Microfluidics-based strategies for molecular diagnostics of infectious diseases

Traditional diagnostic strategies for infectious disease detection require benchtop instruments that are inappropriate for point-of-care testing(POCT). Emerging microfluidics, a highly miniaturized, automatic, and integrated technology,are a potential substitute for traditional methods in performing rapid, low-cost, accurate, and on-site diagnoses.Molecular diagnostics are widely used in microfluidic devices as the most effective approaches for pathogen detection.This review summarizes the latest advances in microfluidics-based molecular diagnostics for infectious diseases from academic perspectives and industrial outlooks. First, we introduce the typical on-chip nucleic acid processes,including sample preprocessing, amplification, and signal read-out. Then, four categories of microfluidic platforms are compared with respect to features, merits, and demerits. We further discuss application of the digital assay in absolute nucleic acid quantification. Both the classic and recent microfluidics-based commercial molecular diagnostic devices are summarized as proof of the current market status. Finally, we propose future directions for microfluidics-based infectious disease diagnosis.

Aptamer-based Membrane Protein Analysis and Molecular Diagnostics

Membrane proteins are vital components of the cell membrane and play crucial roles in various cellular activities.Analysis of membrane proteins is of paramount importance for studying molecular events inside cells and organisms and holds promising prospects for early disease diagnosis and treatment assessment.Benefiting from obvious merits including high affinity,high specificity and ease of modification,aptamers have been regarded as ideal molecular recognition elements in membrane protein analysis and molecular diagnostics strategies.This review summarised recent advances in membrane protein-specific aptamer screening,aptamer-based static and dynamic membrane protein analysis,and aptamer-based molecular diagnostic techniques.Prospects and challenges were also discussed.

A narrative review of cancer molecular diagnostics: past, present, and future

Along with the advances in cancer genomics and the development of targeted therapies, the field of molecular diagnostics has undergone rapid evolution to meet the growing needs associated with patient care. Here, we review the past, present, and possible future of molecular diagnostics, including technologies and testing principles, to provide a comprehensive landscape of molecular diagnostic technologies, testing platforms, and applications. This review is based on the US Food and Drug Administration publications, the National Comprehensive Cancer Network guidelines, and the peer-reviewed English literature published between 2003 and 2021. We conclude that molecular diagnostics has changed dramatically during the past two decades. Next-generation sequencing–based comprehensive genomic profiling has replaced single-gene/single-locus testing for simultaneous detection of mutations, copy number alterations, structural variants, and mutational signatures to facilitate cancer diagnosis, prognosis prediction, targeted therapies, and immunotherapies. Laboratory-developed tests and companion diagnostics approved by the US Food and Drug Administration both play important roles in cancer patient management.

Fast infectious diseases diagnostics based on microfuidic biochip system

Molecular diagnostics is one of the most important tools currently in use for clinical pathogen detection due to its high sensitivity,specificity,and low consume of sample and reagent is keyword to low cost molecular diagnostics.In this paper,a sensitive DNA isothermal amplifi-cation method for fast clinical infectious diseases diagnostics at aM concentrations of DNA was developed using a polycarbonate(PC)microfuidic chip.A portable confocal optical fuo-rescence detector was specifically developed for the microfuidic chip that was capable of highly sensitive real-time detection of amplified products for sequence-specific molecular identification near the optical diffraction limit with low background.The molecular diagnostics of Listeria monocytogenes with nucleic acid extracted from stool samples was performed at a minimum DNA template concentration of 3.65 aM,and a detection limit of less than five copies of genomic DNA.Contrast to the general polymerase chain reaction(PCR)at eppendorf(EP)tube,the detection time in our developed method was reduced from 1.5h to 45 min for multi-target parallel detection,the consume of sample and reagent was dropped from 25μL to 1.45μL.This novel microfuidic chip system and method can be used to develop a micro total analysis system as a clinically relevant pathogen molecular diagnostics method via the amplification of targets,with potential applications in biotechnology,medicine,and clinical molecular diagnostics.

Analytical characteristics of a qPCR-based molecular diagnostic assay-conceptual considerations for laboratory personnel

Dear Editor：Quantitative real-time PCR has revolutionized molecular diagnostics with its ease of use,increased sensitivity and specificity and low turnaround time.PCR/quantitative PCR（qPCR）-based assays offer a distinct advantage over other serological/conventional diagnostic approaches.The ability to diagnose infectious diseases has benefited from the availability of US FDA approved and Conformite Europeenne（CE）-marked qPCR-based in-vitro diagnostic kits from international companies.The high-quality kits are calibrated with the World Health Organization（WHO）reference standards and the National Institute for Biological Standards and Control（NIBSC）standards.

Development of clustered regularly interspaced short palindromic repeats/CRISPR-associated technology for potential clinical applications

The clustered regularly interspaced short palindromic repeats(CRISPR)-CRISPRassociated(Cas)proteins constitute the innate adaptive immune system in several bacteria and archaea.This immune system helps them in resisting the invasion of phages and foreign DNA by providing sequence-specific acquired immunity.Owing to the numerous advantages such as ease of use,low cost,high efficiency,good accuracy,and a diverse range of applications,the CRISPR-Cas system has become the most widely used genome editing technology.Hence,the advent of the CRISPR/Cas technology highlights a tremendous potential in clinical diagnosis and could become a powerful asset for modern medicine.This study reviews the recently reported application platforms for screening,diagnosis,and treatment of different diseases based on CRISPR/Cas systems.The limitations,current challenges,and future prospectus are summarized;this article would be a valuable reference for future genome-editing practices.

Detection of Helicobacter pylori resistance to clarithromycin and fluoroquinolones in Brazil:A national survey

AIM To evaluate bacterial resistance to clarithromycin and fluoroquinolones in Brazil using molecular methods.METHODS The primary antibiotic resistance rates of Helicobacter pylori(H. pylori) were determined from November 2012 to March 2015 in the Southern,South-Eastern,Northern,North-Eastern,and Central-Western regions of Brazil. Four hundred ninety H. pylori patients [66% female,mean age 43 years(range: 18-79)] who had never been previously treated for this infection were enrolled. All patients underwent gastroscopy with antrum and corpus biopsies and molecular testing using Geno Type Helico DR(Hain Life Science,Germany). This test was performed to detect the presence of H. pylori and to identify point mutations in the genes responsible for clarithromycin and fluoroquinolone resistance. The molecular procedure was divided into three steps: DNA extraction from the biopsies,multiplex amplification,and reverse hybridization. RESULTS Clarithromycin resistance was found in 83(16.9%) patients,and fluoroquinolone resistance was found in 66(13.5%) patients. There was no statistical difference in resistance to either clarithromycin or fluoroquinolones(P = 0.55 and P = 0.06,respectively) among the different regions of Brazil. Dual resistance to clarithromycin and fluoroquinolones was found in 4.3%(21/490) of patients. The A2147 G mutation was present in 90.4%(75/83),A2146 G in 16.9%(14/83) and A2146 C in 3.6%(3/83) of clarithromycin-resistant patients. In 10.8%(9/83) of clarithromycin-resistant samples,more than 01 mutation in the 23 S r RNA gene was noticed. In fluoroquinolone-resistant samples,37.9%(25/66) showed mutations not specified by the Geno Type Helico DR test. D91 N mutation was observed in 34.8%(23/66),D91 G in 18.1%(12/66),N87 K in 16.6%(11/66) and D91 Y in 13.6%(9/66) of cases. Among fluoroquinolone-resistant samples,37.9%(25/66) showed mutations not specified by the Geno Type Helico DR test. CONCLUSION The H. pylori clarithromycin resistance rate in Brazil is at the borderline(15%-20%) for applying the standard triple therapy. The fluoroquinolone resistance rate(13.5%) is equally concerning.

Role of hormone receptors and HER2 as prospective molecular markers for breast cancer:An update

This review provides an updated account on the current methods,principles and mechanism of action of therapies for the detection of molecular markers of therapeutic importance in the prognosis of breast cancer progression and recurrence,which includes estrogen receptor(ER),progesterone receptor(PR)and human epidermal growth factor receptor2(HER2).Indeed,hormone-receptors namely,ER,PR,proto-oncogene HER2 are the basic molecular markers that are recognized and established prognostic factors and predictors of response,for therapeutic practice.These markers can be detected by using immunohistochemistry(IHC)and fluorescence in situ hybridization(FISH),which are established,faster and cost effective detection methods.These molecular markers along with clinicopathological prognostic parameters give the best prediction of the prognosis of cancer recurrence and progress.Finally,hormone receptors and HER2 as molecular markers are of prime therapeutic importance and have the capability to take part in future drug development techniques.

Molecular authentication of the traditional Chinese medicine Tongren Dahuoluo Wan and its alternative formulation

Tongren Dahuoluo Wan has been a popular traditional Chinese medicine in international pharmaceutical markets for hundreds of years. Leopard bone powder is the key element in its formulation. However, the leopard has been listed for wildlife conservation, which limits the use of the leopard bone supplies. Therefore, an alternative formulation which substitutes leopard bone with zokor bone in the formula of Tongren Dahuoluo Wan is now manufactured. To develop a simple and reliable molecular method for authenticating the two patent medicines,mitochondrial nucleotide polymorphic sites of 12 S rRNA,COI and Cytb genes were screened in leopard and zokor bones, and nine pairs of species-specific primers were verified for discriminating the two species. For the patent medicine authentication, we set up a molecular diagnostic assay to resolve the difficulties of low concentration of target DNAs and presence of PCR-inhibitory substances in this complex medicine, and successfully confirmed leopard or zokor content using the nine pairs of species-specific primers. We recommend a common technical strategy for authentication of species origins in traditional Chinese medicine, and discuss the experimental solutions for technical problems of molecular diagnostic assays.

Ultrafast photonic PCR

Nucleic acid amplification and quantification via polymerase chain reaction(PCR)is one of the most sensitive and powerful tools for clinical laboratories,precision medicine,personalized medicine,agricultural science,forensic science and environmental science.Ultrafast multiplex PCR,characterized by low power consumption,compact size and simple operation,is ideal for timely diagnosis at the point-of-care(POC).Although several fast/ultrafast PCR methods have been proposed,the use of a simple and robust PCR thermal cycler remains challenging for POC testing.Here,we present an ultrafast photonic PCR method using plasmonic photothermal light-to-heat conversion via photon–electron–phonon coupling.We demonstrate an efficient photonic heat converter using a thin gold(Au)film due to its plasmon-assisted high optical absorption(approximately 65%at 450 nm,the peak wavelength of heat source light-emitting diodes(LEDs)).The plasmon-excited Au film is capable of rapidly heating the surrounding solution to over 150℃ within 3 min.Using this method,ultrafast thermal cycling(30 cycles;heating and cooling rate of 12.7960.93℃ s^(-1) and 6.660.29℃ s^(-1),respectively)from 55℃(temperature of annealing)to 95℃(temperature of denaturation)is accomplished within 5 min.Using photonic PCR thermal cycles,we demonstrate here successful nucleic acid(λ-DNA)amplification.Our simple,robust and low cost approach to ultrafast PCR using an efficient photonic-based heating procedure could be generally integrated into a variety of devices or procedures,including on-chip thermal lysis and heating for isothermal amplifications.

Current Status of Human Papillomavirus-Related Head and Neck Cancer: From Viral Genome to Patient Care

Human papillomavirus(HPV)infection identified as a definitive human carcinogen is increasingly being recognized for its role in carcinogenesis of human cancers.Up to 38%–80%of head and neck squamous cell carcinoma(HNSCC)in oropharyngeal location(OPSCC)and nearly all cervical cancers contain the HPV genome which is implicated in causing cancer through its oncoproteins E6 and E7.Given by the biologically distinct HPV-related OPSCC and a more favorable prognosis compared to HPV-negative tumors,clinical trials on de-escalation treatment strategies for these patients have been studied.It is therefore raised the questions for the patient stratification if treatment de-escalation is feasible.Moreover,understanding the crosstalk of HPV-mediated malignancy and immunity with clinical insights from the proportional response rate to immune checkpoint blockade treatments in patients with HNSCC is of importance to substantially improve the treatment efficacy.This review discusses the biology of HPV-related HNSCC as well as successful clinically findings with promising candidates in the pipeline for future directions.With the advent of various sequencing technologies,further biomolecules associated with HPV-related HNSCC progression are currently being identified to be used as potential biomarkers or targets for clinical decisions throughout the continuum of cancer care.

Oxford Nanopore MinION Sequencing and Genome Assembly

The revolution of genome sequencing is continuing after the successful secondgeneration sequencing （SGS） technology. The third-generation sequencing （TGS） technology, led by Pacific Biosciences （PacBio）, is progressing rapidly, moving from a technology once only capable of providing data for small genome analysis, or for performing targeted screening, to one that promises high quality de novo assembly and structural variation detection for human-sized genomes. In 2014, the MinION, the first commercial sequencer using nanopore technology, was released by Oxford Nanopore Technologies （ONT）. MiniON identifies DNA bases by measuring the changes in electrical conductivity generated as DNA strands pass through a biological pore. Its portability, affordability, and speed in data production makes it suitable for real-time applications, the release of the long read sequencer MiniON has thus generated much excitement and interest in the genomics community. While de novo genome assemblies can be cheaply produced from SGS data, assem- bly continuity is often relatively poor, due to the limited ability of short reads to handle long repeats. Assembly quality can be greatly improved by using TGS long reads, since repetitive regions can be easily expanded into using longer sequencing lengths, despite having higher error rates at the base level. The potential of nanopore sequencing has been demonstrated by various studies in genome surveillance at locations where rapid and reliable sequencing is needed, but where resources are limited.