Molecular Detection Technology Research Articles

[First-line Treatment with Furmonertinib Mesylate in Lung Adenocarcinoma Patient 
with EGFR Exon 20 Insertion Mutantion].

With the continuous innovation of genomics, proteomics and molecular biological detection technology, the treatment of non-small cell lung cancer (NSCLC) has changed from traditional chemotherapy to immunotherapy and targeted therapy. Among them, molecular tumor markers targeting tyrosine kinase pathways play more important roles in clinical practice. For advanced NSCLC patients with positive epidermal growth factor receptor (EGFR) mutations, there are many first-line drugs on the market and they could bring significant efficacy, thus completely subverting the treatment pattern of advanced NSCLC. Common mutations of EGFR in Chinese patients are located on exons 19, 20 and 21, of which exons 19 and 21 mutations are the more common types. Besides, there is also a subtype of EGFR mutations, known as EGFR 20 exon insertion (EGFR 20ins) mutation. The authors summarized the treatment of a lung adenocarcinoma patient with EGFR 20ins mutation accepting Furmonertinib mesylate, in order to provide effective references for clinical diagnosis and treatment.
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Detection of biological loads in sewage using the automated robot‐driven photoelectrochemical biosensing platform

AbstractReal‐time polymerase chain reaction (RT‐PCR) remains the most prevalent molecular detection technology for sewage analysis but is plagued with numerous disadvantages, such as time consumption, high manpower requirements, and susceptibility to false negatives. In this study, an automated robot‐driven photoelectrochemical (PEC) biosensing platform is constructed, that utilizes the CRISPR/Cas12a system to achieve fast, ultrasensitive, high specificity detection of biological loads in sewage. The Shennong‐1 robot integrates several functional modules, involving sewage sampling and pretreatment to streamline the sewage monitoring. A screen‐printed electrode is employed with a vertical graphene‐based working electrode and enhanced with surface‐deposited Au nanoparticles (NPs). CdTe/ZnS quantum dots (QDs) are further fabricated through the double‐stranded DNA (dsDNA) anchored on Au NPs. Using the cDNA template of Omicron BA.5 spike gene as a model, the PEC biosensor demonstrates excellent analytical performance, with a lower detection limit of 2.93 × 102 zm and an outstanding selectivity at the level of single‐base mutation recognition. Furthermore, the rapid, accurate detection of BA.5 in sewage demonstrates the feasibility of the PEC platform for sewage monitoring. In conclusion, this platform allows early detection and tracking of infectious disease outbreaks, providing timely data support for public health institutions to take appropriate prevention and control measures.

Mitochondrial genome provides species-specific targets for the rapid detection of early invasive populations of Hylurgus ligniperda in China

BackgroundHylurgus ligniperda, a major international forestry quarantine pest, was recently found to have invaded and posed a serious threat to the Pinus forests of the Jiaodong Peninsula in China. Continuous monitoring and vigilance of the early population is imperative, and rapid molecular detection technology is urgently needed. We focused on developing a single-gene-based species-specific PCR (SS-PCR) method.ResultsWe sequenced and assembled the mitochondrial genome of H. ligniperda to identify suitable target genes. We identified three closely related species for detecting the specificity of SS-PCR through phylogenetic analysis based on 13 protein-coding genes (PCGs). Subsequently, we analyzed the evolution of 13 PCGs and selected four mitochondrial genes to represent slow-evolving gene (COI) and faster-evolving genes (e.g. ND2, ND4, and ND5), respectively. We developed four species-specific primers targeting COI, ND2, ND4, and ND5 to rapidly identify H. ligniperda. The results showed that the four species-specific primers exhibited excellent specificity and sensitivity in the PCR assays, with consistent performance across a broader range of species. This method demonstrates the ability to identify beetles promptly, even during their larval stage. The entire detection process can be completed within 2–3 h.ConclusionsThis method is suitable for large-scale species detection in laboratory settings. Moreover, the selection of target genes in the SS-PCR method is not affected by the evolutionary rate. SS-PCR can be widely implemented at port and forestry workstations, significantly enhancing early management strategies and quarantine measures against H. ligniperda. This approach will help prevent the spread of the pest and effectively preserve the resources of Chinese pine forests.

Intelligent point of care testing for medicine diagnosis

AbstractIntelligent point‐of‐care testing (iPOCT) is a rapidly advancing technology that combines molecular detection and bio‐sensing techniques to achieve instant and accurate analysis through advanced sensors and intelligent algorithms. Molecular detection technologies, such as nucleic acid isothermal amplification, biochip and microfluidics, enable rapid analysis of the chemical composition and concentration of samples, thereby determining their presence. Bio‐sensing technology utilizes the recognition properties of biomolecules in conjunction with sensors to detect specific biomolecules or biological processes. iPOCT technology holds broad prospects for application in the field of healthcare and is expected to enhance detection sensitivity, accuracy, and intelligence through further development. In this review, we delineate the developmental journey of iPOCT, followed by a discussion on the principles of molecular detection and bio‐sensing technology, as well as the design of iPOCT devices for health monitoring and disease diagnosis. Furthermore, we showcased illustrative examples of iPOCT applications in disease diagnosis, highlighting the integration of iPOCT technology with wearable devices and smartphones for attaining more precise and personalized diagnostic results.

Advances in Detection Techniques for the H5N1 Avian Influenza Virus.

Avian influenza is caused by avian influenza virus infection; the H5N1 avian influenza virus is a highly pathogenic subtype, affecting poultry and human health. Since the discovery of the highly pathogenic subtype of the H5N1 avian influenza virus, it has caused enormous losses to the poultry farming industry. It was recently found that the H5N1 avian influenza virus tends to spread among mammals. Therefore, early rapid detection methods are highly significant for effectively preventing the spread of H5N1. This paper discusses the detection technologies used in the detection of the H5N1 avian influenza virus, including serological detection technology, immunological detection technology, molecular biology detection technology, genetic detection technology, and biosensors. Comparisons of these detection technologies were analyzed, aiming to provide some recommendations for the detection of the H5N1 avian influenza virus.

Identification of the shared gene signatures and molecular pathways in systemic lupus erythematosus and diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) incidence is higher in systemic lupus erythematosus (SLE) patients than the general population, but the molecular mechanisms behind this link remain ambiguous. The aim of this study was to investigate shared gene signatures and molecular pathways between SLE and DLBCL. We procured expression profiles of SLE and DLBCL from public databases and identified common differentially expressed genes (DEGs). Functional pathway enrichment and protein-protein interaction (PPI) analyses were performed on these shared genes. The molecular complex detection technology (MCODE) and eXtreme Gradient Boosting (XGBoost) machine learning algorithm were used to select core shared genes, followed by Gene Set Enrichment Analysis (GSEA) and immune infiltration analysis. We identified 54 DEGs as shared genes, among which CD177, CEACAM1, GPR84 and IFIT3 were identified as core shared genes. These genes showed strong associations with inflammatory and immune response pathways. We found a significant positive correlation between GPR84 and IFIT3 expression levels and the immune microenvironment. Decreased expression levels of GPR84 and IFIT3 were linked to enhanced immune therapy sensitivity, potentially due to lower dysregulation scores during low expression. We also discovered that TP53 mutations might elevate CD177 and GPR84 expression and that reduced expression levels of GPR84 and IFIT3 were linked with better overall survival and progression-free survival in DLBCL patients. Our study provides valuable insights into the shared molecular mechanisms underpinning the pathogenesis of SLE and DLBCL. These findings could potentially offer new biomarkers and therapeutic targets for SLE and DLBCL.

Rapid and non-invasive diagnostic techniques for embryonic developmental potential: a metabolomic analysis based on Raman spectroscopy to identify the pregnancy outcomes of IVF-ET.

The non-invasive and rapid assessment of the developmental potential of embryos is of great clinical importance in assisted reproductive technology (ART). In this retrospective study, we analyzed the metabolomics of 107 samples provided by volunteers and utilized Raman spectroscopy to detect the substance composition in the discarded culture medium of 53 embryos resulting in successful pregnancies and 54 embryos that did not result in pregnancy after implantation. The culture medium from D3 cleavage-stage embryos was collected after transplantation and a total of 535 (107 × 5) original Raman spectra were obtained. By combining several machine learning methods, we predicted the developmental potential of embryos, and the principal component analysis-convolutional neural network (PCA-CNN) model achieved an accuracy rate of 71.5%. Furthermore, the chemometric algorithm was used to analyze seven amino acid metabolites in the culture medium, and the data showed significant differences in tyrosine, tryptophan, and serine between the pregnancy and non-pregnancy groups. The results suggest that Raman spectroscopy, as a non-invasive and rapid molecular fingerprint detection technology, shows potential for clinical application in assisted reproduction.

Recent Advances in Mass Spectrometry-Based Spatially Resolved Molecular Imaging of Drug Disposition and Metabolomics.

Mass spectrometric imaging is a nontargeted, tag-free, high-throughput, and highly responsive analytical approach. The highly accurate molecular visualization detection technology enables qualitative and quantitative analyses of biologic tissues or cells scanned by mass spectrometry in situ, extracting known and unknown multiple compounds, and simultaneously assessing relative contents of targeting molecules by monitoring their molecular ions and pinpointing the spatial locations of those molecules distributed. Five mass spectrometric imaging techniques and their characteristics are introduced in the review, including matrix-assisted laser desorption ionization mass spectrometry, secondary ion mass spectrometry, desorption electrospray ionization mass spectrometry, laser ablation electrospray ionization mass spectrometry, and laser ablation inductively coupled plasma mass spectrometry. The mass spectrometry-based techniques provide the possibility for spatial metabolomics with the capability of high throughput and precision detection. The approaches have been widely employed to spatially image not only metabolome of endogenous amino acids, peptides, proteins, neurotransmitters, and lipids but also the disposition of exogenous chemicals, such as pharmaceutical agents, environmental pollutants, toxicants, natural products, and heavy metals. The techniques also provide us with spatial distribution imaging of analytes in single cells, tissue microregions, organs, and whole animals. SIGNIFICANCE STATEMENT: The review article includes an overview of five commonly used mass spectrometers for spatial imaging and describes the advantages and disadvantages of each. Examples of the technology applications cover drug disposition, diseases, and omics. Technical aspects of relative and absolute quantification by mass spectrometric imaging and challenges for future new applications are discussed as well. The reviewed knowledge may benefit the development of new drugs and provide a better understanding of biochemical processes related to physiology and diseases.

Overview of Molecular Detection Technologies for MET in Lung Cancer.

MET tyrosine kinase receptor pathway activation has become an important actionable target in solid tumors. Aberrations in the MET proto-oncogene, including MET overexpression, the activation of MET mutations, MET mutations that lead to MET exon 14 skipping, MET gene amplifications, and MET fusions, are known to be primary and secondary oncogenic drivers in cancer; these aberrations have evolved as predictive biomarkers in clinical diagnostics. Thus, the detection of all known MET aberrations in daily clinical care is essential. In this review, current molecular technologies for the detection of the different MET aberrations are highlighted, including the benefits and drawbacks. In the future, another focus will be on the standardization of detection technologies for the delivery of reliable, quick, and affordable tests in clinical molecular diagnostics.

A Universal CRISPR/Cas12a‐Assisted Methodology Based on Duplex Switch Structure to Detect Multiple Types of Targets

AbstractRecent years, molecular detection technology has been playing an unprecedentedly important role in disease prevention and public health. Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) systems such as CRISPR/Cas12a and CRISPR/Cas13a, have been increasingly used in the detection of nucleic acid molecules because of its collateral cleavage ability in recent years. Herein, we develop a universal CRISPR/Cas12a‐assisted methodology based on a nucleic acid duplex switch structure that can distinguish different categories of targets, such as DNA, RNA and small molecules. It is worth noting that for nucleic acid detection, this method can significantly identify single base substitutions with high specificity, compared with other Cas12a‐assisted biosensing systems. The experimental results suggest that this method has great specificity for different targets, promising to be applied to rapid molecular diagnosis.

Rapid Identification of Seven Common ABO Alleles by Two-Dimensional Polymerase Chain Reaction Technology

Introduction: The molecular biology detection technology of the human ABO blood group system makes up for the limitations in many aspects compared with conventional serological typing technology. This study aimed to establish a new method to identify seven common ABO alleles (ABO*A1.01, ABO*A1.02, ABO*A2.01, ABO*B.01, ABO*O.01.01, ABO*O.01.02, and ABO*O.02.01) by two-dimensional polymerase chain reaction (2D PCR). 2D PCR can identify multiple target genes in a closed test tube by labeling specific primers with tags homologous to the sequence of fluorescently labeled probes, and melting curve analysis is performed after the fluorescent probes are hybridized with tag complementary sequences in PCR-specific products. In this study, 2D PCR and PCR sequence-specific primer (PCR-SSP) were combined to discriminate different alleles in a single reaction, which has the characteristics of high throughput, and compared with other typing techniques; the typing results can be obtained without additional operations. Methods: The ABO*A1.01 allele genetic sequence was used as the reference sequence. The specific sense and antisense primers for seven common ABO alleles were designed on exons 6 and 7 according to the principle of 2D PCR and PCR-SSP. Single nucleotide polymorphism sites for identifying seven alleles were detected in FAM and HEX channels, respectively. Two hundred sixty DNA samples were enrolled for rapid ABO genotyping by 2D PCR, and 95 of them were selected for Sanger sequencing. The Kappa test was used to analyze the agreement of the methodologies. Results: These 7 alleles each had four characteristic melting valleys at different single nucleotide polymorphism loci. A total of 15 genotypes were detected, including ABO*A1.01/A1.02, ABO*A1.01/O.01.01, ABO*A1.01/O.01.02, ABO*A1.02/A1.02, ABO*A1.02/O.01.01, ABO*A1.02/O.01.02, ABO*B.01/B.01, ABO*B.01/O.01.01, ABO*B.01/O.01.02, ABO*O.01.01/O.01.01, ABO*O.01.01/O.01.02, ABO*O.01.02/O.01.02, ABO*A1.01/B.01, ABO*A1.02/B.01, and ABO*B.01/O.01. v (containing a rare ABO*O allele, based on the sequencing results). The Kappa test showed completely consistent results for 2D PCR and Sanger sequencing (Kappa = 1). Conclusion: The 2D PCR technique could be used for molecular typing of the ABO blood group, which was efficient, rapid, accurate, and economical.

Advances in the application of molecular diagnostic techniques for the detection of infectious disease pathogens (Review).

Infectious diseases are a major global cause of morbidity and mortality, seriously affecting public health and socioeconomic stability. Since infectious diseases can be caused by a wide variety of pathogens with similar clinical manifestations and symptoms that are difficult to accurately distinguish, selecting the appropriate diagnostic techniques for the rapid identification of pathogens is crucial for clinical disease diagnosis and public health management. However, traditional diagnostic techniques have low detection rates, long detection times and limited automation, which means that they do not meet the requirements for rapid diagnosis. Recent years have seen continuous developments in molecular detection technology, which has a higher sensitivity and specificity, shorter detection time and increased automation, and performs an important role in the early and rapid detection of infectious disease pathogens. The present study summarizes recent progress in molecular diagnostic technologies such as PCR, isothermal amplification, gene chips and high‑throughput sequencing for the detection of infectious disease pathogens, and compares the technical principles, advantages and disadvantages, applicability and costs of these diagnostic techniques.

Hatching and development of maize cyst nematode Heteroderazeae infecting different plant hosts1

The occurrence, distribution, and rapid molecular detection technology of Heterodera zeae Koshy, Swarup & Sethi, 1971, have been reported in China. We explored the biological characteristics of H. zeae sampled in Henan Province to understand its interaction with plants. Cysts and second-stage juveniles (J2s) were identified under an optical and scanning electron microscope, internal transcribed spacer (ITS) phylogenetic tree, and sequence characterized amplified region (SCAR)-PCR analyses. The optimum hatching temperatures of H. zeae were 30°C and 28°C, with cumulative hatching rates of 16.5 and 16.1%, respectively, at 30 days post-hatching (dph). The hatching rate of H. zeae eggs was improved by 20- and 50-time maize soil leachate, root juice, and 10-time root exudates. The hatching rate in 10-time root exudates was the highest (25.9%). The 10-time root exudates of maize and millet produced the highest hatching rate at 30 dph (25.9 and 22.9%, respectively), followed by wheat (19.9%), barley (18.3%), and rice (17.6%). H. zeae developed faster in maize than in other crops. Fourth-stage juveniles (J4s) were detected in maize roots 8 days post inoculation (dpi) at 28°C but not in other crops. Combined with hatching tests, the Huang-Huai-Hai summer maize region and the south and central-southwest mountainous maize areas are highly suitable for H. zeae in China. This is the first systematically study of the hatching and infection characteristics on different plant hosts of corn cyst nematode H. zeae in temperate regions. This study laid a theoretical foundation for the rapid spread and high environmental adaptability of corn cyst nematode.

The CRISPR/Cas System: A Customizable Toolbox for Molecular Detection.

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) are promising molecular diagnostic tools for rapidly and precisely elucidating the structure and function of genomes due to their high specificity, programmability, and multi-system compatibility in nucleic acid recognition. Multiple parameters limit the ability of a CRISPR/Cas system to detect DNA or RNA. Consequently, it must be used in conjunction with other nucleic acid amplification techniques or signal detection techniques, and the reaction components and reaction conditions should be modified and optimized to maximize the detection performance of the CRISPR/Cas system against various targets. As the field continues to develop, CRISPR/Cas systems have the potential to become an ultra-sensitive, convenient, and accurate biosensing platform for the detection of specific target sequences. The design of a molecular detection platform employing the CRISPR/Cas system is asserted on three primary strategies: (1) Performance optimization of the CRISPR/Cas system; (2) enhancement of the detection signal and its interpretation; and (3) compatibility with multiple reaction systems. This article focuses on the molecular characteristics and application value of the CRISPR/Cas system and reviews recent research progress and development direction from the perspectives of principle, performance, and method development challenges to provide a theoretical foundation for the development and application of the CRISPR/CAS system in molecular detection technology.

In Situ Proximity Ligation Assay to Visualize Protein-Protein Interactions in Tumor Specimens.

Protein-protein interactions (PPI) are the basis of various biological phenomena, such as intracellular signal transduction, gene transcription, and metabolism. PPI are also considered to be involved in the pathogenesis and development of various diseases, including cancer. PPI phenomenon and their functions have been elucidated by gene transfection and molecular detection technologies. On the other hand, in histopathological analysis, although immunohistochemical analyses provide information pertaining to protein expression and their localization in pathophysiological tissues, it has been difficult to visualize the PPI of these proteins. An in situ proximity ligation assay (PLA) was developed as a microscopic visualization technique for PPI in formalin-fixed, paraffin-embedded (FFPE) tissues as well as in cultured cells and frozen tissues. PLA using histopathological specimens enables cohort studies of PPI, which can clarify the significance of PPI in pathology. We have previously shown the dimerization pattern of estrogen receptors and significance of HER2-binding proteins using breast cancer FFPE tissues. In this chapter, we describe a methodology for the visualization of PPI using PLA in pathological specimens.

Low LOD and Non-Adsorption Probing of Functionalized Magnetic Beads Using Quartz Crystal Microbalance Under Bimodal Magnetic Field

Low limit of detection (LOD) and highly sensitive probing of functional magnetic nanobead labels is the cornerstone of magnetic label molecular detection technology, which is of great significance in analytical chemistry and biomolecular analysis. Here we present a probing technology based on quartz crystal microbalance (QCM) combines bimodal magnetic fields to accurate and rapid probing of magnetic beads. The proposed technique uses QCM to detect the change of viscosity and density of tested solution under a bimodal magnetic field, thereby enabling the quantification of magnetic bead concentration. The experimental findings demonstrate that the technique can detect magnetic beads accurately and rapidly within 0 ng/mL to 100 μg/mL without anchoring magnetic beads on the QCM. Furthermore, when the particle size of magnetic beads is 1 μm and 0.28 μm, the LOD is reached at 4.62 ng/mL and 2.31 ng/mL, respectively. The versatility of the no-modification probing platform yet the wide detection range and low LOD to various magnetic particles, showcase the potential of the proposed technology to enable swift, repeatable, and dependable assays across a broad range of applications, including but not limited to biomolecule analysis, Molecular genetics, pre-symptomatic diagnosis, and biosafety.

Status and Recent Developments of Pantoea stewartii subsp. stewartii Causes the Wilt Disease in Maize in Indonesia: A Review

<em>Pantoea stewartii</em> subsp. <em>stewartii</em> is a pathogen that causes Stewart wilt on maize. This disease is important in maize. Infection early in growth can cause yield losses of up to 100%. This review examines the history, recent developments, economic impacts and developments of detection technology of Stewart wilt diseases. This paper was created by reviewing several articles relevant to the purpose of the topic. The article's results revealed that the pathogen underwent many changes, including its name, taxonomy, physiological abilities, status as an important pathogen in many countries and the development of detection technology. Currently, <em>P. stewartii</em> subsp. <em>stewartii</em> belongs to the Erwiniaceae family and has physiological abilities that can be distinguished from bacteria of the same genus and species. This pathogen has been reported to spread to more than 82 countries, including Indonesia, with 18 host plants. Some areas have reported pathogens in Indonesia, but no vector has been written. The biggest economic threat caused by this disease is the industrial production of corn seeds which require strict phytosanitary requirements and are free from pathogens. To anticipate its spread, there are four methods of detection of pathogenic bacteria that are commonly used, but serological and molecular detection technologies are the main recommendations.

Identification of B(A) Blood Group Serology Combined with Molecular Biology and Blood Transfusion Strategy

To analyze the reasons for ABO blood group forward typing and reverse typing incompatibility of the proband by serological and molecular biological tests, so as to clarify the blood group and genetic rules and determine the reasonable transfusion strategy. On the basis of serological testing results, PCR-SSP method was utilized for the ABO exon sequencing in the peripheral blood of the proband and 5 family members, and blood group results were comprehensively analyzed. The serological results of ABO blood group of the proband were incompatibility, and the molecular biological test results showed that there was a c.700C>G mutation compared with B101, which was consistent with B(A)02 subtype, and the genotype was B(A)02/O02. The results of the elder brother was the same as the proband. The nephew of the proband also detected c.700C>G, genotype A102/B(A) 02. When the result of standard serological test for ABO blood group incompatibility occurs, using molecular biology detection technology to explore mutation is an effective method to confirm ABO subtype, and ensures the safety of clinical blood transfusion.

Research Progress in Detection and Control of Soil Borne Disease Fusarium Root Rot

Fusarium is a variety of species, which is difficult to distinguish in morphology. Traditionally, identification of pathogenic fusarium requires the separation of pathogenic bacteria, purification of pathogenic bacteria, separation and purification after tieback, sequencing and other processes, which are time-consuming and laborious. Molecular detection technology can make pathogen identification more efficient. Fusarium control mainly includes cultivating disease-resistant varieties, chemical control, physical control, biological control and crop rotation. In recent years, various root rot bacteria caused by Fusarium have shown an increasing trend. By reviewing the previous relevant research results, this study summarized the research progress of molecular detection and prevention measures of root rot caused by the soilborne disease fungus Fusarium, and explored the current research status and existing problems of fusarium. In order to provide theoretical reference for further study of soil-borne pathogenic fungi.

The Value of GeneXpert MTB/RIF for Detection in Tuberculosis: A Bibliometrics-Based Analysis and Review.

With the continuous development of medical science and technology, especially with the advent of the era of precision diagnosis and treatment, molecular biology detection technology is widely valued and applied as an aid to early diagnosis of tuberculosis. The GeneXpert Mycobacterium tuberculosis Branching (MTB) technology is a suite of semi-nested real-time fluorescent quantitative PCR in vitro diagnostic technologies developed by Cepheid Inc. It targets the rifampicin resistance gene, rpoB, and can detect both MTB and resistance to rifampicin within 2 h. This review analyzed the papers related to GeneXpert using bibliometric software CiteSpace and Bibliometrix. A total of 151 articles were analyzed, spanning from 2011 to 2021. This bibliometrics-based review summarizes the history of the development of GeneXpert in tuberculosis diagnosis and its current status. Contributions of different countries to the topic, journal analysis, key paper analysis, and clustering of keywords were used to analyze this topic.