Visual Loop-mediated Isothermal Amplification Research Articles

Detection of Vibrio harveyi through RPA combined with CRISPR/Cas12a: Construction of a new method and comparative study

In this study, recombinase polymerase isothermal amplification (RPA) was combined with clustered regularly interspaced short palindromic repeats/associated proteins (CRISPR/Cas) to establish a new method (RPA-CRISPR/Cas12a) for the detection of Vibrio harveyi. We used RPA to amplify the target DNA of V. harveyi and the RPA amplification product as an activator to initiate CRISPR/Cas12a cleavage of the designed ssDNA reporter probe. Then, we used fluorescence intensity as a criterion for the detection results to optimize the reaction conditions and improve the accuracy of the assay. The ToxR-specific gene sequence of V. harveyi was selected, and 3 sets of RPA primers and 2 sets of gRNA were designed for screening to optimize the ratio of LbCas12a protein, gRNA, and reporter. The specificity, sensitivity and detection of the newly developed method on simulated contaminated samples were analyzed and compared with those of the RPA method, fluorescent loop mediated isothermal amplification (LAMP), and visual LAMP methods. The results showed that the newly constructed method exhibited good specificity and no cross-reactivity with other pathogenic bacteria. Maximum fluorescence values were obtained when the molar concentration ratios of LbCas12a protein to reporter and gRNA were 1:0.8 and 1:8, respectively. The sensitivities of RPA-CRISPR/Cas12a for the detection of V. harveyi were 10−5 ng/μL, respectively, and the sensitivities for the simulated contaminated samples were 7 CFU/mL, respectively. The new RPA-CRISPR/Cas12a method provides a new strategy for the immediate detection of foodborne pathogenic microorganisms.

Visual Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid On-Site Detection of Escherichia coli O157: H7 in Milk Products.

(1) Background: Rapid on-site testing is an effective method for the detection of Escherichia 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 of E. coli O157: H7. This study designed new specific LAMP primers for E. coli O157: H7 virulence 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 the Escherichia coli O157: H7 in artificially contaminated milk samples was 5.7 × 102 CFU 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.

A Loop-Mediated Isothermal Amplification Assay for the Rapid Detection of Didymella segeticola Causing Tea Leaf Spot.

Tea leaf spot caused by Didymella segeticola is an important disease that threatens the healthy growth of tea plants (Camellia sinensis) and results in reductions in the productivity and quality of tea leaves. Early diagnosis of the disease is particularly important for managing the infection. Loop-mediated isothermal amplification (LAMP) assay is an efficient diagnostic technique with the advantages of simplicity, specificity, and sensitivity. In this study, we developed a rapid, visual, and high-sensitivity LAMP assay for D. segeticola detection based on sequence-characterized amplified regions. Two pairs of amplification primers (external primers F3 and B3 and internal primers FIP and BIP) were designed based on a specific sequence in D. segeticola (NCBI accession number: OR987684). Compared to common pathogens of other genera in tea plants and other species in the Didymella genus (Didymella coffeae-arabicae, Didymella pomorum, and Didymella sinensis), the LAMP method is specific for detecting the species D. segeticola. The assay was able to detect D. segeticola at a minimal concentration of 1 fg/μL genomic DNA at an optimal reaction temperature of 65 °C for 60 min. When healthy leaves were inoculated with D. segeticola in the laboratory, the LAMP method successfully detected D. segeticola in diseased tea leaves at 72 h post inoculation. The LAMP assays were negative when the DNA samples were extracted from healthy leaves. Leaf tissues with necrotic lesions from 18 germplasms of tea plants tested positive for the pathogen by the LAMP assay. In summary, this study established a specific, sensitive, and simple LAMP method to detect D. segeticola, which provides reliable technical support for estimating disease prevalence and facilitates sustainable management of tea leaf spot.

A Novel Multiplex LAMP Assay for the Detection of Respiratory Human Adenoviruses.

Human adenoviruses (HAdVs) are common pathogens that are associated with a variety of diseases, including respiratory tract infections (RTIs). Without reliable, fast, and cost-effective detection methods for HAdVs, patients may be misdiagnosed and inappropriately treated. To address this problem, we have developed a multiplex loop-mediated isothermal amplification (LAMP) assay for the detection of the species Human adenovirus B (HAdV-B), Human adenovirus C (HAdV-C) and Human adenovirus E (HAdV-E) that cause RTIs. This multiplexing approach is based on the melting curve analysis of the amplicons with a specific melting temperature for each HAdV species. Without the need for typing of HAdVs, the LAMP results can be visually detected using colorimetric analysis. The assay reliably detects at least 375 copies of HAdV-B and -C and 750 copies of HAdV-E DNA per reaction in less than 35 min at 60 °C. The designed primers have no in silico cross-reactivity with other human respiratory pathogens. Validation on 331 nasal swab samples taken from patients with RTIs showed a 90-94% agreement rate with our in-house multiplex quantitative polymerase chain reaction (qPCR) method. Concordance between the quantitative and visual LAMP was 99%. The novel multiplexed LAMP could be an alternative to PCR for diagnostic purposes, saving personnel and equipment time, or could be used for point-of-care testing.

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.

Development of the Visual Loop-Mediated Isothermal Amplification Combined with Lateral Flow Dipstick (LAMP-LFD) Method for Rapid Detection of Vibrio harveyi in Aquatic Animals

Development of the Visual Loop-Mediated Isothermal Amplification Combined with Lateral Flow Dipstick (LAMP-LFD) Method for Rapid Detection of Vibrio harveyi in Aquatic Animals

Development and utilization of a visual loop-mediated isothermal amplification coupled with a lateral flow dipstick (LAMP-LFD) assay for rapid detection of Echinostomatidae metacercaria in edible snail samples

Trematodes belonging to the family Echinostomatidae are food-borne parasites which cause echinostomiasis in animals and humans. This is a global public health issue, particularly in East and Southeast Asia. A method to detect the infective stage of Echinostomatidae species is required to prevent transmission to humans. In this study, a loop-mediated isothermal amplification coupled with a lateral flow dipstick (LAMP-LFD) assay was developed for visual detection of the metacercarial stage in edible snails of the genus Filopaludina from local markets in Thailand. The LAMP-LFD method can be performed within 70 min at a consistent temperature of 66 °C, and the results can be interpreted with the naked eye. The detection limits of the assay using Echinostoma mekongi, E. macrorchis, E. miyagawai and Hypoderaeum conoideum genomic DNA were equal between the four species at 50 pg/μL. A specificity evaluation demonstrated that the LAMP-LFD assay had no cross-reaction with another parasite (Thapariella species) or with the snail host species (Filopaludina martensi martensi, F. sumatrensis speciosa, and F. s. polygramma). Clinical test assessments were compared to microscopic examination in 110 edible snail samples. The clinical sensitivity and specificity of the tests were 84.62 % and 100 %, respectively, with a strong level of agreement based on the kappa statistic and the results of both methods were not significantly different (p > 0.05) per McNemar's test. The test successfully developed in this study may be useful for the detection of the metacercarial stage in edible snails for epidemiological investigations, control, surveillance, and to prevent future echinostomiasis health issues.

Development and evaluation of a rapid visual loop-mediated isothermal amplification assay for the tcdA gene in Clostridioides difficile detection.

The tcdA gene codes for an important toxin produced by Clostridioides difficile (C. difficile), but there is currently no simple and cost-effective method of detecting it. This article establishes and validates a rapid and visual loop-mediated isothermal amplification (LAMP) assay for the detection of the tcdA gene. Three sets of primers were designed and optimized to amplify the tcdA gene in C. difficile using a LAMP assay. To evaluate the specificity of the LAMP assay, C. difficile VPI10463 was used as a positive control, while 26 pathogenic bacterial strains lacking the tcdA gene and distilled water were utilized as negative controls. For sensitivity analysis, the LAMP assay was compared to PCR using ten-fold serial dilutions of DNA from C. difficile VPI10463, ranging from 207 ng/µl to 0.000207 pg/µl. The tcdA gene of C.difficile was detected in 164 stool specimens using both LAMP and polymerase chain reaction (PCR). Positive and negative results were distinguished using real-time monitoring of turbidity and chromogenic reaction. At a temperature of 66 °C, the target DNA was successfully amplified with a set of primers designated, and visualized within 60 min. Under the same conditions, the target DNA was not amplified with the tcdA12 primers for 26 pathogenic bacterial strains that do not carry the tcdA gene. The detection limit of LAMP was 20.700 pg/µl, which was 10 times more sensitive than that of conventional PCR. The detection rate of tcdA in 164 stool specimens using the LAMP method was 17% (28/164), significantly higher than the 10% (16/164) detection rate of the PCR method (X2 = 47, p < 0.01). LAMP method is an effective technique for the rapid and visual detection of the tcdA gene of C. difficile, and shows potential advantages over PCR in terms of speed, simplicity, and sensitivity. The tcdA-LAMP assay is particularly suitable for medical diagnostic environments with limited resources and is a promising diagnostic strategy for the screening and detection of C. difficile infection in populations at high risk.

Rapid and visual detection of Toxoplasma gondii oocyst in cat feces using loop-mediated isothermal amplification (LAMP) assay

Toxoplasma gondii is an obligate parasitic protozoon that transmits to animals and humans via ingested food. Cats that act as T. gondii’s final hosts play a critical role in T. gondii transmission by shedding millions of oocysts. Timely diagnosis of infected cats is essential for preventing toxoplasmosis because oocysts are a putative T. gondii source in epidemiology. We developed a new visual LAMP assay targeting the B1 gene to analyze single oocysts in cat feces in this study. The amplification result could be visually estimated based on the color change. LAMP assay analytical sensitivity was 101 copies/µL for the B1 gene plasmid, which was tenfold better than the PCR reaction. There were no cross-reactions with other parasites. The LAMP assay can detect a single T. gondii oocyst in 200 mg of cat feces. The LAMP assay detected a single oocyst in 200 mg cat feces at a higher rate than the PCR assay (83.3% vs. 50.0%).

Rapid Visual LAMP Method for Detection of Genetically Modified Organisms.

We developed a novel loop-mediated isothermal amplification (LAMP) method using DNA captured on polyacrylamide microparticles (PAMMPs) as templates (PAMMPs@DNA-LAMP) for rapid qualitative detection of genetically modified organisms (GMOs). Here, DNA was extracted by a fast and cost-effective method using PAMMPs. Four LAMP primers were designed for the PAMMPs@DNA-LAMP method to detect the cauliflower mosaic virus 35S (CaMV35S) promotor in GMOs. We thus developed this method for rapid extraction of DNA (5-10 min) and fast amplification of DNA within ∼30 min at a constant temperature of 63 °C. Moreover, the DNA captured by PAMMPs (PAMMPs@DNA) could be effectively detected by both conventional and quantitative PCR (qPCR) and LAMP. The PAMMPs@DNA-LAMP method was validated with high specificity, sensitivity, and performance for practical sample analysis. This assay detected 0.01% target sequences, which had a high specificity like qPCR and better than the conventional PCR (cPCR). Furthermore, PAMMPs@DNA-LAMP was successfully used to extract and detect DNA from food samples of the major crops (soybean, maize, rice, etc.). In summary, a novel PAMMPs@DNA-LAMP assay has been developed, which has higher sensitivity and spends less time than the cPCR detection using the conventional DNA extracted process. This method offers a novel approach for rapid detection of GMOs in the field.

Phenol red as electrochemical indicator for highly sensitive quantification of SARS-CoV-2 by loop-mediated isothermal amplification detection

The current COVID-19 pandemic has made patent the need for rapid and cost-effective diagnostic tests, crucial for future infectious outbreaks. Loop-mediated isothermal amplification (LAMP) is a promising and decentralized alternative to qPCR. In this work we have developed a sensitive, fast, and simple innovative methodology for quantification of SARS-CoV-2 RNA copies, combining reverse-transcription LAMP with electrochemical detection. This is based on the oxidation of phenol red (PR), a visual and electroactive LAMP indicator, which oxidation peak potential (Ep) changes with the progress of the LAMP reaction. Using that Ep shift as analytical signal, a calibration curve was obtained for fragment N1 copies of SARS-CoV2 (which provided better results than N or S fragments), with a potential shift of 16.2 mV per order of magnitude, and a practical limit of detection of 21 copies·μL−1. Moreover, the precision of Ep is excellent (RSD < 2%): 557 ± 5 mV for negative and 602 ± 7 mV for positive (2148 N fragment RNA copies·µL-1·-1) LAMP controls. This methodology has been applied to the analysis of nasopharyngeal swab samples, resulting in total concordance with clinical RT-qPCR results. Advances towards fully decentralization have been achieved by designing and fabricating a small portable heater for isothermal procedures, obtaining comparable results to those from a commercial benchtop thermal cycler.

RNase H2 triggered visual loop-mediated isothermal amplification combining smartphone assisted all-in-one aptamer magnetic enrichment device for ultrasensitive culture-independent detection of Salmonella Typhimurium in chicken meat

Pre-enrichment cultural continues to remain time-consuming and labor-intensive in the detection of low-concentration bacteria in meat samples. Culture-independent detection method is urgently needed in POCT technology. In order to be able to proceed with culture-independent pre-enrichment for low-concentration pathogen detection, a RNase H2 enzyme triggered all-in-one device-based visual Loop-mediated Isothermal Amplification detection method was developed, combining with Salmonella Typhimurium specific Aptamer Magnetic Enrichment steps, termed as the AME-LAMP method. In order to better enable the AME-LAMP method to perform on-site culture-independent detection, a palm-held all-in-one device (PAD) was designed and manufactured. Further, a self-developed smart phone fluorescence analysis app could complete end-point detection, thus eliminating miscalculation. RNase H2 Enzyme triggered AME-LAMP method could detect Salmonella as low as 5.5 CFU/mL while conventional LAMP only detected 550 CFU/mL. By verifying the detection performance of AME-LAMP methods in chicken meat samples, AME-LAMP shows the potential to achieve ultrasensitive Salmonella detection by comparing with ISO isolation method and normal LAMP. With the assistance of smart phone-based PAD, it can be reliably applied for the on-site culture-independent detection of Salmonella Typhimurium.

Integrating microneedle DNA extraction to hand-held microfluidic colorimetric LAMP chip system for meat adulteration detection

Most microfluidic-based “sample-in-result-out” systems suffer sophisticated microfluidic production processes, high-cost chips, and expensive instruments. They cannot be used in the meat market as well as farmer's markets in rural areas. Here, we developed a hand-held microfluidic chip system for on-site meat species qualitative authentication detection which integrated a simple microneedle DNA extraction and a visual loop-mediated isothermal amplification (LAMP). The chip can be used by easily pricking meat samples, simply hand-shaking the chip, and readily available isothermal heating instead of a complicated DNA extraction process and microfluidic control device. The system demonstrates high specificity and sensitivity for selected six species of meat samples and low to 1% simulated adulteration could be detected within 60 min. Besides, the whole cost was less than 1 dollar. The integrated hand-held microfluidic detection system offers a simple, fast, low-cost “sample-in-result-out” point-of-care device which could be extended to medical diagnosis and animal/plant disease identification.

Development of a visual multiplex fluorescent LAMP assay for the detection of foot-and-mouth disease, vesicular stomatitis and bluetongue viruses.

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technique that can be used to amplify target genes at a constant temperature, and it has several advantages, including convenience, specificity and sensitivity. However, due to the special interpretation methods of this technology for reaction results, all the previously reported LAMP detection methods have been restricted to identifying a single target, which limits the application of this technology. In this study, we modified conventional LAMP to include a quencher-fluorophore composite probe complementary to the F1c segment of the inner primer FIP; upon strand separation, a gain in the visible fluorescent signal was observed. The probes could be labeled with different fluorophores, showing different colors at the corresponding wavelengths. Therefore, this multiplex LAMP (mLAMP) assay can simultaneously detect 1-3 target sequences in a single LAMP reaction tube, and the results are more accurate and intuitive. In this study, we comprehensively demonstrated a single-reaction mLAMP assay for the robust detection of three cattle viruses without nonspecific amplification of other related pathogenic cattle viruses. The detection limit of this mLAMP assay was as low as 526-2477 copies/reaction for the recombinant plasmids. It is expected that this mLAMP assay can be widely used in clinical diagnosis.

Visual LAMP method for the detection of Vibrio vulnificus in aquatic products and environmental water

BackgroundA visual, rapid, simple method was developed based on a loop-mediated isothermal amplification (LAMP) assay to detect Vibrio vulnificus in aquatic products and aquaculture waters.ResultsGenomic DNA was extracted from Vibrio vulnificus using the boiling method, and optimized primers were used to detect the gyrB gene using a visual LAMP method. The sensitivity of the assay was 10 fg/μL, and the obtained results were stable and reliable. Out of 655 aquatic product samples and 558 aquaculture water samples, the positive rates of Vibrio vulnificus detection were 9.01% and 8.60%, respectively, which are markedly higher than those of the traditional culture identification methods.ConclusionThe relatively simple technical requirements, low equipment cost, and rapid detection make the visual LAMP method for the detection of Vibrio vulnificus a convenient choice for field detection in the aquaculture industry.

Rapid and visual detection of soybean mosaic virus SC7 with a loop-mediated isothermal amplification strategy

Soybean mosaic virus (SMV) is the most prevalent viral pathogen of soybean worldwide that reduces seed quality and causes serious losses in yields. The accurate, rapid and early detection of SMV strains is crucial for screening SMV-resistant soybean varieties and controlling soybean mosaic disease timely. In this study, we report a loop-mediated isothermal amplification (LAMP) assay for visual and rapid detection of SMV-SC7, the major prevalent SMV strain in China, in soybean leaves. Specific primers were rationally designed and tested to detect the SMV-SC7 strain. The detection limit for SMV-SC7 can be as low as 10−4 ng/μL by the developed LAMP assay. These results were visualized by three indicators (SYBR green I, neutral red and hydroxy naphthol blue) with high fidelity. The high analytical performance of the LAMP assay was demonstrated with 21 soybean cultivars, among which 8 soybean cultivars were further inoculated with consistent results to those achieved by LAMP. This visual and rapid LAMP assay is highly promising for early detection of SMV-SC7-associated plant diseases.

A Naked-Eye Visual Reverse Transcription Loop-Mediated Isothermal Amplification with Sharp Color Changes for Potential Pen-Side Test of Foot-and-Mouth Disease Virus.

Visual loop-mediated isothermal amplification (LAMP) is qualified to be applied in the field to detect pathogens due to its simplicity, rapidity and cost saving. However, the color changes in currently reported visual reverse transcription LAMP (RT-LAMP) for foot-and-mouth disease virus (FMDV) detection are not so obvious to the naked eye, so interpretation of results is troublesome. In this study, a new naked-eye visual RT-LAMP to detect all seven distinct serotypes of FMDV was established based on the 3D genes by using pH-sensitive neutral red as the indicator, rendering a sharp contrast of color changes between the negative (light orange) and the positive (pink). Analytical sensitivity tests showed that the detection limit of the visual RT-LAMP was 104 copies/µL while those were 103 and 104 copies/µL for the RT-qPCR and conventional RT-PCR methods, respectively. Specificity tests proved that the established visual RT-LAMP assay had no cross-reactivity with other common livestock viruses. Furthermore, the analysis of 59 clinical samples showed 98.31% and 100% concordance with the RT-qPCR and the RT-PCR, respectively. The pan-serotypic FMD visual RT-LAMP assay could be suitable for a pen-side test of all seven serotypes of FMDV because the results could be easily distinguished by the naked eye without the requirement of complicated instruments and professional technicians. Hence, the novel method may have a promising prospect in field tests which exert an important role in monitoring, preventing, and controlling FMD, especially in regions with no PCR or qPCR instrument available.

A smartphone-assisted high-throughput integrated color-sensing platform for the rapid detection of Campylobacter coli

Campylobacter is one of the most common foodborne pathogens worldwide. A new smartphone-assisted high-throughput integrated color-sensing platform, called the HICS platform, was developed for the rapid detection of Campylobacter coli. This platform was based on the visual loop-mediated isothermal amplification method. Using this system, as many as 64 samples could be assessed in less than an hour after enrichment. 60 meat samples were tested to compare the performance of the HICS platform and other methods. Having the consistent detection result with TaqMan qPCR (Quantitative Real-time Polymerase Chain Reaction), the HICS platform was able to reliably detect C. coli in meat samples, and its limit of detection is 550 CFU/mL and 120 copies/μL, which was 10-fold higher than that of the PCR (Polymerase Chain Reaction) method. To conclude, considering that our platform showed robust performance and does not require any expensive equipment, it can also be reliably applied for the high-throughput detection of other pathogens.

Clinical Validation of DNA Extraction-Free qPCR, Visual LAMP, and Fluorescent LAMP Assays for the Rapid Detection of African Swine Fever Virus.

The global pig industry and food safety are seriously threatened by outbreaks of African swine fever (ASF). To permit early diagnosis of African swine fever virus (ASFV), prevent its spread, and limit its outbreaks, a highly sensitive diagnostic method that can be performed at pig farms is required. Herein, we established DNA extraction-free real-time PCR (qPCR), visual loop-mediated isothermal amplification (LAMP), and fluorescent LAMP assays, which were compared with the results of World Organization for Animal Health (OIE) qPCR to assess ASFV-infected clinical samples. Based on plasmid DNA, the limit of detection for the three assays and OIE qPCR were 5.8 copies/μL. All four assays had good ASFV specificity and showed no cross-reactivity with other tested viruses. These assays were used to diagnose 100 clinical samples. The assays showed good diagnostic consistency, with kappa values of 1.0, 0.84, and 0.88, respectively. Compared with OIE qPCR, the diagnostic specificity/sensitivity of DNA extraction-free qPCR, visual LAMP, and fluorescent LAMP assays were 100%/100%, 100%/87.1%, and 100%/90.32%, respectively. The assays eliminated the need for DNA extraction and are more suitable for ASF diagnosis by inexperienced farmers in low-resource environments, making them a good choice for on-site monitoring of pig farms.

Development and clinical application of a rapid and visual loop-mediated isothermal amplification test for tetM gene in Clostridioides difficile strains cultured from feces

ObjectivesIn this study, we aimed to develop a rapid and visual loop-mediated isothermal amplification (LAMP) assay targeting the tetM gene in Clostridioides difficile strains cultured from feces. MethodsPrimers were designed to recognize the tetM gene in C. difficile by LAMP, using turbidity and visual detection. The sensitivity and specificity of LAMP primers were determined. In addition, we conducted both LAMP and polymerase chain reaction (PCR) for the tcdA, tcdB, cdtA, cdtB, ermB, and tetM genes in 300 toxigenic C. difficile strains cultured from feces. ResultsThe target DNA was amplified and visualized within 60 minutes at a temperature of 62°C. A total of 26 bacterial strains were found negative for tetM, which manifested high specificity of the primers. The detection limit of LAMP was 36.1 pg/µl, which was 100-fold more sensitive than PCR. The positive rate of tetM in toxigenic C. difficile strains cultured from feces was 93.3% by both LAMP and PCR. The proportion of toxin types in those C. difficile strains was 95.7% for A+B+CDT−, 4% for A−B+CDT−, and 0.3% for A+B+CDT+, respectively. ConclusionThis is the first study examining the tetM gene by LAMP in C. difficile strains cultured from feces. Its high specificity, sensitivity, and visual detection make the new assay a powerful diagnostic tool for rapid testing.