In non-small cell lung cancer, the choice of an effective targeted therapy often depends on the presence of a somatic mutation that alters an amino acid in a key protein. However, if an additional somatic mutation occurs in the same mutant gene, causing an additional amino acid substitution to occur in that protein, the targeted therapy can become ineffective. A PCR assay has now been developed that utilizes two different SuperSelective primers, one primer for the amplification of the DNA strand containing the original somatic mutation, and a second primer for the amplification of the DNA strand containing the additional somatic mutation. Exponential amplification only occurs if these two somatic mutations occur in cis on the DNA molecule present in the same sister chromosome, and does not occur at all if the two different somatic mutations occur in trans on different DNA molecules in two different sister chromosomes. Significantly, if only one of these two mutations is present, amplification does not occur, thereby ensuring that the assay specifically detects the coexistence of both target mutations being present on the same DNA strand. This assay (demonstrated utilizing EGFR exon 20 mutations T790M and C797S) is extraordinarily sensitive, enabling the early substitution of a more effective therapy if the two mutations occur in a cis configuration. Moreover, these SuperSelective PCR assays can utilize DNA fragments isolated from noninvasive liquid biopsy samples, and they can be carried out on widely available spectrofluorometric thermal cyclers.

Escherichia coli and Klebsiella pneumoniae are major hospital-acquired pathogens, posing severe threats to critically ill and immunocompromised patients. Their pathogenicity and virulence factors easily cause invasive infections, endangering patient health and life. This study developed a dual recombinase polymerase amplification combined with Clustered Regularly Interspaced Short Palindromic Repeats-Cas12a assay for rapid, simultaneous, specific detection of E. coli uidA and K. pneumoniae rcsA genes. The assay operated at 37°C, and the total reaction time was approximately 70 min. The analytical sensitivity for E. coli and K. pneumoniae was 5.37 × 10¹ copies/μL and 5.90 × 10¹ copies/μL, respectively. It showed excellent specificity (no cross-reactivity) and 100% concordance with PCR results in clinical sample validation. This assay overcame limitations of traditional methods, such as bacterial culture (time-consuming) and PCR (the dependency on expensive thermal cyclers), and offers advantages of rapidity, high sensitivity, simplicity, and cost-effectiveness, providing a powerful tool for rapid, accurate clinical diagnosis of E. coli and K. pneumoniae infections.IMPORTANCEEscherichia coli and Klebsiella pneumoniae are the main pathogens causing hospital-acquired infections, which can lead to serious complications and pose a significant challenge to public health. Therefore, establishing rapid, sensitive, specific, and reliable detection methods for E. coli and K. pneumoniae is of great significance for promoting accurate early clinical diagnosis and guiding treatment decisions. With the increasing incidence of mixed infections, single-target nucleic acid testing can no longer meet clinical needs. In this study, the efficient recombinase polymerase amplification (RPA) isothermal amplification technique was combined with the highly sensitive Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas12a detection system to successfully develop a duplex RPA-CRISPR-Cas12a method. This system can specifically and simultaneously identify E. coli (targeting the uidA gene) and K. pneumoniae (targeting the rcsA gene) in a single detection process.

To address the issues of operational complexity, long duration association, and reliance on specialized equipment with existing detection methods for Vibrio parahaemolyticus, this study established a rapid detection method for V. parahaemolyticus in exported aquatic products based on the domestically developed Enzymatic Recombinase Amplification (ERA) technology. To target the thermolabile hemolysin gene (tlh) and the iron-regulated virulence regulatory protein gene (irgB) of V. parahaemolyticus, highly specific ERA primers and probes were designed and screened. Two detection platforms, a colorimetric method and a fluorescent method, were developed. Method validation results showed that this detection system achieved specific amplification for all 30 tested V. parahaemolyticus strains, with no cross-reactivity observed with 30 other common foodborne pathogenic bacteria. The detection sensitivity for both the fluorescent and colorimetric methods reached 10-1 ng/μL, with a minimum detection limit of 10 CFU/25 g for artificially contaminated samples. The entire detection process, including sample preparation, requires only approximately 20 min-significantly faster than traditional culture (24-72 h) or even conventional PCR methods. Collaborative validation across five independent laboratories confirmed excellent reproducibility, with inter-laboratory agreement yielding a Kappa coefficient of 0.98. The ERA method operates at a low, constant temperature (37-39 °C), eliminating the need for thermal cyclers. When combined with portable isothermal amplification devices and visual (colorimetric) readout, it offers a distinct advantage in terms of speed, cost-effectiveness, and suitability for resource-limited or field settings compared to existing PCR-based or culture-based platforms. This method is simple to operate, rapid, sensitive, and highly suitable for on-site application, providing a reliable and practical technical solution for the rapid screening and risk monitoring of V. parahaemolyticus in exported aquatic products.

Recent progress in DNA nanotechnology has shown the isothermal assembly of several DNA nanostructures. Isothermal assembly allows DNA nanostructure construction in a variety of ions while simplifying DNA nanotechnology by avoiding the need for thermal cyclers and expands utility by enabling attachment of guest biomolecules on DNA nanostructures at ambient or physiological temperatures. The paranemic crossover (PX) DNA motif has been used in the construction of DNA nanostructures, paranemic cohesion has been used to connect DNA structures as an alternate to sticky end cohesion, and PX DNA has also been implied to have a biological role in homology recognition. In that context, here we demonstrate the successful isothermal assembly of the PX DNA motif in magnesium (Mg2+), calcium (Ca2+), and strontium (Sr2+) at 20 and 37 °C. Using isothermal titration calorimetry, we show that interhelix hybridization of half-PX molecules is favored at higher temperatures, with a heat capacity (ΔCp) of -1.9 kcal/mol·K. To demonstrate a key advantage of isothermal assembly, we show that PX molecules can be designed to contain thrombin-specific aptamers for binding one or two thrombin molecules site specifically in an entirely isothermal procedure. Our work extends isothermal assembly and the use of different counterions for complex DNA motifs while demonstrating the attachment of guest molecules at constant temperatures.

Polymerase Chain Reaction (PCR) requires precise and repeatable thermal cycling to amplify DNA without sample degradation, particularly for microliter-level volumes. This paper presents the design and development of a compact and efficient thermal cycler capable of meeting the crucial temperature requirements of PCR using a PID profiler-based control strategy. The system incorporates a custom fabricated copper sample holder accommodating four conical PCR microtubes. These tubes are having a usable sample volume of 8–10 µL each, optimized for rapid thermal response and uniform heat distribution. A TEC1-12706 Peltier module enables rapid heating and cooling, supported by a heat sink for effective thermal dissipation. Sample temperature is monitored using a miniature K-type thermocouple, calibrated up to 110 °C with an accuracy of ±0.3 °C and a response time of about 2.8 s. A mathematical model of the thermal system, and the Peltier module heat equation, was developed to validate controller performance. A programmable PID profiler is implemented to automatically track the multi-stage PCR temperature profile. Controller parameters were tuned using Ziegler Nichols method, yielding optimized constants Kp = 2.8, Ti = 30 s, and Td = 0.05 min. The system achieved the ramp rate of 8.6 °C/s with minimal overshoot (≤0.4 %), steady-state accuracy within ±0.2 °C, and stable performance. The response demonstrates settling times of 28 s for heating and 18 s for cooling. The developed thermal cycler is portable, cost-effective, and capable of accurately following the PCR temperature profile, confirming its suitability for DNA amplification applications.

Chikungunya virus (CHIKV) and Dengue virus (DENV) infections present with highly similar clinical symptoms but require distinct management strategies, highlighting an urgent need for rapid and accurate differential diagnostic methods. The current gold standard, reverse transcription quantitative real-time PCR (RT-qPCR), has significant limitations: it relies on expensive thermal cyclers, complex laboratory infrastructure, and specialized personnel, making it difficult to implement in resource-limited settings and for on-site screening. To address this technological bottleneck, this study developed a portable, instrument-independent rapid detection technology based on the CRISPR-Cas12a/Cas13a dual-enzyme cleavage system. By precisely designing specific crRNAs targeting the CHIKV E1 gene and the DENV 3′-UTR region, and integrating them with reverse transcription recombinase-aided amplification (RT-RAA) technology, we established an integrated reaction system that performs nucleic acid amplification and detection under isothermal conditions. The core mechanism of the technology is as follows: upon recognition of the corresponding viral targets, Cas12a (targeting CHIKV) and Cas13a (targeting DENV) become activated and cleave fluorescent reporters or lateral flow strip reporter probes, enabling visual detection. Gradient-diluted viral nucleic acid tests indicated that the lowest detectable concentration that generated a signal reached 10² copies/mL, which is comparable to qPCR. Clinical simulated sample validation showed 100% overall agreement with qPCR, and the system accurately distinguished single infections from co-infections.The CRISPR dual-enzyme cleavage technology established in this study effectively circumvents the reliance of qPCR on sophisticated equipment, providing a rapid, simple, and low-cost solution for CHIKV/DENV differential diagnosis. It holds significant practical value for the early prevention and control of arboviral diseases.

Tomato chlorosis virus (ToCV) represents a major threat to the tomato industry in China. Conventional Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR)assays require expensive thermal cyclers and are poorly suited to on-site screening in fields or at ports of entry. Here, two multi-enzyme isothermal rapid amplification (MIRA) systems targeting the highly conserved minor capsid protein (CPm) region of ToCV were developed: (i) a basic MIRA-electrophoresis assay (B-MIRA), and (ii) a fluorescent MIRA-real-time assay (F-MIRA). Primers/probes, reaction temperature, sensitivity and specificity were systematically optimized, and performance was validated with field samples. The basic assay completed amplification at 41 °C within 30 min, generating a single 250 bp band; the limit of detection (LOD) was 2.78 × 10-1 pg/μL. The fluorescent assay reached its endpoint in 20 min at 37 °C using the optimal primer/probe set F2/R1 + P (Ct = 12.4 ± 0.3 min); LOD was 2.78 × 10-1 pg/μL. The B-MIRA and F-MIRA assays developed in this study can reliably detect ToCV within 20 min with high sensitivity. Free of bulky instrumentation or professional laboratories, the protocols can be directly applied to seedling quarantine, field-side early inspection and border control, offering an accurate and ready-to-use diagnostic tool for the green management of ToCV. © 2026 Society of Chemical Industry.

The aim of this study was to investigate the impact of the T-786C polymorphism of the NOS3 gene on the onset and progression of renal dysfunction in patients of the Uzbek population with chronic heart failure (CHF). The study included 200 patients of Uzbek nationality diagnosed with CHF. Among them, 110 patients had a glomerular filtration rate (eGFR) ≥ 60 mL/min/1.73 m², while in 90 patients this indicator was lower. The control group consisted of 120 conditionally healthy donors of Uzbek nationality. Analysis of the NOS3 T-786C polymorphism was performed using commercially available test kits developed by NPF Litex LLC (Moscow, Russia) in accordance with the manufacturer's standard protocol. Amplification of the polymorphic region of the NOS3 promoter was carried out using a Rotor-Gene Q thermal cycler (QIAGEN, Hilden, Germany). Polymerase chain reaction (PCR) was performed in a total volume of 25 µL under the following cycling conditions: initial denaturation at 95°C for 5 minutes; 35 cycles of denaturation at 95°C for 30 seconds, primer annealing at 60°C for 30 seconds, and DNA extension at 72°C for 1 minute; followed by a final extension at 72°C for 10 minutes. The resulting data were analyzed using the SPSS statistical package (IBM Corp., Armonk, NY, USA) and OpenEpi v9.2 (OpenEpi, Emory University, Atlanta, GA, USA). Differences were observed in the distribution of genotypic and allelic variations. In the main group, the frequency of the C allele was 35.5%, compared to 28.3% in the control group. Patients with eGFR < 60 mL/min/1.73 m² were more likely to have the C/C genotype (15.6% versus 10.8% in the control group). The T-786C polymorphism may exacerbate renal impairment by reducing NOS3 activity and lowering nitric oxide (NO) production. The genetic variant C of the T-786C NOS3 polymorphism is associated with impaired renal function in patients with CHF.

INTRODUCTION: Yellow fever is undergoing a resurgence in the Americas, with 235 human cases and a 41% case fatality rate reported as of May 25, 2025. OBJECTIVE: To review the current epidemiological landscape of yellow fever and evaluate the limitations of conventional diagnostic methods versus emerging molecular technologies for optimizing outbreak surveillance and response. METHODS: Narrative review of scientific literature (2015-2025) across PubMed, Scopus, Web of Science, LILACS, and SciELO, including 55 validated primary studies organized into four thematic axes. RESULTS: Conventional diagnostics are limited by IgM/IgG serological cross-reactivity with flaviviruses, post-vaccination antibody persistence up to 4 years, and RT-PCR’s ≤14-day detection window requiringspecialized laboratories. Emerging solutions include: digital PCR for absolute quantification of viral RNA at ultra-low loads in epizootics with incipient viremia; RT-LAMP for field-deployable detection within 1 hour with 10-fold higher sensitivity (LOD 0.29-12 PFU/mL) without thermal cyclers; CRISPR-dx (SHERLOCK/DETECTR) for ultra-rapid sequencespecific detection with low-cost potential, though still experimental for flaviviruses; and NGS/metagenomics for strain characterization and differentiation of wild-type from vaccine-derived viruses. CONCLUSIONS: Urgent diagnostic modernization is needed. Emerging technologies address critical sensitivity and specificity gaps, but require strengthened Latin American laboratory networks, validated integrated algorithms, and coordinated crossborder responses to contain yellow fever re-emergence.

The protocol describes the removal of embryonic cells using a simple manual method that eliminates the need for micromanipulation equipment, followed by sample processing for whole genome amplification, up to the point at which the samples are sent to the genotyping laboratory. Thus, steps required for genomic selection of bovine embryos-from the in vitro fertilization (IVF) laboratory to the genotyping service-are encompassed.Grade I embryos at the blastocyst stage, preferably on day 7 (154-168h.p.i.), are selected and transferred to biopsy plates containing identified microdroplets, with one embryo per droplet. Using an appropriate microblade, the operator cuts a portion of the trophectoderm from the embryo. The embryo is then cryopreserved, and the excised sample is transferred to a 0.2mL tube and stored. The second stage of the protocol involves processing the sample by lysing cell membranes to release and denature DNA, followed by amplification of the DNA through an enzymatic reaction. These operations are carried out using a thermal cycler and, by the end of the protocol, yield a sufficient amount of DNA for submission to the genotyping laboratory.

ABSTRACTIntroduction:Severe Acute Respiratory Illness (SARI) is primarily caused by influenza viruses (types A and B) and coronaviruses, notably SARS-CoV-2, which causes COVID-19. The emergence of SARS-CoV-2 has complicated respiratory disease management globally, especially as coinfections with influenza lead to more severe illness, particularly in vulnerable populations like the elderly and children.Methods:This study was conducted at the Virology Research and Diagnostic Laboratory, Department of Microbiology, MGM Medical College, Indore, from August to January 2023. Respiratory specimens from SARI patients were processed for RNA extraction using the Thermo Scientific Kingfisher Flex system and RT-PCR to detect influenza and SARS-CoV-2. A multiplex real-time RT-PCR assay kit from National Institute of Virology, Pune, identified influenza subtypes. The Biorad C1000 Thermal Cycler with CFX96 Real-Time System was used for the PCR analysis.Results:Out of 347 samples tested, maximum infectivity was found for Inf A (H1N1) and SARS-CoV-2, and only two cases showed coinfection. Inf A and SARS-CoV-2 positivity was more in females, but Inf B infection was observed only in males. Inf A infection was significantly higher in 46–60 years age group, and coinfection was observed only in >60 years. The majority of the Influenza and SARS-CoV-2 infections were observed in Indore district.Conclusion:This study found prevalent influenza subtypes in our region but low coinfection with SARS-CoV-2. Detecting coinfections is difficult due to differences in viral incubation and shedding times. Early and accurate detection is key to preventing transmission, guiding treatment, and improving patient care.

This study introduces a low-cost, portable DNA amplification kit that performs a modified loop-mediated isothermal amplification (LAMP) reaction, which produces DNA nanoballs and combines it with a previously developed microfluidic impedance-based digital assay to deliver a potential all-in-one, point-of-care (POC) diagnostic platform for the detection of target nucleic acids. The device combines sample processing and detection in a single streamlined workflow, utilizing induction heating and Arduino-based temperature control, along with several engineering innovations, including a custom-designed polycarbonate microtube holder and an optimized thermocouple-based temperature-control feedback system, ensuring stable reaction conditions for reproducible amplification. System performance was validated through the detection of a synthesized β-lactamase target DNA gene block, including samples with additional non-target background DNA. In addition to a qualitative colorimetric readout, label-free impedance-based quantification confirmed the robust production of DNA nanoballs with high specificity and minimal background interference. The amplification quality was revealed to be comparable to that of a commercial thermal cycler. Subsequent sensitivity testing using serial dilutions of the target DNA (between 101-105 copies per μl) in a complex background DNA mixture demonstrated detection results that strongly correlated with quantitative PCR (qPCR). These findings demonstrate that the amplification kit achieves performance parity with gold-standard nucleic acid detection methods while offering portability, affordability, and ease of use. By enabling accurate, rapid, and decentralized diagnostics without reliance on laboratory infrastructure, this combined workflow holds promise for advancing infectious disease monitoring and antimicrobial resistance surveillance, among other applications, at the point of care.

Malaria is an infectious disease that remains a global public health problem, especially in tropical and subtropical countries such as Indonesia. This disease is caused by the Plasmodium parasite, which is transmitted through the bite of infected female Anopheles mosquitoes. According to the 2024 World Malaria Report, there were approximately 249 million cases of malaria and 597,000 deaths worldwide, with Indonesia accounting for approximately 1.8 million cases or 46% of the total cases in Southeast Asia. This condition shows that malaria is still a major challenge in the national health system, especially in endemic areas such as Papua, Nusa Tenggara, and parts of Kalimantan. Rapid and accurate diagnosis of malaria is crucial in reducing morbidity and mortality rates. Peripheral blood microscopy is still considered the gold standard because it can identify Plasmodium species and assess the degree of parasitemia, but its sensitivity decreases in infections with low parasite density. Advances in diagnostic methods have led to the development of Rapid Diagnostic Tests (RDTs), which detect specific parasite antigens and provide rapid results, although the results can be affected by HRP2 gene mutations and reagent storage conditions. Furthermore, molecular methods such as Polymerase Chain Reaction (PCR) offer the highest sensitivity with the ability to detect up to 0.25–5 parasites/µL, but require advanced laboratory facilities. The latest innovation, Loop-Mediated Isothermal Amplification (LAMP), can amplify parasite DNA at a constant temperature of 60–65°C without a thermal cycler, with sensitivity and specificity reaching 95–99%. Therefore, this literature review highlights that a combination of conventional and molecular methods is essential to improve diagnostic accuracy and support malaria elimination efforts in Indonesia.

Development of a high-throughput centrifugal thermal cycler by an innovative non-contact induction heating mechanism for rapid diagnosis of respiratory infectious viruses

Polymerase chain reaction (PCR) is a highly sensitive and accurate technique for amplifying and detecting nucleic acids (NA). Its extensive applications in clinical diagnostics, environmental monitoring, agricultural, and forensic science highlight the critical need for translating this laboratory-based technique into a point-of-care (POC) quantitative PCR (qPCR) platform. The transition to POC qPCR faces significant challenges, including the need to streamline sample preparation, miniaturize devices without compromising their analytical performance, and achieve cost-effective and standardized platforms suitable for use by non-experts. This review discusses the challenges and approaches involved in developing simplified sample-preparation methods and miniaturized qPCR devices suitable for on-site implementation. The speed, efficiency, and simplicity of recent sample preparation methods that utilize microfluidics and nanoparticles are highlighted as pivotal innovations for NA extraction. Approaches for developing thermal cyclers and NA detection methods, emphasizing the hurdles and limitations that impede effective device miniaturization and field implementation, are then examined. This review also discuss strategies to promote widespread adoption of POC qPCR devices, with a focus on cost reduction and standardization as critical enabling factors. Finally, the key opportunities to address these challenges are outlined through practical considerations, thereby supporting the development of rapid, precise, handheld, and low-cost POC qPCR devices.

Demand for less labor-intensive in vitro assays of theactivityof CRISPR/Cas proteins is rising to extend the potential applicationsof CRISPR in the field of diagnostics. RNA guided DNA endonucleasesof the Cas family generate double-strand breaks in the target DNA,which results in two shorter DNA fragments. We hypothesized that thiscleavage event could be studied using melting curve analysis, andusing SpyCas9, we demonstrate that it is possible to evaluate theactivity of Cas proteins by measuring the melting curves of theirproducts. We present here a novel assay for the in vitro activityof Cas9 that exploits melting curve analysis (MCA) to be fast, inexpensive,and widely accessible. The assay can, in fact, be performed with readilyavailable componentsin its simplest form a real-time thermalcycler and an intercalating dye (SYBR Green I)and producesreliable results with a run-time of 15 min. It does not require externalintervention to stop the reaction, which is done by thermal denaturationof the protein directly in the thermal cycler machine. The describedadvantages, combined with the provided data analysis package, makethe assay robust and amenable to high-throughput applications. Toincrease the accessibility of our assay, we provided an R packagethat simplifies the analytical process.

Relevance. Recent data from the World Health Organization highlight the high global prevalence of periodontal diseases. Among middle-aged adults in European countries, the prevalence ranges from 50% to 76%, while in Russia it reaches 86.2% in this group and approaches 100% by the age of 60–65. The diagnosis and prognosis of chronic periodontitis remain pressing challenges in clinical periodontology. While periodontal diseases have multifactorial etiology, microbial factors are primary. In particular, bacteria of the red complex – Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola – are considered key contributors to the progression of inflammation and tissue destruction in the periodontium. Objective. To detect and quantify red complex bacteria in patients with varying severity of chronic periodontitis. Materials and methods. The study included 126 patients diagnosed with chronic periodontitis, categorized as mild (n = 39), moderate (n = 42), and severe (n = 45). A control group consisted of 39 periodontally healthy individuals. Subgingival plaque samples (from periodontal pockets in patients and from gingival sulci in controls) were analyzed using real-time polymerase chain reaction (PCR) on a DT-96 thermal cycler (DNA-Technology, Russia) with the ParodontoScreen diagnostic kit. Results . The severity of periodontitis strongly correlated with the presence of P. gingivalis, T. forsythia and T. denticola , with correlation coefficients of 0.997, 0.929, and 0.888, respectively (p &lt; 0.05). The number of genomic equivalents of these microorganisms also correlated with disease severity, with coefficients of 0.948, 0.984, and 0.889, respectively (p &lt; 0.05). Conclusion . The study confirms the significant role of red complex bacteria in the pathogenesis and progression of chronic periodontitis, supporting their relevance for diagnostic and prognostic applications in periodontal care.

Abstract Background Pneumocystis jirovecii pneumonia (PCP) is an opportunistic infection commonly affecting immunocompromised individuals. Invasive procedures are usually required to obtain lower respiratory specimens such as bronchial wash and bronchoalveolar lavage to diagnose PCP. However, this invasive procedure is sometimes not possible in severely ill patients especially patients who are thrombocytopenic. Hence, there is a need for a less invasive sample to diagnose PCP. The majority of previous studies on PCP diagnosis have been conducted mostly among HIV-infected patient population. This study compared the use of induced sputum and bronchial wash in diagnosing PCP in oncology patients. Methods Induced sputum and bronchial wash specimens were collected from patients presenting to a cancer hospital in New York between October 2019 and November 2021. DNA extraction from induced sputum and bronchial wash was done using the BioMerieux eMag Nucleic Acid Extraction System. The Roche Light-cycler 2.0 Real-Time PCR Thermal Cycler system was used to detect P. jirovecii DNA. Results In total, 324 PCP from 289 unique patients were tested in sputum (n = 324; 289 unique patients) and bronchial wash (n = 69; 66 unique patients). Of these, 30 (30/324, 9.3%; 29/289, 10% unique patients) and 9 (9/69, 13%; 8/66, 12% unique patients) PCP positives were detected in sputum and bronchial wash respectively. Further analyses of patient-matched sputum and bronchial wash specimens showed that the induced sputum sensitivity was 72.2%, specificity was 92.9%, positive predictive value was 66.7%, negative predictive value was 94.5% and the accuracy was 89.6%, for the diagnosis of PCP, compared with bronchial wash. Conclusion Detection of P. jirovecii in lower respiratory tract samples especially using minimally invasive collected samples like induced sputum is crucial to the timely diagnosis of PCP and monitoring of patients with chronic infections. Our results suggest that induced sputum is a useful minimally invasive sample for diagnosing PCP infection in our patient population and this has potential application in routine diagnostic laboratory. Comparing PCP diagnosis in lower respiratory samples using a larger patient population (&amp;gt;3500 tests from &amp;gt;2000 unique patients tested in 6 years) to further our understanding of the utility of induced sputum in PCP diagnosis is the current focus of our laboratory.

The detection of mutations in circulating tumor DNA (ctDNA) is challenging due to the significant fragmentation of ctDNA and the high prevalence of the wild-type template. Additionally, variant detection through qPCR is typically dependent on target-specific fluorescence probes, and no more than five targets can be identified in a single reaction due to the limited fluorescence colors in thermal cyclers. To address these limitations, we introduce the Dual-Role Mediator Blocker Amplification (DMBA) strategy, enabling sensitive and multiplex mutation detection without reliance on specific fluorescence probes. This strategy is applicable in both qPCR and melting curve analysis (MCA) platforms. The mediator blockers in DMBA play dual roles: enhancing discrimination between wild-type and mutant DNA and releasing mediator primers. These mediator primers extend the helper target and cleave universal fluorescence probes in qPCR, enabling the detection of mutations at variant allele fractions (VAFs) as low as 0.01%. The DMBA MCA method can identify multiple mutations, overcoming limitations in fluorescence channels by using mediator primers to extend universal fluorescence probes, producing fluorescent double strands with different Tm's and colors. Multiplexed DMBA-MCA was developed to detect seven variants at 0.1-0.5% VAF in one tube. Our innovative method offers advantages including exceptional sensitivity, elimination of the requirement for specific fluorescence probes, shorter amplicons, and high multiplexing capacity, potentially revolutionizing clinical practice and precision medicine.

The integrity of herbal products is frequently undermined by both intentional and unintentional adulteration, leading to substantial health risks and economic losses. Loop-mediated isothermal amplification (LAMP), a DNA-based molecular technique, has emerged as a formidable solution due to its simplicity, specificity, sensitivity, and ability to operate under isothermal conditions. The review critically evaluates the application of LAMP in authenticating herbal materials, showcasing its enhanced efficiency and user-friendliness compared to conventional techniques. The LAMP technique employs four to six primers that target six to eight distinct regions of the target DNA, ensuring unparalleled specificity. The amplification at a constant temperature negates the need for thermal cyclers, thus rendering it highly suitable for point-of-care applications and field-based authentication. The article presents case studies that illustrate LAMP's efficacy in detecting adulteration across traditional medicines, dietary supplements, and crude drug materials. Visualization methods in LAMP, such as turbidity, colorimetry, and fluorescence, greatly enhance its accessibility and ease of use, making it well-suited for both laboratory and field applications. Although there are limitations, such as primer design complexity and contamination risks, recent innovations, including the use of lyophilized reagents, multiplexing capabilities, and integration with mobile detection platforms, are significantly advancing the practicality of LAMP assays. This review underscores the potential of LAMP in both regulatory and commercial contexts, promoting the authenticity, safety, and quality of herbal products, thereby making a vital contribution to consumer protection and the sustainability of the herbal medicine trade.