Current and future molecular diagnostics in colorectal cancer and colorectal adenoma

Colorectal cancer(CRC)is one of the most prevalent cancers in developed countries.On the other hand,CRC is also one of the most curable cancers if it is detected in early stages through regular colonoscopy or sigmoidoscopy.Since CRC develops slowly from precancerous lesions,early detection can reduce both the incidence and mortality of the disease.Fecal occult blood test is a widely used non-invasive screening tool for CRC.Although fecal occult blood test is simple and cost-effective in screening CRC,there is room for improvement in terms of the accuracy of the test.Genetic dysregulations have been found to play an important role in CRC development.With better understanding of the molecular basis of CRC,there is a growing expectation on the development of diagnostic tests based on more sensitive and specific molecular markers and those tests may provide a breakthrough to the limitations of current screening tests for CRC.In this review,the molecular basis of CRC development,the characteristics and applications of different non-invasive molecular biomarkers,as well as the technologies available for the detection were discussed.This review intended to provide a summary on the current and future molecular diagnostics in CRC and its pre-malignant state,colorectal adenoma.

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.

Advancing public health preparedness:Establishment of Nipah virus molecular diagnostic test at the National Reference Laboratory,Sri Lanka

Objective:To establish Nipah virus diagnostic capabilities at the National Reference Laboratory in Sri Lanka using the NIV Pune real-time PCR kit.Methods:Strict safety precautions were adhered during testing due to the high pathogenicity of the Nipah virus,with all diagnostics conducted in a BSL2+laboratory at the Medical Research Institute in Sri Lanka.RNA extraction was performed using the QIAamp Viral RNA Mini kit.The NIV Pune in-house real-time PCR kit was employed,following established primer/probe sequences and controls.The assay was validated using the Rotor-Gene Q Series Real-time PCR platform.Results:The validation run of the Nipah virus real-time PCR test demonstrated robust performance,with positive controls consistently detecting Nipah RNA at a Ct value of 21.50±0.01.Negative controls confirmed assay specificity with an external negative control which was also used as an extraction control and showed no interference.The internal control exhibited stable behavior,enhancing confidence in PCR results.The qPCR analysis graph illustrated the successful detection of internal and positive controls,validating the reliability of the assay.Conclusions:Establishing Nipah virus diagnostic capabilities in Sri Lanka signifies a proactive and collaborative response to the persistent global health threat.

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.

Non-canonical BRAF variants and rearrangements in hairy cell leukemia

Hairy cell leukemia(HCL)is an uncommon mature B-cell malignancy characterized by a typical morphology,immunophenotype,and clinical profile.The vast majority of HCL patients harbor the canonical BRAF V600E mutation which has become a rationalized target of the subsequently deregulated RAS-RAF-MEK-MAPK signaling pathway in HCL patients who have relapsed or who are refractory to front-line therapy.However,several HCL patients with a classical phenotype display non-canonical BRAF mutations or rearrangements.These include sequence variants within alternative exons and an oncogenic fusion with the IGH gene.Care must be taken in the molecular diagnostic work-up of patients with typical HCL but without the BRAF V600E to include investigation of these uncommon mechanisms.Identification,functional characterization,and reporting of further such patients is likely to provide insights into the pathogenesis of HCL and enable rational selection of targeted inhibitors in such patients if required.

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.

Next generation sequencing in oral disease diagnostics

DNA sequencing is the method of identifying the precise order of DNA nucleotides within a molecule. The information of DNA sequencing is of prime requisite for basic biological research as well as in various clinical specialties.They can be used to determine the individual genetic sequence, larger genetic regions, chromosomes as well as to sequence RNA and proteins. Since the first DNA sequencing in 1970s, there has been tremendous advancements in the technologies aimed to determine the entire human genome. The need for rapid and accurate sequencing of human genome has resulted in the introduction of next generation sequencing(NGS) technology. NGS refers to the secondgeneration DNA sequencing technologies where millions of DNA can be sequenced simultaneously. Some of the next gen sequencing methods employed are Roche/454 life science, Illumina/Solexa, SOLiD system and HeliScope.Application of NGS in decoding the genomic database of various oral diseases may possess therapeutic and prognostic value. This presentation provides an overview of the basics of NGS and their potential applications in oral disease diagnostics.

Molecular diagnosis and therapy for occult peritoneal metastasis in gastric cancer patients

To apply an individualized oncological approach to gastric cancer patients,the accurate diagnosis of disease entities is required.Peritoneal metastasis is the most frequent mode of metastasis in gastric cancer,and the tumor-node-metastasis classification includes cytological detection of intraperitoneal cancer cells as part of the staging process,denoting metastatic disease.The accuracy of cytological diagnosis leaves room for improvement;therefore,highly sensitive molecular diagnostics,such as an enzyme immunoassay,reverse transcription polymerase chain reaction,and virusguided imaging,have been developed to detect minute cancer cells in the peritoneal cavity.Molecular targeting therapy has also been spun off from basic research in the past decade.Although conventional cytologyis still the mainstay,novel approaches could serve as practical complementary diagnostics to cytology in near future.

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.

Protein-nucleic acid hybrid nanostructures for molecular diagnostic applications

Molecular diagnostic technologies empower new clinical opportunities in precision medicine.However,existing approaches face limitations with respect to performance,operation and cost.Biological molecules including proteins and nucleic acids are being increasingly adopted as tools in the development of new molecular diagnostic technologies.In particular,leveraging their complementary properties—the functional diversity of proteins and the precision programmability of nucleic acids—a wide range of protein–nucleic acid hybrid nanostructures have been developed.These hybrid structures take diverse forms,ranging from one-dimensional to three-dimensional hybrids,as static assemblies to dynamic machines,and possess myriad functions to recognize target biomarkers,encode vast information and execute catalytic activities.Motivated by recent advances in this area of molecular nanotechnology,we review the state-of-art design and application of various types of protein–nucleic acid hybrid nanostructures for molecular diagnostics,and present an outlook on the challenges and opportunities for emerging pre-clinical and clinical applications,highlighting the promise for earlier detection,more refined diagnosis and highly tailored treatment decision that ultimately lead to improved patient outcomes.

Occult hepatitis B virus infection among Mexican human immunodeficiency virus-1-infected patients

AIM: To determine the frequency of occult hepatitis B infection (OHBI) in a group of human immunodeficiency virus (HIV)-1+/ hepatitis B surface antigen negative (HBsAg)- patients from Mexico.

Prevalence of HIV and HCV infections in two populations of Malian women and serological assays performances

AIM: To estimate the prevalence of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections in women in Mali and to evaluate the performance of serological assays.

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.

Current practices and guidelines for clinical next-generation sequencing oncology testing

Next-generation sequencing(NGS) has been rapidly integrated into molecular pathology, dramatically increasing the breadth genomic of information available to oncologists and their patients. This review will explore the ways in which this new technology is currently applied to bolster care for patients with solid tumors and hematological malignancies, focusing on practices and guidelines for assessing the technical validity and clinical utility of DNA variants identified during clinical NGS oncology testing.

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.

Multiplex ligation-dependent probe amplification for rapid detection of deletions and duplications in the dystrophin gene

Objective：Duchenne muscular dystrophy （DMD） and Becker muscular dystrophy （BMD） are X-linked disorders caused by mutations in the dystrophin gene. The majority of recognized mutations are copy number changes of individual exons. The objective of the present study was to assess the multiplex ligation-dependent probe amplification （MLPA） effects of detection of gene mutations. Methods： Samples of 20 control males and 80 males and their mothers referred to our diagnostic facility on the clinical suspi- cion of DMD or BMD were tested by MLPA and multiplex PCR. Results ： The mean DQs for all peak of 20 control male samples was 1.02 （range from 0.83 to 1.21） by MLPA. Deletions or duplications were iden- tified in 6 out of 31 families that had been previously tested as negative by multiplex PCR. One case of complex rearrangement involving a duplication of two regions： dupEX3-9 and dupEX 17-41 were found by MLPA. Conclusions： MLPA is a highly sensitive method and rapid alternative to multiplex PCR for detec- tion of DMD and BMD.

A novel diagnostic gene region for distinguishing between two pest fruit flies:Bactrocera tryoni(Froggatt)and Bactrocera neohumeralis(Hardy)(Diptera:Tephritidae)

Bactrocera tryoni and Bactrocera neohumeralis are morphologically similar sibling pest fruit fly species that possess different biological attributes,geographic distributions,and host ranges.The need to differentiate between the two species is critical for accurate pest status assessment,management,biosecurity,and maintenance of reference colonies.While morphologically similar,adults may be separated based on subtle characters;however,some characters exhibit intraspecific variability,creating overlap between the two species.Additionally,there is currently no single molecular marker or rapid diagnostic assay that can reliably distinguish between B.neohumeralis and B.tryoni;therefore,ambiguous samples remain undiagnosed.Here we report the first molecular marker that can consistently distinguish between B.tryoni and B.neohumeralis.Our diagnostic region consists of two adjacent single nucleotide polymorphisms(SNPs)within the pangolin(pan)gene region.We confirmed the genotypes of each species are consistent across their distributional range,then developed a tetra-primer amplification refractory mutation system(ARMS)PCR assay for rapid diagnosis of the species.The assay utilizes four primers in multiplex,with two outer universal primers,and two internal primers:one designed to target two adjacent SNPs(AA)present in B.tryoni and the other targeting adjacent SNPs present in B.neohumeralis(GG).The assay accurately discriminates between the two species,but their SNP genotypes are shared with other nontarget tephritid fruit fly species.Therefore,this assay is most suited to adult diagnostics where species confirmation is necessary in determining ambiguous surveillance trap catches;maintaining pure colony lines;and in Sterile Insect Technique management responses.

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.