Isothermal Amplification Method Research Articles

Development and evaluation of a loop-mediated isothermal amplification (LAMP) method for Candida glabrata detection.

Loop-mediated isothermal amplification (LAMP) is a simple and rapid nucleic acid method for DNA amplification at a constant temperature. The "gold standard" culture method for yeast detection, has low sensitivity with severe consequences, increasing morbidity and mortality rates. Here, we report the development of a LAMP method for the specific detection of C. glabrata. The specific LAMP primers for C. glabrata detection were designed and evaluated. The LAMP assay accurately detected C. glabrata with no cross-reactivity with other Candida species. The developed molecular method would be a promising tool in the management of invasive candidiasis.

Development of a highly specific LAMP assay for detection of Sarcocystis tenella and Sarcocystis gigantea in sheep.

Sarcocystis infection in sheep has caused significant economic losses in the livestock industry, and the genetic similarity among Sarcocystis species highlights the need for precise diagnostic methods in sheep. This study developed a loop-mediated isothermal amplification (LAMP) method targeting COX-1 and 28S rRNA genes to detect Sarcocystis tenella and Sarcocystis gigantea, respectively. The LAMP method exhibited high specificity, selectively amplifying target DNA sequences without cross-reactivity with closely related protozoa, such as Toxoplasma gondii and Neospora caninum. Detection limits were determined as 3 × 105 copies/L for S. tenella and 6 × 104 copies/L for S. gigantea, enabling sensitive identification of low-level infections. Comparative analysis with conventional PCR on sheep cardiac tissues demonstrated a higher LAMP detection rate (80.0% vs 66.7%). In conclusion, the LAMP method offers superior sensitivity to conventional PCR, allows visual confirmation of results, and provides a rapid diagnostic tool for identifying S. tenella and S. gigantea infection in sheep. However, due to the limitation of sample availability, we were unable to assess all Sarcocystis species that use sheep as intermediate hosts, which warrants further research.

Development and application of a rapid visual detection technique for VanA gene in vancomycin-resistant Enterococcus faecium.

One of the key approaches to treating and controlling vancomycin-resistant Enterococcus faecium (VREFm) is an accurate and rapid diagnosis. To achieve this goal, a simple and rapid method must be constructed for immediate detection in the field. Multienzyme isothermal rapid amplification (MIRA) is an isothermal rapid amplification method that allows amplification reactions to be completed under room temperature conditions. When combined with lateral flow strips (LFSs), MIRA-LFS enables the rapid detection of pathogenic microorganisms. However, the MIRA method often produces false signals. These false signals are eliminated by using base mismatches introduced in primers and probes. The MIRA-LFS system was constructed with high specificity and sensitivity for the detection of VREfm, without the limitation of sophisticated instruments. This enables the prompt formulation of diagnostic and therapeutic decisions.

A new exploration of the amplification mechanism of saltatory rolling circle amplification (SRCA) for food safety detection

A new property of Bst DNA polymerase was discovered by our team, which led to the establishment of a new isothermal amplification method for SRCA nucleic acid, which was applied to food safety detection, investigated the amplification mechanism, and the SRCA version 1.0 amplification mechanism was proposed. This study proposes a new SRCA amplification mechanism (version 2.0) by using the autosynthetic sequences CDG2040 and CDG2068, as well as the Listeria monocytogenes hlyA gene and Vibrio parahaemolyticus toxR gene as target sequences to further investigate the amplification mechanism. The initiation of the SRCA reaction depends primarily on the spatial structure of the non-closed circle formed by the discontinuous complementation of the bases on both sides of the target sequence. The DNA fragments that connect the target sequences in the SRCA reaction are the sum of the complementary sequences of adjacent partial sequences on both sides of the target sequence.

Advances in plant pathogen detection: integrating recombinase polymerase amplification with CRISPR/Cas systems.

Plant pathogens are causing substantial economic losses and thus became a significant threat to global agriculture. Effective and timely detection methods are prerequisite for combating the damages caused by the plant pathogens. In the realm of plant pathogen detection, the isothermal amplification techniques, e.g., recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP), have emerged as a fast, precise, and most sensitive alternative to conventional PCR but they often comprise high rates of non-specific amplification and operational complexity. In recent advancements, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease Cas systems, particularly Cas12, have emerged as powerful tools for highly sensitive, specific, and rapid pathogen detection. Exploiting the collateral activities of Cas12, which selectively cleaves single-stranded DNA (ssDNA), novel detection platforms have been developed. The mechanism employs the formation of a triple complex molecule comprising guide RNA, Cas12 enzyme, and the substrate target nucleotide sequence. Upon recognition of the target, Cas12 indiscriminately cleaves the DNA strand, leading to the release of fluorescence from the cleaved ssDNA reporter. Integration of isothermal amplification methods with CRISPR/Cas12 enables one-step detection assays, facilitating rapid pathogen identification within 30min at a single temperature. This integrated RPA-CRISPR/Cas12a approach eliminates the need for RNA extraction and cDNA conversion, allowing direct use of crude plant sap as a template. With an affordable fluorescence visualization system, this portable method achieves 100-fold greater sensitivity than conventional techniques. This review summarizes recent advances in RPA-CRISPR/Cas12a for detecting plant pathogens, covering primer design, field-level portability, and enhanced sensitivity.

Rapid and simultaneous detection of common childhood diarrhea viruses by microfluidic-FEN1-assisted isothermal amplification with ultra-high specificity and sensitivity

Rapid and accurate diagnostic methods are crucial for managing viral gastroenteritis in children, a leading cause of global childhood morbidity and mortality. This study introduces a novel microfluidic-Flap endonuclease 1 (FEN1)-assisted isothermal amplification (MFIA) method for simultaneously detecting major viral pathogens associated with childhood diarrhea—rotavirus, norovirus, and adenovirus. Leveraging the specificity-enhancing properties of FEN1 with a universal dspacer-modified flap probe and the adaptability of microfluidic technology, MFIA demonstrated an exceptional detection limit (5 copies/μL) and specificity in the simultaneous detection of common diarrhea pathogens in clinical samples. Our approach addresses the limitations of current diagnostic techniques by offering a rapid (turn around time <1 h), cost-effective, easy design steps (universal flap design), and excellent detection performance method suitable for multiple applications. The validation of MFIA against the gold-standard PCR method using 150 actual clinical samples showed no statistical difference in the detection performance of the two methods, positioning it as a potential detection tool in pediatric diagnostic virology and public health surveillance. In conclusion, the MFIA method promises to transform pediatric infectious disease diagnostics and contribute significantly to global health efforts combating viral gastroenteritis.

Multiplex competitive annealing mediated isothermal amplification with high fidelity DNA polymerase (HiFi-CAMP)

Various isothermal amplification methods have been developed for point-of-care testing (POCT) of various infectious diseases. Here, we proposed a novel isothermal amplification method, named as 5′-half complementary primers mediated isothermal amplification (HCPA). Because of the similarity of our method to the previous method competitive annealing mediated isothermal amplification (CAMP) in primer design, we also use the name CAMP for our method. We demonstrated that CAMP is mediated by both a linear isothermal amplification pattern and a loop-mediated isothermal amplification pattern. To improve the specificity and enable multiplex detection, we further developed HiFi-CAMP method that uses a small amount of high-fidelity DNA polymerase to cut HFman probe to release fluorescent signal. The HiFi-CAMP method was demonstrated to have a good specificity and sensitivity, and fast amplification speed in detection of three human respiratory viruses, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), respiratory syncytial virus A (RSV-A) and influenza A viruses (IAV). When compared with gold standard RT-qPCR assays, the HiFi-CAMP assays showed sensitivities of 90.0 %, 71.4 % and 78.1 %, specificities of 100 %, 100 % and 95.5 %, and consistencies of 93.0 %, 93.3 % and 88.2 % for SARS-CoV-2, RSV-A and IAV, respectively. Furthermore, a duplex HiFi-CAMP assay was also developed to simultaneously detect RSV-A and SARS-CoV-2. The HiFi-CAMP will provide a promising candidate for POCT diagnosis in resource-limited settings.

Identification of Pathogen Causing Bulb Rot in Fritillaria taipaiensis P. Y. Li and Establishment of Detection Methods.

Fritillaria taipaiensis P. Y. Li (F. taipaiensis) is a traditional Chinese herbal medicine that has been used for over two millennia to treat cough and expectoration. However, the increasing cultivation of F. taipaiensis has led to the spread of bulb rot diseases. In this study, pathogens were isolated from rotten F. taipaiensis bulbs. Through molecular identification, pathogenicity testing, morphological assessment, and microscopy, Fusarium solani was identified as the pathogen causing bulb rot in F. taipaiensis. The colonization of F. solani in the bulbs was investigated through microscopic observation. The rapid and accurate detection of this pathogen will contribute to better disease monitoring and control. Loop-mediated isothermal amplification (LAMP) and qPCR methods were established to quickly and specifically identify this pathogen. These results provide valuable insights for further research on the prediction, rapid detection, and effective prevention and control of bulb rot in F. taipaiensis.

Rapid Detection of Staphylococcus Epidermidis Using Electric-Double-Layer (EDL) Field-Effect Transistor (FET) Biosensors with Loop-Mediated Isothermal Amplification

Staphylococcus epidermidis is a Gram-positive bacterium which commonly grows in human skin, nasal passages, respiratory tract, intestinal tract, air, and water. Although it is harmless to healthy individuals, immunocompromised patients are at risk of infection, which can lead to severe symptoms. With the increased reliance on antibiotics in hospitals, the antimicrobial resistance of Staphylococcus epidermidis has also risen, pointing out a challenge in clinical treatment.Therefore, the diagnosis of this bacterium has gained significant attention. The aim of this experiment is to detect bloodstream infections (BSIs) in the shortest possible time. The study is intended to be implemented in hospitals to provide a more convenient method for medical professionals to conduct screenings. The approach involves screening for the most common disease sequence, ssDNA, followed by sequential detection of corresponding cDNA, plasmids, and bacteria. To detect bacteria with the least amount of bacteerial load, a novel isothermal amplification method called Loop-mediated isothermal amplification (LAMP) is employed to increase the concentration of DNA of BSIs bacteria before measurement. The overall time required for this process doesn’t exceed one day, significantly increasing speed for the detection of blood-borne bacteria in hospitals. The core mechanism of this platform involves the use of electric-double-layer-gated field-effect-transistors (EDL-gated FETs) to amplify the gate voltage changes generated when the ssDNA binds with complementary DNA. These voltage changes are then converted into numerical values for analysis and interpretation.Currently, the research results show that this biomedical sensor can detect concentrations ranging from 1fM to 1pM. With the assistance of LAMP, it has successfully achieved a concentration of approximately 1yM (10-24 M) and detected with EDL-gated FETs. The experiment was also carried out step by step on simulated clinical specimens. The experiments have found successful serum nucleic acid amplification methods that provide excellent detection limits. Compared with other biomedical sensors in the past, it not only has lower detection limits but also brings features such as faster, more convenient, and more portable.Keywords: EDL, FET, Staphylococcus epidermidis, Biosensor, LAMP Figure 1

EasyNAT Malaria: a simple, rapid method to detect Plasmodium species using cross-priming amplification technology.

This study established and validated EasyNAT Malaria Assay as a promising molecular detection tool for malaria screening of high-risk populations in clinical practice. This novel isothermal amplification method may effectively facilitate the rapid diagnosis of malaria and prevent its transmission.

Rapid cyprinid herpesvirus 3 detection using loop-mediated isothermal amplification (LAMP) combined with gold nanoprobes and SYBR safe

As an etiological agent of a notifiable and emerging disease, Cyprinid herpesvirus 3 (CyHV-3 or koi herpesvirus, KHV) is a highly risky factor that affects koi and common carp yield through increasing the mortality rates. In the current study, a highly rapid and sensitive technique was established to detect CyHV-3 with the help of loop-mediated isothermal amplification (LAMP) method with dual visualizing indicators (SYBR safe and gold nanoparticle probes; AuNP-probes). Six specific primers were used to amplify the thymidine kinase (TK) of CyHV-3 using a LAMP reaction of 45 min at 65 °C along with detecting the products through visual inspection via hybridization at 65 °C for 20 min with a functionalized thiol-AuNP probe. A closed-tube LAMP-SYBR safe assay was also developed under the same condition without AuNP probe. The amplification of KHV-LAMP products were evaluated by the naked eye, through fluorescent emission of LAMP-SYBR safe dye-complex and colorimetric aggregate of LAMP product in the presence of AuNP probe. Furthermore, quantitative measurement was applied to the LAMP-AuNPs assay, using the spectral shift assay. The detection limit was 1.25 fentograms DNA which can compete the latest techniques available. The proposed technique yielded negative outcomes with DNA template from other viruses such as CyHV-1 and CEV. The LAMP-SYBR safe and LAMP-AuNPs assays introduced in this article were very sensitive, fast, and reliable diagnostic tools for the detection of CyHV-3.

SERS biosensors based on catalytic hairpin self-assembly and hybridization chain reaction cascade signal amplification strategies for ultrasensitive microRNA-21 detection.

A highly sensitive surface-enhanced Raman scattering (SERS) biosensor has been developedfor the detection of microRNA-21 (miR-21) using an isothermal enzyme-free cascade amplification method involving catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR). The CHA reaction is triggered by the target miR-21, which causes hairpin DNA (C1 and C2) to self-assemble into CHA products. After AgNPs@Capture captures the resulting CHA product, the HCR reaction is started, forming long-stranded DNA on the surface of AgNPs. A strong SERS signal is generated due to the presence of a large amount of the Raman reporter methylene blue (MB) in the vicinity of the SERS "hot spot" on the surface of AgNPs. The monitoring of the SERS signal changes of MB allows for the highly sensitive and specific detection of miR-21. In optimal conditions, the biosensor exhibits a satisfactory linear range and a low detection limit for miR-21 of 42.3 fM. Additionally, this SERS biosensor shows outstanding selectivity and reproducibility. The application of this methodology to clinical blood samples allows for the differentiation of cancer patients from healthy controls. As a result, the CHA-HCR amplification strategy used in this SERS biosensor could be a useful tool for miRNA detection and early cancer screening.

Traditional to technological advancements in Ganoderma detection methods in oil palm.

Ganoderma sp., the fungal agent causing basal stem rot (BSR), poses a severe threat to global oil palm production. Alarming increases in BSR occurrences within oil palm growing zones are attributed to varying effectiveness in its current management strategies. Asymptomatic progression of the disease and the continuous monoculture of oil palm pose challenges for prompt and effective management. Therefore, the development of precise, early, and timely detection techniques is crucial for successful BSR management. Conventional methods such as visual assessments, culture-based assays, and biochemical and physiological approaches prove time-consuming and lack specificity. Serological-based diagnostic methods, unsuitable for fungal diagnostics due to low sensitivity, assay affinity, cross-contamination which further underscores the need for improved techniques. Molecular PCR-based assays, utilizing universal, genus-specific, and species-specific primers, along with functional primers, can overcome the limitations of conventional and serological methods in fungal diagnostics. Recent advancements, including real-time PCR, biosensors, and isothermal amplification methods, facilitate accurate, specific, and sensitive Ganoderma detection. Comparative whole genomic analysis enables high-resolution discrimination of Ganoderma at the strain level. Additionally, omics tools such as transcriptomics, proteomics, and metabolomics can identify potential biomarkers for early detection of Ganoderma infection. Innovative on-field diagnostic techniques, including remote methods like volatile organic compounds profiling, tomography, hyperspectral and multispectral imaging, terrestrial laser scanning, and Red-Green-Blue cameras, contribute to a comprehensive diagnostic approach. Ultimately, the development of point-of-care, early, and cost-effective diagnostic techniques accessible to farmers is vital for the timely management of BSR in oil palm plantations.

Direct detection of Aeromonas hydrophila from water and tissue samples by loop-mediated isothermal amplification (LAMP) method

Direct detection of Aeromonas hydrophila from water and tissue samples by loop-mediated isothermal amplification (LAMP) method

Development and Comparison of Visual LAMP and LAMP-TaqMan Assays for Colletotrichum siamense.

Strawberry anthracnose caused by Colletotrichum spp. has resulted in significant losses in strawberry production worldwide. Strawberry anthracnose occurs mainly at the seedling and early planting stages, and Colletotrichum siamense is the main pathogen in North China, where mycelia, anamorphic nuclei, and conidia produced in the soil are the main sources of infection. The detection of pathogens in soil is crucial for predicting the prevalence of anthracnose. In this study, a visualized loop-mediated isothermal amplification (LAMP) assay and a loop-mediated isothermal amplification method combined with a TaqMan probe (LAMP-TaqMan) assay were developed for the β-tubulin sequence of C. siamense. Both methods can detect Colletotrichum siamense genomic DNA at very low concentrations (104 copies/g) in soil, while both the visualized LAMP and LAMP-TaqMan assays exhibited a detection limit of 50 copies/μL, surpassing the sensitivity of conventional PCR and qPCR techniques, and both methods showed high specificity for C. siamense. The two methods were compared: LAMP-TaqMan exhibited enhanced specificity due to the incorporation of fluorescent molecular beacons, while visualized LAMP solely necessitated uncomplicated incubation at a constant temperature, with the results determined by the color change; therefore, the requirements for the instrument are relatively straightforward and user-friendly. In conclusion, both assays will help monitor populations of C. siamense in China and control strawberry anthracnose in the field.

The Establishment of the Multi-Visual Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Vibrio harveyi, Vibrio parahaemolyticus, and Singapore grouper iridovirus

Groupers are valuable economic fish in the southern sea area of China, but the threat of disease is becoming more and more serious. Vibrio harveyi, V. parahaemolyticus, and Singapore grouper iridovirus (SGIV) are three important pathogens that cause disease in groupers, and infection with either a single one or a mix of these pathogens poses a serious threat to the healthy development of grouper culture. To enhance the rapid diagnosis and screening in the early stages, it is necessary to develop rapid detection methods of these pathogens. To simultaneously and rapidly detect the three pathogens, in this study, we utilized the TolC of V. harveyi, DNAJ of V. parahaemolyticus, and RAD2 of SGIV as the target genes and established a triple visual loop-mediated isothermal amplification (LAMP) method. This LAMP method showed a detection time as fast as 30 min and a high sensitivity of 100 fg/μL. Moreover, this method exhibited strong specificity and no cross-reaction with seven types of Vibrio and Staphylococcus aureus, as well as five common viruses in aquatic animals. Then, the LAMP products were enzymically cut, and three characteristic strips were used to identify the pathogen species. The results of the clinical trials demonstrated that the method could accurately and specifically detect V. harveyi, V. parahaemolyticus, and SGIV in grouper tissues. In summary, this study successfully established a triple visual LAMP rapid detection method for V. harveyi, V. parahaemolyticus, and SGIV. The method offers several advantages including simple equipment, easy operation, rapid reaction, high specificity, high sensitivity, and visual results. It is suitable for the early and rapid diagnosis of groupers infected with V. harveyi, V. parahaemolyticus, and SGIV, thereby providing useful technical support for further application in the large-scale disease surveillance of aquaculture animals.

Two-Stage Mixed-Dye-Based Isothermal Amplification with Ribonuclease-Cleavable Enhanced Probes for Dual-Visualization Detection of SARS-CoV-2 Variants of Interest.

Rapid and visual detection of SARS-CoV-2 variants is vital for timely assessment of variant transmission in resource-limited settings. Here, a closed-tube, two-stage, mixed-dye-based isothermal amplification method with ribonuclease-cleavable enhanced probes (REP), termed REP-TMAP, for dual-visualization detection of SARS-CoV-2 variants including JN.1, BA.2, BA.4/5, and Delta is introduced. The first stage of REP-TMAP is reverse transcription recombinase polymerase amplification and the second stage is dual-visualization detection synergistically mediated by the REP and the mixed dyes of cresol red and hydroxy naphthol blue. In REP-TMAP reaction, the color change under ambient light indicates SARS-CoV-2 infection, while the fluorescence change under blue light excitation specifies variant type. On detecting transcribed RNA of SARS-CoV-2 spike gene, this assay is rapid (within 40min), highly sensitive (10-200 copies per reaction), and highly specific (identification of single-base mutations). Furthermore, the assay has been clinically validated to accurately detect JN.1, BA.2, and BA.4/5 variants from 102 human oropharyngeal swabs. The proposed assay therefore holds great potentials to provide a rapid, dual-visualization, sensitive, specific, point-of-care detection of SARS-CoV-2 variants and beyond.

Authentication of the Apis cerana honey with the proofreading enzyme-mediated probe cleavage combined with ladder-shape melting temperature isothermal amplification (Proofman-LMTIA) method

Honey adulteration has been on the rise with the increasing demand for high-quality honey, and the authentication of A. cerana honey is a major problem that the current bee industry needs to solve. The aim of this research was to establish the proofreading enzyme-mediated probe cleavage combined with ladder-shape melting temperature isothermal amplification (Proofman-LMTIA) method for the authentication of Apis cerana honey. The LMTIA primers were designed to target the specific 18S rRNA sequence of A. cerana, and the conditions for Proofman-LMTIA amplification were optimized. The specificity and sensitivity of the method were determined and compared with that of the droplet digital polymerase chain reaction (PCR). The optimal temperature of the established Proofman-LMTIA method was 62 °C. The method can specially to detect the A. cerana gene without cross-reactivity with the genomes from other animal and plant species, and the amplification reaction can be finished within 20 min with the sensitivity of 10 pg A. cerana genomic DNA, which was more sensitive than the droplet digital PCR method. The obtained results demonstrate that the established Proofman-LMTIA procedure was specific, sensitive, and rapid, and can be used for authentication of A. cerana honey.

Development of a Simple Method to Detect the Carbapenemase-Producing Genes blaNDM, blaOXA-48-like, blaIMP, blaKPC, and blaVIM Using a LAMP Method with Lateral Flow DNA Chromatography.

Infections by carbapenemase-producing Enterobacterales constitute a global public health threat. The rapid and efficient diagnosis of Enterobacterales infection is critical for prompt treatment and infection control, especially in hospital settings. We developed a novel loop-mediated isothermal amplification (LAMP) method combined with DNA chromatography to identify five major groups of carbapenemase-producing genes (blaNDM, blaOXA-48-like, blaIMP, blaKPC, and blaVIM). This method uses DNA-DNA hybridization-based detection in which LAMP products can be easily visualized as colored lines. No specific technical expertise, expensive equipment, or special facilities are required for this method, allowing its broad application. Here, 73 bacteria collections including strains with carbapenemase-producing genes were tested. Compared to sequencing results, LAMP DNA chromatography for five carbapenemase-producing genes had a sensitivity and specificity of 100% and >97%, respectively. This newly developed method can be a valuable rapid diagnostic test to guide appropriate treatments and infection control measures, especially in resource-limited settings.

Development of a multiplex Loop-Mediated Isothermal Amplification (LAMP) for the diagnosis of bacterial periprosthetic joint infection.

Periprosthetic joint infection (PJI) is one of the most serious and debilitating complications that can occur after total joint arthroplasty. Therefore, early diagnosis and appropriate treatment are important for a good prognosis. Recently, molecular diagnostic methods have been widely used to detect the causative microorganisms of PJI sensitively and rapidly. The Multiplex Loop-Mediated Isothermal Amplification (LAMP) method eliminates the complex temperature cycling and delays caused by temperature transitions seen in polymerase chain reaction (PCR) methods, making it faster and easier to perform compared to PCR-based assays. Therefore, this study developed a multiplex LAMP assay for diagnosing bacterial PJI using LAMP technology and evaluated its analytical and clinical performance. We developed a multiplex LAMP assay for the detection of five bacteria: Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae, Pseudomonas aeruginosa, and Escherichia coli, frequently observed to be the causative agents of PJI. The method of analytical sensitivity and cross-reactivity were determined by spiking standard strains into the joint synovial fluid. The analytical sensitivity of the multiplex LAMP assay was compared with that of a quantitative real-time PCR (qPCR) assay. Clinical performance was evaluated using 20 joint synovial fluid samples collected from patients suspected of having bacterial PJI. The analytical sensitivity of the gram-positive bacterial multiplex LAMP assay and qPCR were 105/104 CFU/mL, 103/103 CFU/mL, and 105/104 CFU/mL against S. agalactiae, S. epidermidis, and S. aureus, respectively. For P. aeruginosa and E. coli, the analytical sensitivity of the multiplex LAMP and qPCR assays were 105/104 and 106/104 CFU/mL, respectively. The multiplex LAMP assay detects target bacteria without cross-reacting with other bacteria, and exhibited 100% sensitivity and specificity in clinical performance evaluation. This multiplex LAMP assay can rapidly detect five high-prevalence bacterial species causing bacterial PJI, with excellent sensitivity and specificity, in less than 1 h, and it may be useful for the early diagnosis of PJI.