Foods
·
2025
·Vol. 14
·期 13
·2321
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IF 5.1
Xin Li,Mingxue Zhu,Siyuan Wang,Weijia Li,Baohong Ren,Lingbo Qu,Xiaoling Zhang
Listeria monocytogenes,Staphylococcus aureus,Salmonella enterica, andEscherichia coliO157:H7 are four major foodborne pathogenic bacteria found in meat and meat products, which pose significant threats to human health. In this study, we developed specific loop-mediated isothermal amplification (LAMP) primers targeting these four pathogenic bacteria. Following the optimization of system components and reaction parameters, four rapid and simplified LAMP-based detection assays were established, which enabled the visual detection of these four pathogenic bacteria within 40–50 min. The three established LAMP assays targetingL. monocytogenes,S. aureus, andE. coliO157:H7 achieved species-level discrimination, whereas the LAMP method forSalmonellaexhibited genus-level specificity. The detection limits of the LAMP assays were determined as follows: 1.8 × 101colony forming units (CFU)/mL forL. monocytogenes, 5.1 × 101CFU/mL forS. aureus, 1.2 × 101CFU/mL forS. enterica, and 3.3 × 103CFU/mL forE. coliO157:H7, with sensitivity improved by 10–1000-fold compared to conventional PCR. The developed LAMP assays were used to analyze 52 meat and meat product samples, and 7 samples were positive, which was consistent with the results of the conventional PCR and culture-based methods, demonstrating an accuracy rate of 100% for the LAMP methods. In conclusion, the established LAMP assays exhibit high specificity, enhanced sensitivity, and result visualization, making them suitable for on-site rapid detection in food safety monitoring.
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CLINICA CHIMICA ACTA
·
2025
·
IF 2.9
Yanghe Liu,Sijie Yin,Shiyue Chen,Chunlei Zhang,Nasifu Lubanga,Ping Tai,Mengqiu Xiong,Boyue Fan,Xincheng Yang,Xinyi Xia,PanFei Hou,Bangshun He
Helicobacter pylori ( H. pylori ) is a risk factor of gastrointestinal diseases. Vacuolating cytotoxin A (VacA) is a key component of the pathogenicity of H. pylori for gastric diseases , and detection for virulence genotypes facilitates precise clinical diagnosis and individual treatment. Hence, a more accurate, convenient, and highly sensitive detection method is urgently needed. In this study, we developed a method based on fluorescence-colorimetric dual-indicator loop-mediated isothermal amplification (FC-LAMP), allowing for naked-eye readout, using a constant temperature water bath within 40 min in clinical settings. Such an excellent sensitivity and specificity, and user-friendly operation approach achieved a limit of 10 −6 ng/μl for 16 s rRNA detection of H. pylori , and 100 % specificity for 55 clinical gastric fluid samples compared with the immunofluorescence staining (IFS) and quantitative polymerase chain reaction (qPCR) as reference methods. In short, this study offers a new strategy for detection of H. pylori and its virulence genotype in primary hospitals and in limited resources settings, facilitating individualized precision eradication of H. pylori .
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Biosensors-Basel
·
2023
·Vol. 13
·期 2
·180
·
IF 5.4
Sheng Li,Wenlong Guo,Minmin Xiao,Yulin Chen,Xinyi Luo,Wenfei Xu,Jianhua Zhou,Jiasi Wang
Rapid and sensitive detection of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for early diagnosis and effective treatment. Nucleic acid testing has been considered the gold standard method for the diagnosis of COVID-19 for its high sensitivity and specificity. However, the polymerase chain reaction (PCR)-based method in the central lab requires expensive equipment and well-trained personnel, which makes it difficult to be used in resource-limited settings. It highlights the need for a sensitive and simple assay that allows potential patients to detect SARS-CoV-2 by themselves. Here, we developed an electricity-free self-testing system based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) that allows for rapid and accurate detection of SARS-CoV-2. Our system employs a heating bag as the heat source, and a 3D-printed box filled with phase change material (PCM) that successfully regulates the temperature for the RT-LAMP. The colorimetric method could be completed in 40 min and the results could be read out by the naked eye. A ratiometric measurement for exact readout was also incorporated to improve the detection accuracy of the system. This self-testing system is a promising tool for point-of-care testing (POCT) that enables rapid and sensitive diagnosis of SARS-CoV-2 in the real world and will improve the current COVID-19 screening efforts for control and mitigation of the pandemic.Keywords:SARS-CoV-2;self-testing;reverse transcription loop-mediated isothermal amplification
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Plants-Basel
·
2021
·Vol. 10
·期 2
·253
·
IF 3.935
Qin Xiong,Linlin Zhang,Xinyue Zheng,Yulin Qian,Yaxin Zhang,Lijuan Zhao,Qiang Cheng
Marssonina brunneais the main pathogen that causes poplar black spot disease, which leads to the decrease of the photosynthetic efficiency and significantly affects the production and quality of timber. Currently, no in-field diagnostic exists forM. brunnea. Here, we described a loop-mediated isothermal amplification (LAMP) assay for the rapid and sensitive detection ofM. brunnea. A set of six oligonucleotide primers was designed to recognize eight distinct sequences of the internal transcribed spacer (ITS) region ofM. brunnea. The LAMP assay was optimized by the combination of high specificity, sensitivity, and rapidity for the detection of less than 10 pg/μL of target genomic DNA in 60 min per reaction at 65 °C, whereas with PCR, there was no amplification of DNA with concentration less than 1 ng/μL. Among the genomic DNA of 20 fungalisolates, only the samples containing the genomic DNA ofM. brunneachanged from violet to sky blue (visible to the naked eye) by using hydroxynaphthol blue (HNB) dye. No DNA was amplified from the eight other fungus species, including two otherMarssoninapathogens, three other foliar fungi pathogens of poplar, and three common foliar fungal endophytes of poplar. Moreover, the detection rates ofM. brunneafrom artificially and naturally infected poplar leaves were 10/16 (62.5%) and 6/16 (37.5%) using PCR, respectively, while the positive-sample ratios were both 16/16 (100%) using the LAMP assay. Overall, theITSLAMP assay established here can be a better alternative to PCR-based techniques for the specific and sensitive detection ofM. brunneain poplar endemic areas with resource-limited settings.Keywords:Marssonina brunnea;rapid detection;loop-mediated isothermal amplification;poplar black spot
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SENSORS AND ACTUATORS B-CHEMICAL
·
2023
·
IF 8.4
Li Meng,Mingzhong Li,Zhenyu Xu,Aman Lv,Yanwei Jia,Meiwan Chen,Pui-In Mak,Rui P. Martins,Man-Kay Law
ABSTRACT Digital microfluidics (DMF) shows great potential in the manipulation of individual droplets. However, the limited number of electrode control wirings for droplet control intrinsically restrains the massive generation of pL/nL individual droplets, fundamentally hindering the realization of high throughput applications on DMF such as digital nucleic acid amplification and analysis. This work demonstrates on-chip high throughput digital loop-mediated isothermal amplification (dLAMP) on a DMF platform based on a functional substrate with super-wettability contrast. Exploiting the passive dispensing technique as empowered by electrowetting on dielectric (EWOD) effect, a total of 1818 individual droplets with high uniformity are generated on the super-hydrophilic microarray within 1 minute. Separation within the oil phase without tubes and valves can avoid evaporation, ensuring a precise total reaction volume. Absolute quantification of λDNA with a dynamic range of 1-1000 copies/μL is achieved by counting the amount of positive microdroplets after on-chip incubation, demonstrating the strong potential for rapid, low-cost, reliable, and quantitative nucleic acid analysis with high accuracy.
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ANALYTICA CHIMICA ACTA
·
2023
·
IF 6.2
Ling Deng,Xiaoyi He,Ke Liu,Yuanli Li,Han Xia,Hang Qian,Xiaoxue Lu,Xuhu Mao,Yang Xiang
Burkholderia pseudomallei is the causative agent of melioidosis, a potentially life-threatening infectious disease, and poses public health risks in endemic areas. Due to the high mortality, intrinsic antibiotic resistance , and atypical manifestations, establishing a rapid, accurate, and sensitive identification of B. pseudomallei enables earlier diagnosis, proper treatments, and better outcomes of melioidosis. Herein, we present a O ne- P ot C RISPR-integrated assay for I nstant and V isual D etection (termed OPC-IVD) of B. pseudomallei . The integration of recombinase polymerase amplification and CRISPR-Cas12a recognition-activated trans -cleavage, achieved a true all-in-one single-tube reaction system, initiating the amplification and cleavage simultaneously, which realized a facile sample-to-answer assay. This approach could be performed with simplified DNA extraction and completed around 30 min by holding the reaction tube in the hand. The detection limit of our OPC-IVD was determined to be 2.19 copy/uL of plasmid DNA, 12.5 CFU/mL of B. pseudomallei , and 61.5 CFU/mL of bacteria in spiked blood samples, respectively. Furthermore, the introduction of internal amplification control effectively reduced the occurrence of false negatives, which was incorporated in the reaction system, and amplified simultaneously with the target and read by naked eyes. The assay exhibited 100% accuracy when evaluated in clinical isolates and samples. The streamlined workflow of our OPC-IVD of B. pseudomallei enables a field-deployable, instrument-free, and ultra-fast approach that can be utilized by non-expert personnel in the field of molecular diagnosis of melioidosis especially in under-resourced setting.
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FOOD CONTROL
·
2024
·
IF 5.6
Yingying Chen,Zipeng Guo,Zhaoqing Sun,Xinyu Wu,Yifan Xu,Zhengye Jin,Yuying Cheng,Zhen She,Qianqian Jiang,Yuxin Wo,Ting Wu,Hongwang Liu,Meihui Liu,Yulu Liu,Enjie Diao,Dengchao Li,Ruifeng Mao
Cashew nut allergy shows an increasing prevalence along with its high consumption. Nowadays, food allergy still has no cure, and allergen avoidance is still the standard management, urging a sensitive and convenient cashew nut detection method. Herein, saltatory rolling circle amplification (SRCA), loop-mediated isothermal amplification (LAMP), as well as polymerase chain reaction (PCR) targeting the Ana o 2 gene were proposed and compared. In addition, a closed-tube SRCA assay using hydroxynaphthol blue (HNB) as a color indicator (HNB-SRCA) was developed to realize visual on-site detection of cashew nut. SRCA and HNB-SRCA can detect 75.3 fg of cashew nut DNA, which is 10- and 100-times more sensitive than LAMP and PCR, respectively. SRCA and HNB-SRCA exhibited a relative sensitivity of 0.01% in a binary mixture, which is comparable to LAMP and 10-times higher than that of PCR. When combined with a rapid DNA extraction method, SRCA and HNB-SRCA performed better applicability compared to LAMP and PCR. Additionally, compared to SRCA using electrophoresis to analyze the amplification products, the visual closed-tube HNB-SRCA combined with the rapid DNA extraction protocol is a better potential cashew nut on-site detection strategy in foods.
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Foods
·
2024
·Vol. 13
·期 13
·2143
·
IF 4.7
Shuangshuang Cui,Yong Wei,Can Li,Jian Zhang,Yunfeng Zhao,Xiayu Peng,Fengxia Sun
(1) Background: Rapid on-site testing is an effective method for the detection ofEscherichia coli O157: H7(E. coli O157: H7) in food ingredients and the environment. (2) Methods: In this study, we developed colorimetric loop-mediated isothermal amplification (LAMP) and immunochromatographic test strips (ICTs) for the rapid and visual detection ofE. coli O157: H7. This study designed new specific LAMP primers forE. coli O157: H7virulence island genes. After the LAMP amplification, the double-stranded DNA target sequence labeled with digoxin and fluorescein isothiocyanate (FITC) at both ends was bound to the anti-digoxin antibody on the gold nanoparticles. Subsequently, it was further bound to the anti-FITC antibody at the T line of the ICTs, forming a positive test result. Hydroxynaphthyl blue dye was directly added to the LAMP amplification product. A blue color indicated positive results, while a purple color indicated negative results. (3) Results: Two visualization methods showed high specificity for the target strains. The visualization tests had sensitivities of 5.7 CFU mL−1, and the detection limit of theEscherichia coli O157: H7in artificially contaminated milk samples was 5.7 × 102CFU mL−1, which was consistent with the results of the standard method (LAMP-electrophoresis method) used in commercial inspection. (4) Conclusions: Both methods could be useful in remote and under-resourced areas.
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SENSORS AND ACTUATORS B-CHEMICAL
·
2025
·
IF 7.7
Li Meng,Mingzhong Li,Zhenyu Xu,Tianlan Chen,Meiwan Chen,Joana Vaz Pinto,Bruno Veigas,Man-Kay Law
Precise temperature control and measurement are crucial for in vitro nucleic acid denaturation, especially in melting curve analysis (MCA). However, the inherent cumbersome multi-step calibration process required to obtain an accurate spatial temperature gradient can be a major limitation for the study of MCA on existing microfluidic chips. This work proposes to obtain a highly linear temperature gradient with a functional substrate for achieving high-resolution MCA on a digital microfluidics (DMF) platform. Taking advantage of the high thermal conductivity of aluminum, we employ a thin trapezoidal aluminum plate as the DMF substrate to compensate for the uneven heat transfer caused by temperature differences at different substrate positions and the ambient environment. We further pattern a banded superhydrophilic thread on the superhydrophobic surface to passively achieve a liquid thread by driving the mother droplet through the electrowetting on dielectric (EWOD) effect. With a heater and a cooler positioned at the opposite ends of the substrate, we can create a highly linear (simulated R 2 = 1 and experimental R 2 = 0.9998) temperature gradient from 40°C to 90°C across the liquid thread, requiring only temperature calibration at the two end points. The elimination of laborious temperature calibration steps can effectively enable rapid, continuous and reliable nucleic acid amplification as well as MCA on DMF. The high temperature accuracy also ensures simultaneous multiple hybrid DNA detection for rapid one-time discrimination of single nucleotide mutations in KRAS gene targets. This innovation underscores the DMF platform's potential in the realms of biomedical research and clinical diagnostics.
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AUSTRALASIAN PLANT PATHOLOGY
·
2025
·
IF 1.1
Chen Jiajia,Huang Xiaoxiao,Peng Jinfeng,Chen Yuanyuan,Gafforov Yusufjon,Xing Yifan,Yu Jian
Globisporangium recalcitrans is an important phytopathogenic oomycete that causes soybean root rot. To develop a loop-mediated isothermal amplification (LAMP)-based system for rapid and specific detection of G. recalcitrans , four LAMP primers and two loop primers were designed with the Ribosomal DNA transcribed spacer 2 sequence (ITS2) as the target gene. The specificity and sensitivity of these primers were validated, and the system was also successfully applied to detect G. recalcitrans in soybean tissues after artificial inoculation and natural infection. The nucleic acid amplification reaction was performed under isothermal conditions at 63 °C for 60 min. Specificity was compared with those for 71 strains of G. recalcitrans , other Globisporangium spp. The minimum detection limit of the system was 100 fg·μL −1 for detecting fungal genomic DNA. This LAMP-based system provides a technique for specific detection of G. recalcitrans and rapid diagnosis of the disease it caused.
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Frontiers in Cellular and Infection Microbiology
·
2025
·
IF 4.8
Xiangrong Zheng,Jian Yu,Jinfeng Peng,Xiuwen Qiu,Yaya Sun,Danyang Fu,Yusufjon Gafforov,Ali Chenari Bouket,Jiajia Chen
Damping-off has caused huge losses in soybean production areas of China. Based on multi-locus phylogenetic analysis and Koch's Postulates, the causal agent of soybean damping-off was identified as Globisporangium intermedium. Given the potential danger of G. intermedium, early and precise detection methods are needed for both disease management and prevention. In this study, a LAMP assay utilizing a new target gene Rpbrpb1 was developed and evaluated for the detection of G. intermedium. This Rpbrpb1 LAMP assay was found highly specific to G. intermedium. All nine tested isolates of G. intermedium yielded positive results, whereas 30 non-target isolates belonging to Globisporangium spp., Pythium spp., Phytophthora spp., Phytopythium spp., and soil-borne fungi lacked detection. The LAMP method can identify G. intermedium at DNA concentrations as low as 10 pg/μL. In terms of on-site disease diagnosis, the LAMP assay could detect G. intermedium from artificially inoculated soybean tissues and naturally infested rhizosphere soil collected from fields in soybean production regions. Consequently, this study not only stands as the first record of G. intermedium as a new soybean pathogen in China, but also provides an efficient LAMP field detection method that could significantly contribute to the management and prevention of soybean damping-off.
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