Cell-SELEX and application research of a DNA aptamer against esophageal squamous cell carcinoma (ESCC) cell line TE-1.

Esophageal cancer is a common cancer with high morbidity and mortality that severely threatens the safety and quality of human life. The strong metastatic nature of esophageal cancer enables it to metastasize more quickly and covertly, making it difficult for current diagnostic and treatment methods to achieve efficient early screening, as well as timely and effective treatment. As a promising solution, nucleic acid aptamers, a kind of special single-stranded DNA or RNA oligonucleotide selected by the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technology, can specifically bind with different molecular targets. In this paper, random DNA single-stranded oligonucleotides were used as the initial library. Using TE-1 cells and HEEC cells as targets, specific binding sequences were selected by 15 rounds of the cell-SELEX method, and the aptamer sequence that binds to TE-1 cells with the most specificity was obtained and named Te4. The Te4 aptamer was further validated for binding specificity, binding affinity, type of target, in vitro cytotoxicity when conjugated with DOX(Te4-DOX), and in vivo distribution. Results of in vitro validation showed that Te4 has outstanding binding specificity with a Kd value of 51.16 ± 5.52 nM, and the target type of Te4 was preliminarily identified as a membrane protein. Furthermore, the cytotoxicity experiment showed that Te4-DOX has specific cytotoxicity towards cultured TE-1 cells. Finally, the results of the in vivo distribution experiment showed that the Te4 aptamer is able to specifically target tumor regions in nude mice, showing great potential to be applied in future diagnosis and targeted therapy of esophageal cancer.

Viral aptamer screening and aptamer-based biosensors for virus detection: A review.

Virus-induced infectious diseases have a detrimental effect on public health and exert significant influence on the global economy. Therefore, the rapid and accurate detection of viruses is crucial for effectively preventing and diagnosing infections. Aptamer-based detection technologies have attracted researchers' attention as promising solutions. Aptamers, small single-stranded DNA or RNA screened via systematic evolution of ligands by exponential enrichment (SELEX), possess a high affinity towards their target molecules. Numerous aptamers targeting viral marker proteins or virions have been developed and widely employed in aptamer-based biosensors (aptasensor) for virus detection. This review introduces SELEX schemes for screening aptamers and discusses distinctive SELEX strategies designed explicitly for viral targets. Furthermore, recent advances in aptamer-based biosensing methods for detecting common viruses using different virus-specific aptamers are summarized. Finally, limitations and prospects associated with developing of aptamer-based biosensors are discussed.

From biogenesis to aptasensors: advancements in analysis for tumor-derived extracellular vesicles research.

Extracellular vesicles (EVs) are enclosed by a nanoscale phospholipid bilayer membrane and typically range in size from 30 to 200 nm. They contain a high concentration of specific proteins, nucleic acids, and lipids, reflecting but not identical to the composition of the parent cell. The inherent characteristics and variety of EVs give them extensive and unique advantages in the field of cancer identification and treatment. Recently, EVs have been recognized as potential tumor markers for the detection of cancer. Aptamers, which are molecules of single-stranded DNA or RNA, demonstrate remarkable specificity and affinity for their targets by adopting distinct tertiary structures. Aptamers offer various advantages over their protein counterparts, such as reduced immunogenicity, the ability for convenient large-scale synthesis, and straightforward chemical modification. In this review, we summarized EVs biogenesis, sample collection, isolation, storage and characterization, and finally provided a comprehensive survey of analysis techniques for EVs detection that are based on aptamers.

Multi-omics study reveals different pathogenesis of the generation of skin lesions in SLE and IDLE patients.

Lupus erythematosus (LE) is a heterogeneous, antibody-mediated autoimmune disease. Isolate discoid LE (IDLE) and systematic LE (SLE) are traditionally regarded as the two ends of the spectrum, ranging from skin-limited damage to life-threatening multi-organ involvement. Both belong to LE, but IDLE and SLE differ in appearance of skin lesions, autoantibody panels, pathological changes, treatments, and immunopathogenesis. Is discoid lupus truly a form of LE or is it a completely separate entity? This question has not been fully elucidated. We compared the clinical data of IDLE and SLE from our center, applied multi-omics technology, such as immune repertoire sequencing, high-resolution HLA alleles sequencing and multi-spectrum pathological system to explore cellular and molecular phenotypes in skin and peripheral blood from LE patients. Based on the data from 136 LE patients from 8 hospitals in China, we observed higher damage scores and fewer LE specific autoantibodies in IDLE than SLE patients, more uCDR3 sharing between PBMCs and skin lesion from SLE than IDLE patients, elevated diversity of V-J recombination in IDLE skin lesion and SLE PBMCs, increased SHM frequency and class switch ratio in IDLE skin lesion, decreased SHM frequency but increased class switch ratio in SLE PBMCs, HLA-DRB1*03:01:01:01, HLA-B*58:01:01:01, HLA-C*03:02:02:01, and HLA-DQB1*02:01:01:01 positively associated with SLE patients, and expanded Tfh-like cells with ectopic germinal center structures in IDLE skin lesions. These findings suggest a significant difference in the immunopathogenesis of skin lesions between SLE and IDLE patients. SLE is a B cell-predominate systemic immune disorder, while IDLE appears limited to the skin. Our findings provide novel insights into the pathogenesis of IDLE and other types of LE, which may direct more accurate diagnosis and novel therapeutic strategies.

Thymus as Incontrovertible Target of Future Immune Modulatory Therapeutics.

Thymus plays a crucial role in cellular immunity by acting as a warehouse for proliferating and differentiating lymphocytes. Thymic stromal cells educate T-cells to differentiate self from non-self antigens while nurse cells and thymoproteasome play a major role in the maturation and differentiation of T-cells. The thymic conditions dictate T-cells to cope with the risk of cancer development. A study was designed to demonstrate potential mechanisms behind the failure to eliminate tumors and impaired immune surveillance as well as the impact of delay in thymus regression on cancer and autoimmune disorders. Scientific literature from Pubmed; Scopus; WOS; JSTOR; National Library of Medicine Bethesda, Maryland; The New York Academy of Medicine; Library of Speech Rehabilitation, NY; St. Thomas' Hospital Library; The Wills Library of Guys Hospital; Repository of Kings College London; and Oxford Academic repository was explored for pathological, physiological, immunological and toxicological studies of thymus. Studies have shown that systemic chemotherapy may lead to micro inflammatory environment within thymus where conventionally and dynamically metastasized dormant cells seek refuge. The malfunctioning of the thymus and defective T and Treg cells, bypassing negative selection, contributes to autoimmune disorders, while AIRE and Fezf2 play significant roles in thymic epithelial cell solidity. Different vitamins, TCM, and live cell therapy are effective therapeutics. Vitamin A, C, D, and E, selenium and zinc, cinobufagin and dietary polysaccharides, and glandular extracts and live cell injections have strong potential to restore immune system function and thymus health. Moreover, the relationship between different ages/stages of thymus and their corresponding T-cell mediated anti-tumor immune response needs further exploration.

Gravity-driven flow control in a fully integrated microfluidic cartridge for molecular point-of-care testing.

Molecular point-of-care testing (POCT) system is crucial for the timely prevention and control of infectious diseases. We recently proposed a gravity-driven microfluidic cartridge for molecular POCT detection, without the need for external sources or actuators, demonstrating the advantages in terms of the reduced cartridge size and low development costs. How to achieve precise control of liquid flow behavior is challenging for the gravity-driven cartridge. In this work, we explored the underlying mechanism of flow control in the cartridge and offered optimized solutions for our cartridge design to achieve precise control of dynamic flow rates and enhance pumping efficiency significantly. Through the computational fluid dynamics simulations, we demonstrated that adopting an asymptotic contraction chamber geometry design and a closed-loop air flow channel design with the cartridge inlet can facilitate stable laminar flow of the liquid in our microfluidic cartridge, enabling precise control of flow velocity. We further optimized the microchannel diameter and the contact angle of the liquid with the microchannel wall. The effectiveness of the optimized cartridge for POCT detection was well validated by the accurate detection of the human papillomavirus type 16 virus in the 120 clinical swab samples.

Research on a Magnetic Separation-Based Rapid Nucleic Acid Extraction System and Its Detection Applications.

Nucleic acid extraction represents the "first step" in molecular diagnostic experiments. The quality of this extraction serves as a fundamental prerequisite for ensuring the accuracy of nucleic acid detection. This article presents a comprehensive design scheme for a rapid automated nucleic acid extraction system based on magnetic separation. The design and implementation of the system are analyzed and investigated in-depth, focusing on the core methods, hardware control, and software control of the automated nucleic acid extraction system. Additionally, a study and evaluation were carried out concerning the nucleic acid extraction and detection aspects encompassed by the system. The results demonstrate that the temperature deviation in the lysis and elution fluids is approximately ±1 °C, the positioning accuracy of the system's movement is ±0.005 mm, the average magnetic bead recovery rate is 94.98%, and the average nucleic acid recovery rate is 91.83%. The developed automated system and manual methods are employed for sample extraction, enabling the isolation of highly pure nucleic acids from bacteria, blood, and animal tissues for RT-PCR detection. The instrument employs lysis temperatures ranging from 70-80 °C, elution temperature of 80 °C, and drying time of 5-10 min, with a total extraction time of less than 35 min for different sample types. Overall, the system yields high nucleic acid concentration and purity, exhibits stable instrument operation, good repeatability, high efficiency, and low cost. It meets the requirements of genetic-level research and is worthy of clinical promotion and usage.

A Chamber-Based Digital PCR Based on a Microfluidic Chip for the Absolute Quantification and Analysis of KRAS Mutation.

The Kirsten rat sarcoma virus gene (KRAS) is the most common tumor in human cancer, and KRAS plays an important role in the growth of tumor cells. Normal KRAS inhibits tumor cell growth. When mutated, it will continuously stimulate cell growth, resulting in tumor development. There are currently few drugs that target the KRAS gene. Here, we developed a microfluidic chip. The chip design uses parallel fluid channels combined with cylindrical chamber arrays to generate 20,000 cylindrical microchambers. The microfluidic chip designed by us can be used for the microsegmentation of KRAS gene samples. The thermal cycling required for the PCR stage is performed on a flat-panel instrument and detected using a four-color fluorescence system. "Glass-PDMS-glass" sandwich structure effectively reduces reagent volatilization; in addition, a valve is installed at the sample inlet and outlet on the upper layer of the chip to facilitate automatic control. The liquid separation performance of the chip was verified by an automated platform. Finally, using the constructed KRAS gene mutation detection system, it is verified that the chip has good application potential for digital polymerase chain reaction (dPCR). The experimental results show that the chip has a stable performance and can achieve a dynamic detection range of four orders of magnitude and a gene mutation detection of 0.2%. In addition, the four-color fluorescence detection system developed based on the chip can distinguish three different KRAS gene mutation types simultaneously on a single chip.

Aptamers in cancer therapy: problems and new breakthroughs.

Aptamers, a class of oligonucleotides that can bind with molecular targets with high affinity and specificity, have been widely applied in research fields including biosensing, imaging, diagnosing, and therapy of diseases. However, compared with the rapid development in the research fields, the clinical application of aptamers is progressing at a much slower speed, especially in the therapy of cancer. Obstructions including nuclease degradation, renal clearance, a complex selection process, and potential side effects have inhibited the clinical transformation of aptamer-conjugated drugs. To overcome these problems, taking certain measures to improve the biocompatibility and stability of aptamer-conjugated drugs in vivo is necessary. In this review, the obstructions mentioned above are thoroughly discussed and the methods to overcome these problems are introduced in detail. Furthermore, landmark research works and the most recent studies on aptamer-conjugated drugs for cancer therapy are also listed as examples, and the future directions of research for aptamer clinical transformation are discussed.

Chitosan-based packaging films with an integrated antimicrobial peptide: Characterization, in vitro release and application to fresh pork preservation.

Chitosan (CS) films were developed incorporating peptide HX-12C. The films were studied to determine their microstructures, physical properties, release properties of peptide HX-12C and functional properties. The results indicated that there may be hydrogen bonding interactions between CS and peptide HX-12C, thereby creating a homogeneous internal microstructure and lower crystallinity (10.8-12.8 %). Compared with CS film, CS-HX-12C films displayed lower light transmission, MC (20.8-19.9 %), WVP (8.82-8.59 × 10-11·g·m-1·s-1·Pa-1), OTR (0.015-0.037 cc/(m2.day)) and higher WS (15.7-32.4 %) values. Moreover, controlled-release experiments showed that pH, ionic strength and temperature could all significantly affect the release of peptide HX-12C from the films. Finally, the increase of pH value and TVC and lipid oxidation of fresh pork were delayed due to the treatment with CS-2%HX-12C film. However, incorporating peptide HX-12C into CS films did not improve the mechanical properties of the films and their effects against protein oxidation. Our results suggest that the CS-based antimicrobial packaging films integrated with peptide HX-12C exhibit the potential for fresh pork preservation.

Advancements, challenges and future perspectives on peptide-based drugs: Focus on antimicrobial peptides.

Among other health related issues, the rising concerns on drug resistance led to look for alternative pharmaceutical drugs that are effective both against infectious and noninfectious diseases. Antimicrobial peptides (AMPs) emerged as potential therapeutic molecule with wide range of applications. With their limitations, AMPs have gained reputable attentions in research as well as in the pharmaceutical industry. This review highlighted the historical background, research trends, technological advancements, challenges, and future perspectives in the development and applications of peptide drugs. Some vital questions related with the need for pharmaceutical production, factors for the slow and steady journey, the importance of oral bioavailability, and the drug resistance possibilities of AMPs were raised and addressed accordingly. Therefore, the current study is believed to provide a profound understanding in the past and current scenarios and future directions on the therapeutic impacts of peptide drugs.

Novel aerosol detection platform for SARS­CoV­2: Based on specific magnetic nanoparticles adsorption sampling and digital droplet PCR detection.

The SARS­CoV­2 virus is released from an infectious source (such as a sick person) and adsorbed on aerosols, which can form pathogenic microorganism aerosols, which can affect human health through airborne transmission. Efficient sampling and accurate detection of microorganisms in aerosols are the premise and basis for studying their properties and evaluating their hazard. In this study, we built a set of sub-micron aerosol detection platform, and carried out a simulation experiment on the SARS­CoV­2 aerosol in the air by wet-wall cyclone combined with immunomagnetic nanoparticle adsorption sampling and ddPCR. The feasibility of the system in aerosol detection was verified, and the influencing factors in the detection process were experimentally tested. As a result, the sampling efficiency was 29.77%, and extraction efficiency was 98.57%. The minimum detection limit per unit volume of aerosols was 250 copies (102 copies/mL, concentration factor 2.5).

Identification of PHB2 as a Potential Biomarker of Luminal A Breast Cancer Cells Using a Cell-Specific Aptamer.

Precise diagnosis of breast cancer molecular subtypes remains a great challenge in clinics. The present molecular biomarkers are not specific enough to classify breast cancer subtypes precisely, which requests for more accurate and specific molecular biomarkers to be discovered. Aptamers evolved by the cell-systematic evolution of ligands by exponential enrichment (SELEX) method show great potential in the discovery and identification of cell membrane targets via aptamer-based cell membrane protein pull-down, which has been regarded as a novel and powerful weapon for the discovery and identification of new molecular biomarkers. Herein, a cell membrane protein PHB2 was identified as a potential molecular biomarker specifically expressed in the cell membranes of MCF-7 breast cancer cells using a DNA aptamer MF3Ec. Further experiments demonstrated that the PHB2 protein is differentially expressed in the cell membranes of MCF-7, SK-BR-3, and MDA-MB-231 breast cancer cells and MCF-10A cells, and the binding molecular domains of aptamer MF3Ec and anti-PHB2 antibodies to the PHB2 protein are different due to there being no obvious competitions between aptamer MF3Ec and anti-PHB2 antibodies in the binding to the cell membranes of target MCF-7 cells. Due to those four cells belonging to luminal A, HER2-positive, and triple-negative breast cancer cell subtypes and human normal mammary epithelial cells, respectively, the PHB2 protein in the cell membrane may be a potential biomarker for precise diagnosis of the luminal A breast cancer cell subtype, which is endowed with the ability to differentiate the luminal A breast cancer cell subtype from HER2-positive and triple-negative breast cancer cell subtypes and human normal mammary epithelial cells, providing a new molecular biomarker and therapeutic target for the accurate and precise classification and diagnostics and personalized therapy of breast cancer.

Soft integration of a neural cells network and bionic interfaces.

Both glial cells and neurons can be considered basic computational units in neural networks, and the brain-computer interface (BCI) can play a role in awakening the latency portion and being sensitive to positive feedback through learning. However, high-quality information gained from BCI requires invasive approaches such as microelectrodes implanted under the endocranium. As a hard foreign object in the aqueous microenvironment, the soft cerebral cortex's chronic inflammation state and scar tissue appear subsequently. To avoid the obvious defects caused by hard electrodes, this review focuses on the bioinspired neural interface, guiding and optimizing the implant system for better biocompatibility and accuracy. At the same time, the bionic techniques of signal reception and transmission interfaces are summarized and the structural units with functions similar to nerve cells are introduced. Multiple electrical and electromagnetic transmissions, regulating the secretion of neuromodulators or neurotransmitters via nanofluidic channels, have been flexibly applied. The accurate regulation of neural networks from the nanoscale to the cellular reconstruction of protein pathways will make BCI the extension of the brain.

Lung Cancer Nodules Detection via an Adaptive Boosting Algorithm Based on Self-Normalized Multiview Convolutional Neural Network.

Lung cancer is the deadliest cancer killing almost 1.8 million people in 2020. The new cases are expanding alarmingly. Early lung cancer manifests itself in the form of nodules in the lungs. One of the most widely used techniques for both lung cancer early and noninvasive diagnosis is computed tomography (CT). However, the intensive workload of radiologists to read a large number of scans for nodules detection gives rise to issues like false detection and missed detection. To overcome these issues, we proposed an innovative strategy titled adaptive boosting self-normalized multiview convolution neural network (AdaBoost-SNMV-CNN) for lung cancer nodules detection across CT scans. In AdaBoost-SNMV-CNN, MV-CNN function as a baseline learner while the scaled exponential linear unit (SELU) activation function normalizes the layers by considering their neighbors' information and a special drop-out technique (α-dropout). The proposed method was trained and tested using the widely Lung Image Database Consortium and Image Database Resource Initiative (LIDC-IDRI) and Early Lung Cancer Action Program (ELCAP) datasets. AdaBoost-SNMV-CNN achieved an accuracy of 92%, sensitivity of 93%, and specificity of 92% for lung nodules detection on the LIDC-IDRI dataset. Meanwhile, on the ELCAP dataset, the accuracy for detecting lung nodules was 99%, sensitivity 100%, and specificity 98%. AdaBoost-SNMV-CNN outperformed the majority of the model in accuracy, sensitivity, and specificity. The multiviews confer the model's good generalization and learning ability for diverse features of lung nodules, the model architecture is simple, and has a minimal computational time of around 102 minutes. We believe that AdaBoost-SNMV-CNN has good accuracy for the detection of lung nodules and anticipate its potential application in the noninvasive clinical diagnosis of lung cancer. This model can be of good assistance to the radiologist and will be of interest to researchers involved in the designing and development of advanced systems for the detection of lung nodules to accomplish the goal of noninvasive diagnosis of lung cancer.

A miniaturized and integrated dual-channel fluorescence module for multiplex real-time PCR in the portable nucleic acid detection system.

A portable nucleic acid detection (PNAD) system based on real-time polymerase chain reaction (real-time PCR) has been developed for point-of-care testing (POCT) of infectious disease pathogens. In order to achieve "sample-in, result-out" while keeping the system compact, the hardware system integrates optical, thermal and motion control modules in a limited space for nucleic acid extraction, purification, amplification and detection. Among these hardware modules, the fluorescence module is one of the most important modules, because its performance directly affects the accuracy and sensitivity of the testing results. In this paper, a miniaturized, high-sensitivity and integrated dual-channel fluorescence module have been proposed for the homemade PNAD system. Based on the principle of confocal optical path, two group of excitation-emission optical paths of different wavelengths are integrated in a small space. In terms of circuitry, a current-light dual negative feedback light emitting diode (LED) drive circuit is applied to improve the stability of the excited light source. All optical and electronic components are integrated in a metal box of 55 mm × 45 mm × 15 mm, that helps miniaturize the detection system. Two different modules have been assembled to fit various fluorescent dyes or probes with the set of excitation and emission as follow: module 1#: 470 nm/525 nm, 570 nm/630 nm; module 2#: 520 nm/570 nm, 630 nm/690 nm. Finally, hepatitis B virus (HBV) concentration gradient detection and multiplex detection of different gene targets of SARS-CoV-2 are carried out on the PNAD system equipped with these two fluorescence modules for evaluating their performances. Compared with the commercial real-time PCR instrument, our fluorescence module has good stability and detection sensitivity.

A Detection-Service-Mobile Three-Terminal Software Platform for Point-of-Care Infectious Disease Detection System.

The traditional infectious disease detection process is cumbersome, and there is only a single application scenario. In recent years, with the development of the medical industry and the impact of the epidemic situation, the number of infectious disease detection instruments based on nursing point detection has been increasing. Due to this trend, many detection instruments and massive detection data urgently need to be managed. In addition, the experiment failed due to the abnormal fluorescence curve generated by a human operator or sample impurities. Finally, the geographic information system has also played an active role in spreading and preventing infectious diseases; this paper designs a "detection-service-mobile" three-terminal system to realize the control of diagnostic instruments and the comprehensive management of data. Machine learning is used to classify the enlarged curve and calculate the cycle threshold of the positive curve; combined with a geographic information system, the detection results are marked on the mobile terminal map to realize the visual display of the positive results of nucleic acid amplification detection and the early warning of infectious diseases. In the research, applying this system to portable field pathogen detection is feasible and practical.

A Highly Integrated and Diminutive Fluorescence Detector for Point-of-Care Testing: Dual Negative Feedback Light-Emitting Diode (LED) Drive and Photoelectric Processing Circuits Design and Implementation.

As an important detection tool in biochemistry, fluorescence detection has wide applications. Quantitative detection can be achieved by detecting fluorescence signals excited by excitation light at a specific wavelength range. Therefore, the key to fluorescence detection is the stable control of the excitation light and the accurate acquisition of weak photoelectric signals. Moreover, to improve portability and instantaneity, devices are developing in miniaturization and integration. As the core of such devices, fluorescence detectors should also have these features. Under this circumstance, we designed a highly integrated and diminutive fluorescence detector and focused on its excitation light driving and photoelectric signal processing. A current-light dual negative feedback light-emitting diode (LED) driving circuit was proposed to obtain constant current and luminance. In addition, a silicon photodiode (PD) was used to receive and convert the fluorescence signal to an electric signal. Then, amplifying, filtering, and analog-to-digital (A/D) converting were applied to make the detection of weak fluorescence signals possible. The test results showed that the designed circuit has wonderful performance, and the detector shows good linearity (R2 = 0.9967) and sensitivity (LOD = 0.077 nM) in the detection of fluorescein sodium solution. Finally, a real-time fluorescence polymerase chain reaction (real-time PCR) of Legionella pneumophila was carried out on a homemade platform equipped with this detector, indicating that the detector met the requirements of real-time PCR detection.

Immune repertoire analysis of normal Chinese donors at different ages.

OBJECTIVES: This study investigated the characteristics of the immune repertoire in normal Chinese individuals of different ages. MATERIALS AND METHODS: In this study, all seven receptor chains from both B and T cells in peripheral blood of 16 normal Chinese individuals from two age groups were analyzed using high-throughput sequencing and dimer-avoided multiplex PCR amplification. Normal in this study is defined as no chronic, infectious or autoimmune disease within 6 months prior to blood draw. RESULTS: We found that compared with the younger group, the clonal expression of T-cell receptor repertoire increased in the older group, while diversity decreased. In addition, we found that the T-cell receptor repertoire was more significantly affected by age than the B-cell receptor repertoire, including significant differences in the use of the unique TCR-alpha and TCR-beta V-J gene combinations, in the two groups of normal participants. We further analyzed the degree of complementarity determining region 3 sequence sharing between the two groups, and found shared TCR-alpha, TCR-gamma, immunoglobulin-kappa and immunoglobulin-lambda chain complementarity determining region 3 sequences in all subjects. CONCLUSION: Taken together, our study gives us a better understanding of the immune repertoire of different normal Chinese people, and these results can be applied to the treatment of age-related diseases. Immune repertoire analysis also allows us to observe participant's wellness, aiding in early-stage diagnosis.

A sample-to-answer, quantitative real-time PCR system with low-cost, gravity-driven microfluidic cartridge for rapid detection of SARS-CoV-2, influenza A/B, and human papillomavirus 16/18.

The pandemic of coronavirus disease 2019 (COVID-19), due to the novel coronavirus (SARS-CoV-2), has created an unprecedented threat to the global health system, especially in resource-limited areas. This challenge shines a spotlight on the urgent need for a point-of-care (POC) quantitative real-time PCR (qPCR) test for sensitive and rapid diagnosis of viral infections. In a POC system, a closed, single-use, microfluidic cartridge is commonly utilized for integration of nucleic acid preparation, PCR amplification and florescence detection. But, most current cartridge systems often involve complicated nucleic acid extraction via active pumping that relies on cumbersome external hardware, causing increases in system complexity and cost. In this work, we demonstrate a gravity-driven cartridge design for an integrated viral RNA/DNA diagnostic test that does not require auxiliary hardware for fluid pumping due to adopted extraction-free amplification. This microfluidic cartridge only contains two reaction chambers for nucleic acid lysis and amplification respectively, enabling a fast qPCR test in less than 30 min. This gravity-driven pumping strategy can help simplify and minimize the microfluidic cartridge, thus enabling high-throughput (up to 12 test cartridges per test) molecular detection via a small cartridge readout system. Thus, this work addresses the scalability limitation of POC molecular testing and can be run in any settings. We verified the analytical sensitivity and specificity of the cartridge testing for respiratory pathogens and sexually transmitted diseases using SARS-CoV-2, influenza A/B RNA samples, and human papillomavirus 16/18 DNA samples. Our cartridge system exhibited a comparable detection performance to the current gold standard qPCR instrument ABI 7500. Moreover, our system showed very high diagnostic accuracy for viral RNA/DNA detection that was well validated by ROC curve analysis. The sample-to-answer molecular testing system reported in this work has the advantages of simplicity, rapidity, and low cost, making it highly promising for prevention and control of infectious diseases in poor-resource areas.