we summarize existing challenges with photonic PCR and hypothesize its promising future research directions.

Gene synthesis demystified.

DNA fabrication of genetic cassettes at base-level precision is transforming genetic engineering from a laborious art to an information-driven discipline. Although substantial advances have been made in the development of DNA fabrication, the methods employed vary widely based on the length of the DNA. All of these methods are available commercially, but can also be performed at the molecular biology bench using typical reagents and procedures. Because the technology is not mature and is still evolving rapidly, it is helpful to gain some understanding of the different steps in this process and the associated technical challenges to successfully take advantage of DNA fabrication in a research project.

Miniaturized PCR chips for nucleic acid amplification and analysis: latest advances and future trends.

The possibility of performing fast and small-volume nucleic acid amplification and analysis on a single chip has attracted great interest. Devices based on this idea, referred to as micro total analysis, microfluidic analysis, or simply 'Lab on a chip' systems, have witnessed steady advances over the last several years. Here, we summarize recent research on chip substrates, surface treatments, PCR reaction volume and speed, architecture, approaches to eliminating cross-contamination and control and measurement of temperature and liquid flow. We also discuss product-detection methods, integration of functional components, biological samples used in PCR chips, potential applications and other practical issues related to implementation of lab-on-a-chip technologies.

[Quality assessment of HCV RNA detection by PCR method in Federal System of External Quality Assessment in 2005 - 2007].

Part of the Federal System of External Quality Assessment of Clinical Laboratory Tests named "PCR-identification of hepatitis C virus (HCV)" and aimed at detection of HCV RNA by polymerase chain reaction (PCR) assay is functioning from 2000. Ninety, 98, and 112 laboratories from more than 50 regions of Russian Federation participated in it in 2000, 2006, and 2007 respectively. Analysis of results of control samples tests, which were performed by participated laboratories, showed increasing proportion of method of amplification in the presence of fluorescent-marked probes both in real-time and in end-point regimens. In these circumstances, significant increase of specificity and sensitivity of tests was observed. In 2007, proportion of correct results obtained by the participants for tests with negative control samples was 96 - 97%, whereas during detection of HCV RNA in concentration 10(3) IU/ml the proportion of correct results was 80 - 83%. Significant proportion of laboratories, which did not detect HCV RNA in concentration 10(3) IU/ml, points on the necessity to improve sensitivity of this important method of laboratory tests.

[Real-time polymerase chain reaction--applications in research and clinical molecular diagnostics]. / Lancuchowa reakcja polimerazy w czasie rzeczywistym--zastosowanie w badaniach naukowych i diagnostyce medycznej.

Resent years have brought fast development of one of the molecular biology methodology, real time polymerase chain reaction. It has been introduced in common use. Real time PCR allows to detect and qualify very small amounts of specific nucleic acid sequences. As a research tool, a major application of this method is the rapid and accurate detection of genes expression changes as a result of physiological and pathophysiological mechanisms. In clinical diagnostics, this method is used to measure viral or bacterial load or to assess cancer status.

Clinical implementation of molecular methods in detection of microorganisms from blood with a special focus on PCR electrospray ionization mass spectrometry.

INTRODUCTION: The ongoing improvement and development of state-of-the-art diagnostic methods indicate that we are in an era of revolution in clinical microbiological diagnosis of infectious diseases. Non-culture-based methods have the possibility to play a central role in delivering personalized microbiological diagnoses of severe infections. The PCR electrospray ionization mass spectrometry (PCR/ESI-MS) system is built on the principle of universal detection and specific identification. The performance studies using PCR/ESI-MS on whole blood samples, as well as our experiences, indicate that this method provides useful clinical information. These types of modern molecular methods deserve further development for broad implementation into clinical practices. Areas covered: The review describes briefly hitherto developed molecular assays in detection of microorganisms directly from whole blood and focuses on the clinical implementation of PCR/ESI-MS. Expert opinion: The detection of an extensive broad-spectrum of microorganisms directly from whole blood samples with a series of tests that are run automatically with a turn-around time of 8 h would be a desirable diagnostic tool for the clinical microbiology laboratories. We believe that the clinical experience with PCR-ESI MS may guide the development and establishment of similar state-of-the-art diagnostic technologies in medicine in the future.