BACKGROUND:Photothermal PCR based on specific nanomaterials has attracted attention due to their efficient photothermal conversion. However, these nanomaterials absorb polymerases, thereby inhibiting the PCR. Owing to their small size and highly active surface atoms, they are prone to aggregation and sedimentation, resulting in uneven heating within the reaction system. In addition, the fluorescent groups used for detection are susceptible to photobleaching under excitation light. Therefore, there is a need for stable photothermal nanomaterials and compatible photothermal PCR detection strategies. RESULTS:This paper presents a coated-type nanoplasmas-based photothermal PCR method and platform for ultra-fast nucleic acid analysis. Pullulan-coated AuNRs were introduced, and pullulan-AuNRs nanoplasmas were prepared to prevent the aggregation of AuNRs and the adsorption of the polymerase. A photoelectric platform for ultrafast photothermal PCR was built. The photothermal effect of AuNRs was excited by an infrared laser, causing the PCR solution to rapidly heat up. By regulating the on-off cycle of the excitation light source and the fan through temperature feedback, the photothermal conversion of AuNRs was alternately excited, achieving ultra-fast photothermal PCR within 6 min. The CRISPR/Cas detection technology was coupled with the photothermal PCR to detect the amplified products and output fluorescence signals within 4 min, enabling ultra-fast detection of Salmonella DNA as low as 38 copies/reaction. SIGNIFICANCE:The pullulan-coated photothermal nanomaterials were introduced, which solved the problems of instability and incompatibility with the PCR system. The one-tube photothermal PCR-CRISPR assay maintains airtight conditions while avoiding interference between the excitation light source and the detection light source. This method achieves ultrafast detection within 10 min in a simple, rapid, accurate, and contamination-free manner.

Ultrasensitive protein detection is essential for early disease diagnosis and biomedical research, where many clinically relevant biomarkers exist at extremely low abundance. To address this challenge, discretization-based digital protein detection has emerged as a robust analytical paradigm through the conversion of continuous biochemical signals into countable digital events. This review systematically summarizes recent advances in digital protein detection from the perspectives of discretization mechanisms and signal output modalities. Both hard discretization strategies, including microwell-, droplet-, and bead-based systems, and soft discretization approaches based on proximity effects and single-molecule resolution are discussed. In addition, major signal readout modalities, including fluorescence, electrochemical, and Raman-based methods, are compared in terms of their working principles and analytical performance. The integration of discretization, amplification, and signal transduction facilitates enhanced sensitivity, improved quantitative accuracy, and flexible assay design. Ultimately, current challenges and future opportunities are highlighted, emphasizing the potential of digital protein detection in clinical diagnostics and precision medicine.

Proteins are the main executors of life activities and often exist at ultra-trace levels in biological matrices. Developing ultrasensitive protein detection methods is of great significance in clinical diagnosis, food safety assessment, and environmental monitoring. Listeria monocytogenes is one of the most serious foodborne pathogens worldwide. Herein, an A/T extension-triggered proximity-activated magnetic immuno-PCR (ATP-IPCR) was developed for the ultrasensitive detection of Listeria monocytogenes p60 protein. To improve the sensitivity of protein detection, antibody–DNA conjugates were used in ATP-IPCR to convert the protein signal into a detectable nucleic acid signal. To minimize background signals, oligo(dT) magnetic beads were employed to capture the sandwich immune complex, an A/T extension reaction using only dATP and dTTP as substrates was applied to release the immune complex from the beads, and a proximity-activated extension reaction supplemented with dCTP and dGTP was used to generate the template for PCR amplification. The resulting PCR template is quantified by real time fluorescence PCR. ATP-IPCR achieved a detection limit of 6.8 × 101 CFU mL−1 for Listeria monocytogenes and showed no cross-reactivity with six non-target bacteria, demonstrating excellent sensitivity and specificity. In applicability validation with pork, lettuce, and skim milk samples, ATP-IPCR showed high recovery rates of 92.4%–107.0%. Overall, ATP-IPCR uses magnetic beads to capture immune complexes and employs a two-step extension to separately perform the release of the complexes from the beads and the proximity-activated generation of PCR templates, thereby minimizing background signals. This design offers new insights into ultrasensitive protein detection method.

Personalized neoantigen cancer mRNA vaccines are promising candidates for precision medicine. However, the difficulty of identifying neoantigens heavily hinders their broad applicability. This study developed a universal strategy of anti-tumor mRNA vaccine by harnessing "off-the-shelf" immunity to known antigens. First, the model antigen ovalbumin (OVA) is used for mRNA vaccine design. In vitro test indicated that this mRNA vaccine reprogrammed tumor cells that can be recognized and killed by OVA-specific cytotoxic T lymphocytes (CTLs). In situ mRNA vaccine notably inhibited tumor growth across three subcutaneous solid tumor models in mice. Further single-cell sequencing analyses revealed that mRNA vaccination act to reshape the immunosuppressive tumor microenvironment (TME) toward more proinflammatory characteristics. Strikingly, this framework of mRNA-based strategy can be applied to two clinical pathogen antigens, hepatitis B surface antigen (HBsAg), and SARS-CoV-2 spike receptor-binding domain (SRBD). Interestingly, the mRNA-based strategy largely recapitulated the scenario of spontaneous cancer regression following pathogen infection or vaccination. Collectively, this study provides not only proof of concept for universal anti-tumor mRNA therapy, but also mechanistic insights in echoing the long-standing puzzle of spontaneous cancer regression.

Urolithiasis is a prevalent urological condition that poses a significant public health challenge worldwide. This study investigates the impact of urolithiasis in China and globally between 1990 and 2021, with projections through 2030. Data on incidence and disability-adjusted life years (DALYs), along with age-standardized rates (ASR), were obtained from the Global Burden of Disease (GBD) 2021. Temporal trends were assessed by utilizing the Estimated Annual Percentage Change (EAPC) and Joinpoint regression analysis. Decomposition analyses were performed to assess the contributions of population growth, aging, and epidemiological changes. Future age-standardized incidence rates (ASIR) were forecast using the Auto-Regressive Integrated Moving Average (ARIMA) model. In 2021, the global number of urolithiasis cases reached 73.12 million, with China accounting for over 18 million. The condition primarily affects males and individuals aged 50–59 years. In both China and globally, the ASR of incidence and DALYs showed a downward trend from 1990 to 2021, with a more pronounced decline in China. However, the total number of incident cases continued to rise. Decomposition analysis revealed that population aging was a major contributor to the increase in cases in China, while global increases were largely driven by population growth. Projections from the ARIMA model indicate a slight further decrease in ASIRs by 2030 for both China and globally. These findings highlight the need for future efforts to focus on optimizing healthcare resources and strengthening primary prevention strategies to mitigate the public health impact of urolithiasis.

Abnormal tryptophan metabolism is closely linked with neurological disorders. Research has shown that indoleamine 2,3-dioxygenase 1 (IDO-1), the first rate-limiting enzyme in tryptophan degradation, is upregulated in Parkinson's disease (PD). However, the precise role of IDO-1 in PD pathogenesis remains elusive. In this study, we administered 1-methyl-tryptophan (1-MT), an IDO-1 inhibitor, intraperitoneally at 15 mg/kg daily for 21 days to PD mice induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) at 30 mg/kg daily for 5 days. Our results show that IDO-1 inhibition improves behavioral performance, reduces dopaminergic neuron loss, and decreases serum quinolinic acid (QA) content and the aryl hydrocarbon receptor (AHR) expression in the striatum and colon. It also alleviates glial-associated neuroinflammation and mitigates colonic inflammation (decreasing iNOS, COX2) by suppressing the Toll-like receptor 4/nuclear factor-κB (TLR4/NF-κB) pathway. Furthermore, IDO-1 inhibition promotes hippocampal neurogenesis (increasing doublecortin positive (DCX+) cells and SOX2+ cells), which have recently been recognized as key pathological features and potential therapeutic targets in PD, likely through the activation of the BDNF/TrkB pathway. We further explored the gut-brain connection by depleting the gut microbiota in mice using antibiotics. Notably, the neuroprotective effects of IDO-1 inhibition were completely abolished in pseudo-germ-free mice (administrated an antibiotic mixture orally for 14 days prior to 1-MT treatment), highlighting the dependency of 1-MT's neuroprotective effects on the presence of gut microbiota. Finally, we found IDO-1 inhibition corrects the abnormal elevation of fecal short chain fatty acids (SCFAs). Collectively, these findings suggest that IDO-1 inhibition may represent a promising therapeutic approach for PD.

The wound healing process in diabetic patients is often complex and prolonged, frequently complicated by persistent bacterial infections that can develop into multidrug-resistant infections, posing significant challenges for treatment. However, traditional hydrogel dressings often exhibit limited efficacy against complex wounds, primarily because therapeutic molecules are confined within the cross-linked matrix and exert nonselective antibacterial effects. This study developed a novel polyrotaxane-based hydrogel (FDS) against diabetic wounds complicated by drug-resistant bacterial infections. By assembling SNO-modified β-cyclodextrin onto the F-127DA copolymer backbone and copolymerized with dimethylamino propyl methacrylamide (DMAPMA), FDS hydrogel was endowed with conformational freedom of NO (Nitric Oxide) donor through rotatable and slidable motions and acid-responsive antibacterial properties simultaneously. This FDS hydrogel exhibited excellent antibacterial (both Staphylococcus aureus and methicillin-resistant S. aureus) and biofilm-dispersing effects, NO-enhanced angiogenesis, significantly reduced inflammatory response, and accelerated healing of chronic diabetic wounds in vitro and in vivo. By virtue of movable molecular anchoring facilitated NO delivery and microenvironment-activatable antibacterial activity, this research offers new hope for addressing the challenges of wound healing in diabetic patients accompanied by bacterial infections.

Breast cancer (BC) has the highest prevalence among cancers specific to women, and its incidence rates are increasing in many countries. Subtypes of BC, including HER2-positive or triple-negative BC, exhibit differing treatment responses; consequently, demand for personalized therapy is increasing, and relevant precision medicine strategies are under development. Aerobic glycolysis in cancer cells can lead to excessive lactate production, which in turn promotes lactylation and influences tumor cell behavior. Epigenetic alterations and metabolic reprogramming are prominent characteristics of tumors. Because lactate and lactylation are important in cancer, further investigation of the mechanisms underlying lactate metabolism and lactylation, and the development of therapeutic strategies targeting these processes, are topics of increasing interest. This review describes current research on lactate metabolism and lactylation in BC, thus offering new perspectives for advancing treatment and management toward more precise and personalized approaches that will ultimately increase BC survival rates and patient quality of life.

The inadequate body donors have restricted the development of medical sciences in some ways in mainland China. However, the investigation of potential donors may change the status quo of body donation. From July 10th to September 15th, 2021, we conducted a cross-sectional, multi-stage sampling study collected demographic data and individuals' willingness to accept body donation from 120 cities in mainland China. A stepwise linear regression analysis was adopted. 11,031 valid samples were collected for this survey. The willingness to donate body among Chinese residents averaged 53.66 points. To be specific, patients with a different number of children (1: β=-0.05; 2: β=-0.04; ≥3: β= -0.03) are less willing to donate their organs while respondents who live in an urban area (β = 0.03), have higher education level (high school or junior college: β = 0.04, such as a bachelor degree or above: β = 0.09), feel anxious (mild, moderate β = 0.02), feel pressured (moderate: β = 0.08; severe: β = 0.09), have higher scores of the Short-Form Health Literacy Instrument (HLS-SF12) (β = 0.30), Chronic Disease Self-Management Study Measures (CDSMS) (β = 0.18) and EuroQol Visual Analogue Scale (EQ-VAS) (β = 0.23), are more positive to donate. In this study, we discovered that Chinese residents' acceptance level of body donation is affected by age, gender, housing, educational levels, anxiety, pressure, social support, and health literacy, among which health literacy plays a key role in residents' attitudes towards body donation. This study firstly discusses the public acceptance of body donation through a nationwide sample around mainland China.

The dissemination of quarantine pathogens poses a significant risk to global crop production, threatening crop health and disrupting agroecosystems. Timely and accurate on-site detection is crucial to prevent outbreaks. Recombinase polymerase amplification (RPA) and multicomponent DNAzyme (MNAzyme) are promising isothermal detection technologies; however, their integration has been hindered by the requirement of single-stranded DNA (ssDNA) for MNAzyme activation, as RPA generates double-stranded DNA (dsDNA). To address this, we developed the OAR-MNA biosensor, an innovative one-pot platform coupling asymmetric RPA (aRPA) with MNAzyme-assisted target recycling. The biosensor was applied to detect Acidovorax citrulli, the causative agent of bacterial fruit blotch (BFB) in cucurbits. By adjusting RPA primer concentrations, aRPA generates abundant ssDNA, enabling visual detection of A. citrulli with a detection limit of 20 copies/μL and perfect specificity, surpassing the performance of fluorescent RPA. The biosensor demonstrated 100% concordance with qPCR in seed samples and maintained robust performance in spiked melon homogenate. Our comparative analysis revealed advantages of the OAR-MNA system, including reduced detection costs and enhanced field applicability. When coupled with a portable DNA analyser, the biosensor provides user-friendly results (+/-), making it ideal for on-site deployment. Beyond BFB detection, OAR-MNA has been successfully applied to the detection of Cucumber green mottle mosaic virus (CGMMV), an RNA quarantine pathogen that causes severe losses to cucurbit crop production. By enabling early, accurate on-site diagnosis, the OAR-MNA biosensor represents an advancement in plant pathogen detection technology, offering a promising tool for safeguarding global crop production.

BACKGROUND:Despite the insights into the role of aldehyde dehydrogenase 1 family member A1 (ALDH1A1) in various liver diseases, the expression and its prognostic significance in patients with hepatitis E virus-related acute liver failure (HEV-ALF) remain unclear. This study delved into the assessment of serum exosome-derived ALDH1A1 expression and its prognostic implications for HEV-ALF patients. METHODS:Between January 2018 and December 2023, a total of 226 individuals with acute hepatitis E (AHE) and 210 patients with HEV-ALF were recruited from member units of Chinese Consortium for the Study of Hepatitis E. According to the number of organ failure, we categorized 210 HEV-ALF patients into three groups: two organs failure (n = 131), three organs failure (n = 46), and more than three organs failure (n = 33). In addition, 200 health controls from Suzhou Municipal Hospital were included. RESULTS:The levels of serum exosome-derived ALDH1A1 in HEV-ALF patients were significantly higher than those in AHE patients and health controls (both P < 0.05). Furthermore, the levels of serum exosome-derived ALDH1A1 were the highest in more than three organs failure group, followed by three organs failure group and two organs failure group (all P < 0.001). Moreover, serum exosome-derived ALDH1A1 was positively correlated with total bilirubin in HEV-ALF patients (r = 0.315, P < 0.001). The comparisons of serum exosome-derived ALDH1A1 levels in treatment response showed that serum exosome-derived ALDH1A1 levels were decreased in the improvement group, while increased in the fluctuation and deterioration groups (all P < 0.001). Moreover, serum exosome-derived ALDH1A1 was an independent risk factor for predicting the 30-day mortality (P < 0.001). Furthermore, the area under the receiver operating characteristic curve was 0.943, with the sensitivity of 94.87% and specificity of 87.72%, indicating the robust decision-making ability. However, no significant differences were found in serum exosome-derived ALDH1A1 levels between patients aged < 60 and ≥ 60 years old (P = 0.131). CONCLUSIONS:Serum exosome-derived ALDH1A1 can greatly predict the prognosis of HEV-ALF patients.

BACKGROUND:Aquatic animal diseases cause significant economic losses in aquaculture. Effective pathogen diagnostics enable targeted treatment measures. Traditional detection methods primarily rely on polymerase chain reaction. However, due to the bulky and expensive nature of the equipment, on-site testing is impractical. Furthermore, the process requires low-temperature preservation, transportation to centralized laboratories, and analysis by trained personnel. The combination of loop-mediated isothermal amplification (LAMP) and lateral flow assay (LFA) has promoted on-site detection with greater convenience. However, there are still some shortcomings with the current LAMP-LFA technology. RESULTS:A micro-volume LAMP-LFA platform (μLAMP-LFA) was developed for on-site diagnosis of aquatic animal pathogens. The platform comprised a microfluidic chip and dedicated equipment (mini LAMPer). The μLAMP-LFA chip integrated LAMP and LFA technologies. After rotating the chip, the amplification solution was automatically transferred into the LFA chamber. The hermetically sealed chip effectively prevented false-positive results caused by aerosols. Additionally, the chip included a dedicated compartment for separate storage of dilution solutions, facilitating micro-volume LAMP-LFA. The μLAMP-LFA chip demonstrated excellent flow control, fluidity, and mixing capabilities. The rapid heating rate and stable temperature of the mini LAMPer ensured optimal conditions for LAMP reactions. The platform detected white spot syndrome virus and Salmonella at concentrations as low as 28 copies/μL and 15 copies/μL, respectively. The dual pathogen detection capability of the platform was validated using real shrimp samples. SIGNIFICANCE:Compared to conventional LAMP-LFA, μLAMP-LFA minimizes reagent usage, drastically lowering detection costs. More importantly, the pre-stored dilution solution in the chip plays a critical role in cooling the amplified products, preventing potential test strip failure. Its simple operation, portability and low cost grant the platform significant potential for field detection of pathogens in aquatic animals. In addition, it can also be applied to food safety, agricultural monitoring, environmental analysis, and other on-site detection applications.

As a complicated and heterogeneous condition, breast cancer (BC) has posed a tremendous public health challenge across the world. Recent studies have uncovered the crucial effect of human microbiota on various perspectives of health and disease, which include cancer. The oral-gut microbiome axis, particularly, have been implicated in the occurrence and development of colorectal cancer through their intricate interactions with host immune system and modulation of systemic inflammation. However, the research concerning the impact of oral-gut microbiome axis on BC remains scarce. This study focused on comprehensively reviewing and summarizing the latest ideas about the potential bidirectional relation of the gut with oral microbiota in BC, emphasizing their potential impact on tumorigenesis, treatment response, and overall patient outcomes. This review can reveal the prospect of tumor microecology and propose a novel viewpoint that the oral-gut microbiome axis can be a breakthrough point in future BC studies.

Fluorescence dye-based loop-mediated isothermal amplification (LAMP) is a sensitive nucleic acid detection method, but is limited to single-plex detection and may yield non-specific signals. In this study, we propose a bifunctional probe-based real-time LAMP amplification method for single-plexed or multiplexed detection. The bifunctional probe is derived by modifying the 5′ end of the fluorophore and an internal quencher on one of the LAMP primers; therefore, it can simultaneously be involved in the LAMP process and signal amplification. The fluorescence intensity undergoes a cumulative exponential increase during the incorporation of the bifunctional probe into double-stranded DNA amplicons. The bifunctional probe-based LAMP method is simplified and cost-effective, as the primer design and experimental operations align entirely with the ordinary LAMP. Different from other current probe-based methods, this method does not require additional enzymes, sequences, or special probe structures. Also, it is 10 min faster than several other probe-based LAMP methods. The bifunctional probe-based LAMP method allows the simultaneous detection of the target Vibrio parahaemolyticus DNA and the internal amplification control in a one-pot reaction, demonstrating its potential for multiplexed detection.

Point-of-care nucleic acid detection is essential for diagnosis and food safety, especially in resource-limited areas. This study reports a gravity-driven and rotation-controlled (GR) chip-coupled lateral flow-based assay (LFA) for point-of-care nucleic acid detection. The sample solution is added to the inlet of the GR chip and flows into the loop-medicated isothermal amplification (LAMP) chamber by the action of gravity. After the LAMP reaction, the GR chip is manually rotated to allow the amplified solution to flow into the LFA chamber for result readout. The GR chip integrates the LAMP reaction and LFA in a fully closed environment, avoiding the aerosol contamination problem. The GR chip's simple and ingenious structure simplifies the fabrication and reduces its cost, making it possible for point-of-care and even home self-testing.

Rapid and accurate detection of genetically modified crops (GMC) is significant for crop supervision and genetically modified product labeling. For field detection of GMC, the rapidity and simplicity of the sample pretreatment and detection process are very important. Simplifying the extraction process of DNA in crops and speeding up the detection process are the technical problems that need to be solved. Therefore, this paper provides a field detection method for GMC. First, the selected universal LAMP primers of the chloroplast gene can be used to detect reference genes in nine crops with the most transgenic detection needs. The universal primers of reference genes can not only improve the accuracy of detection, but also omit the design and synthesis of reference gene primers in different crops. Second, a high concentration of DNA can be obtained using simple and fast DNA extraction methods within 5 min. The specific concentration of GuHCl in lysis buffer can accelerate the subsequent nucleic acid amplification reaction. Third, a new probe-LAMP method was established, with high reaction speed and specificity. Using the dual probe-LAMP for transgenic and reference genes in a handheld nucleic acid detector, the detection of GMC can be completed within 25 min. Two different fluorescence signals generated from the LAMP reaction are processed through a special logic gate, and finally three kinds of results of "Positive", "Negative" and "Invalid" are output on a smartphone. This method from sample processing to intelligent result output can achieve accurate, rapid, simple, low-cost, pollution-free field detection of GMC.

Primary sclerosing cholangitis (PSC) is an autoimmune cholangiopathy characterized by chronic inflammation of the biliary epithelium and periductal fibrosis, with no curative treatment available, and liver transplantation is inevitable for end-stage patients. Human placental mesenchymal stem cell (hpMSC)-derived exosomes have demonstrated the ability to prevent fibrosis, inhibit collagen production and possess immunomodulatory properties in autoimmune liver disease. Here, we prepared hpMSC-derived exosomes (ExoMSC) and further investigated the anti-fibrotic effects and detailed mechanism on PSC based on Mdr2−/− mice and multicellular organoids established from PSC patients. The results showed that ExoMSC ameliorated liver fibrosis in Mdr2−/− mice with significant collagen reduction in the preductal area where Th17 differentiation was inhibited as demonstrated by RNAseq analysis, and the percentage of CD4+IL-17A+T cells was reduced both in ExoMSC-treated Mdr2−/− mice (Mdr2−/−-Exo) in vivo and ExoMSC-treated Th17 differentiation progressed in vitro. Furthermore, ExoMSC improved the hypersecretory phenotype and intercellular interactions in the hepatic Th17 microenvironment by regulating PERK/CHOP signaling as supported by multicellular organoids. Thus, our data demonstrate the anti-fibrosis effect of ExoMSC in PSC disease by inhibiting Th17 differentiation, and ameliorating the Th17-induced microenvironment, indicating the promising potential therapeutic role of ExoMSC in liver fibrosis of PSC or Th17-related diseases.

Current research endeavors have focused on the combination of various isothermal nucleic acid amplification methods with CRISPR/Cas systems, aiming to establish a more sensitive and reliable molecular diagnostic approach. Nevertheless, most assays adopt a two-step procedure, complicating manual operations and heightening the risk of contamination. Efforts to amalgamate both assays into a single-step procedure have faced challenges due to their inherent incompatibility. Furthermore, the presence of the protospacer adjacent motif (PAM) motif (e.g., TTN or TTTN) in the target double-strand DNA (dsDNA) is an essential prerequisite for the activation of the Cas12-based method. This requirement imposes constraints on crRNA selection. To overcome such limitations, we have developed a novel PAM-free one-step asymmetric recombinase polymerase amplification (RPA) coupled with a CRISPR/Cas12b assay (OAR-CRISPR). This method innovatively merges asymmetric RPA, generating single-stranded DNA (ssDNA) amenable to CRISPR RNA binding without the limitations of the PAM site. Importantly, the single-strand cleavage by PAM-free crRNA does not interfere with the RPA amplification process, significantly reducing the overall detection times. The OAR-CRISPR assay demonstrates sensitivity comparable to that of qPCR but achieves results in a quarter of the time required by the latter method. Additionally, our OAR-CRISPR assay allows the naked-eye detection of as few as 60 copies/μL DNA within 8 min. This innovation marks the first integration of an asymmetric RPA into one-step CRISPR-based assays. These advancements not only support the progression of one-step CRISPR/Cas12-based detection but also open new avenues for the development of detection methods capable of targeting a wide range of DNA targets.

Genetic factors and infectious pathogens that cause plant diseases have a major impact on agricultural production. In recent years, the potential of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system in nucleic acid analysis and plant disease diagnostics has been demonstrated. We highlight progress of CRISPR/Cas technology that is significant for monitoring plant growth and preventing diseases.

Nucleic acid quantification, allowing us to accurately know the copy number of target nucleic acids, is significant for diagnosis, food safety, agricultural production, and environmental protection. However, current digital quantification methods require expensive instruments or complicated microfluidic chips, making it difficult to popularize in the point-of-care detection. Paper is an inexpensive and readily available material. In this study, we propose a simple and cost-effective paper membrane-based digital loop-mediated isothermal amplification (LAMP) method for nucleic acid quantification. In the presence of DNA fluorescence dyes, the high background signals will cover up the amplicons-formed bright spots. To reduce the background fluorescence signals, a quencher-fluorophore duplex was introduced in LAMP primers to replace non-specific fluorescence dyes. After that, the amplicons-formed spots on the paper membrane can be observed; thus, the target DNA can be quantified by counting the spots. Take Vibrio parahaemolyticus DNA detection as an instance, a good linear relationship is obtained between the light spots and the copy numbers of DNA. The paper membrane-based digital LAMP detection can detect 100 copies target DNA per reaction within 30 min. Overall, the proposed nucleic acid quantification method has the advantages of a simple workflow, short sample-in and answer-out time, low cost, and high signal-to-noise, which is promising for application in resourced limited areas.