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Background: Trypanosoma evansi, the causative agent of surra, poses a major veterinary concern in tropical regions, particularly affecting cattle and buffalo. The disease leads to reproductive failures, including abortion, and significant economic losses. Early and accurate diagnosis is crucial for effective control, especially in endemic, resource-limited areas. Objectives: This study aimed to develop a loop-mediated isothermal amplification (LAMP) assay for rapid detection of T. evansi and evaluate its diagnostic performance in comparison with conventional polymerase chain reaction (PCR) and the CATT/T. evansi card agglutination test. Materials and methods: Four LAMP primers were designed to target the RoTat 1.2 variant surface glycoprotein (VSG) gene of T. evansi. Optimal reaction parameters, including temperature and incubation time, were established. The LAMP assay, conventional PCR, and the CATT/T. evansi card agglutination test were performed on 79 blood samples collected from cattle with suspected trypanosomiasis in northern Thailand (Lamphun and Chiang Mai). Diagnostic sensitivity, specificity, and agreement between tests were statistically analyzed. Results: The LAMP assay detected T. evansi in 32 (40.5%, 95% CI: 29.8-51.9%) samples, slightly outperforming PCR, which detected 30 (37.9%, 95% CI: 27.6-49.0%). However, this difference was not statistically significant (McNemar’s test, p=0.724). The CATT/T. evansi test yielded 45 (56.9%) positives but lacked the ability to differentiate active infection from prior exposure. Conclusion: The LAMP assay demonstrated high sensitivity, specificity, and rapid detection capabilities under simplified conditions, making it highly suitable for field applications. When paired with colorimetric or lateral flow readouts, LAMP offers a promising point-of-care diagnostic tool for improving trypanosomiasis control in endemic regions.

DNA detection of Paragonimus westermani: Diagnostic validity of a new assay based on loop-mediated isothermal amplification (LAMP) combined with a lateral flow dipstick

BackgroundLeptospirosis is a zoonotic disease caused by bacteria of the genus Leptospira that affects both humans and animals worldwide. Early detection of the pathogen in humans is crucial for early intervention and control of the progression of the disease to a severe state. It is also vitally important to be able to detect the presence of the pathogen in carrier animals to control the spread of the disease from the environment. Here we developed a simple and rapid loop-mediated isothermal amplification (LAMP) assay targeting the leptospiral secY gene.ResultsSeveral reaction conditions of the LAMP reaction were optimized to ensure efficient amplification of the target DNA. The sensitivity of the developed LAMP assay obtained using a pure Leptospira culture was 2 × 104 copies of genomic DNA per reaction (equivalent to 0.1 ng) for a 40-minute reaction time. No cross-reactions were observed in the LAMP reaction against a series of non-leptospiral bacteria, indicating a specific reaction. The applicability of the LAMP assay was demonstrated on human blood and urine specimens collected from suspected leptospirosis patients and rat kidney specimens collected from suspected leptospirosis outbreak areas and high-risk areas. The developed LAMP assay demonstrated a higher detection rate for leptospiral DNA compared with the polymerase chain reaction (PCR) assay, possibly due to the presence of inhibitory substances, especially in rat kidney specimens, to which the PCR method is more susceptible. The present findings also highlight the importance of urine sample collection from patients for routine monitoring of the disease.ConclusionsIn short, the developed LAMP assay can serve as a feasible alternative tool for the diagnosis of leptospirosis and be used for epidemiological and environmental surveillance of the disease, considering its robustness, rapidity, sensitivity, and specificity, as demonstrated in this study.

Staphylococcus pseudintermedius is a global animal pathogen. Traditional identification methods are time-consuming necessitating a more efficient approach. This study validated and enhanced the loop-mediated isothermal amplification (LAMP) technique by integration it with a lateral flow dipstick (LFD) assay for the detection of S. pseudintermedius and methicillin-resistant S. pseudintermedius (MRSP) strains. Conventional identification methods were compared with LAMP and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The isolates were tested for MRSP detection using oxacillin and cefoxitin disk diffusion tests alongside the LAMP assay targeting the mecA gene, a marker for methicillin resistance. Results showed that LAMP combined with LFD effectively detected S. pseudintermedius and MRSP. This study identified 53 isolates as S. pseudintermedius by conventional and LAMP methods, with MALDI-TOF MS correctly identifying 39.62% (21/53). The mecA gene, crucial for methicillin resistance, was detected in all PCR-positive isolates (n = 33) by LAMP, while the disk diffusion method identified 69.70% (23/33) of mecA-positive strains. LAMP and conventional methods exhibited superior accuracy, sensitivity, and specificity (all 100%) compared to MALDI-TOF MS, which showed lower sensitivity (39.62%) for S. pseudintermedius identification. Similarly, the LAMP assay demonstrated higher accuracy, sensitivity, and specificity (all 100%) for MRSP detection compared to the disk diffusion method (83.33%, 69.70%, and 94.87%, respectively). The LAMP assay coupled with the LFD method proved suitable for routine bacterial identification in laboratories, offering adequate sensitivity and specificity with simple steps and short reaction time.

Enteric fever is one of the major causes of death and morbidity around the world, especially in resource limited health care facility. The primary reason for empirical therapy in enteric fever management is a lack of rapid diagnostic testing. To improve typhoid fever diagnosis and treatment, as well as to reduce antibiotic overuse, we attempted to develop a loop-mediated isothermal amplification (LAMP) for rapid detection of enteric fever. We designed and evaluated a LAMP assay that targets the STY2879 gene, which is found only in S. Typhi and encodes for reverse transcriptase protein. LAMP utilises three sets of primers to complete the reaction in 60 minutes at 65°C. The LAMP assay procedure in our study for detecting S. Typhi by targeting the STY2879 gene was rapid and more sensitive than the Polymerase Chain Reaction (PCR) method. Among the 107 blood samples that have been tested, the sensitivity and specificity of the assay we obtained were 100% and 87.65% respectively. Also, we demonstrated lower limit of detection (LOD) of target DNA concentration ranging from 10 pg to 5 ng that could be readily detected by a LAMP assay within 60 min. The findings of our study suggest that the LAMP assay is superior to PCR and can be used as a rapid alternative diagnostic tool for the diagnosis of enteric fever in the aspects of specificity and sensitivity. As a result, with some additional enhancements and modifications, this reliable and cost-effective assay can be promptly used to enhance disease management and surveillance.

Mycoplasma gallisepticum infections impose a significant economic burden on the poultry industry. In the current study, a loop-mediated isothermal amplification (LAMP) assay was developed and optimized to detect M. gallisepticum based on a gene within the pyruvate dehydrogenase complex, the pdhA gene, which codes for the major subunit (E1α) in the complex. The reaction conditions were optimized, and the specificity was confirmed by successful amplification of several M. gallisepticum strains, while no amplification was detected with 20 other major bacterial and viral pathogens of poultry. Additionally, the LAMP assay achieved 10-fold higher sensitivity than an existing polymerase chain reaction (PCR) method. The LAMP assay was applied to swab samples collected from poultry farms and compared with PCR. The positive detection rate was 20.2% (37/183) by LAMP and 13.1% (24/183) by PCR. The LAMP assay could provide a cost-effective, quick, and sensitive method for the detection of M. gallisepticum.

The aim of the study was to compare Polymerase Chain Reaction (PCR) and Loop Mediated Isothermal Amplification (LAMP) assay for detection of Salmonella in Chicken Meat. Food safety remains a major concern worldwide, with outbreaks of foodborne illness resulting in significant costs to individuals and to the food industry. The Salmonella genus is the most prevalent group of foodborne pathogens that are regularly isolated from food-producing animals and thus are one of the leading causes of food borne diseases. Out of total 40 Salmonella isolates, LAMP and PCR assay detected 36 (90%) and 32 (80%) isolates, respectively. The sensitivity of the LAMP assay was noted to be 10-fold higher than that of PCR whereas the specificity of both LAMP and PCR was same.

Trichomonas tenax is a flagellated protozoan that inhabits the human and canine oral cavity in patients with poor oral hygiene and periodontal disease. The loop-mediated isothermal amplification (LAMP) assay could provide clinicians with a quick, cheap and reliable diagnostic test used for the detection of T. tenax in various settings. In this study, we aimed to develop a LAMP assay that can detect T. tenax with high sensitivity and specificity. A set of LAMP primers were specifically designed to detect the ITS and 5.8S rRNA gene of T. tenax. The newly developed LAMP assay was 1000 times more sensitive than conventional PCR. The limit of detection of the LAMP assay was 10 fg of genomic DNA, or 0.2–1 cell. Moreover, the LAMP assay was specific, resulting in no cross-reaction even with a closely related protozoan T. vaginalis or other microorganisms (Streptococcus pyogenes, Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, and Candida albicans) used. The present LAMP assay can be performed directly without prior DNA extraction, making the assay an easy, fast, cheap, specific and sensitive diagnostic tool for the detection of T. tenax at the point-of-care of both medical and veterinary clinics in developed and developing countries.

Loop mediated isothermal amplification (LAMP) assays as a rapid diagnostic for COVID-19

Meloidogyne partityla is the dominant root-knot nematode (RKN) species parasitizing pecan in Georgia. This species is known to cause a reduction in root growth and a decline in the yields of mature pecan trees. Rapid and accurate diagnosis of this RKN is required to control this nematode disease and reduce losses in pecan production. In this study, a loop-mediated isothermal amplification (LAMP) method was developed for simple, rapid, and on-site detection of M. partityla in infested plant roots and validated to detect the nematode in laboratory and field conditions. Specific primers were designed based on the sequence distinction of the internal transcribed spacer (ITS)-18S/5.8S ribosomal RNA gene between M. partityla and other Meloidogyne spp. The LAMP detection technique could detect the presence of M. partityla genomic DNA at a concentration as low as 1 pg, and no cross reactivity was found with DNA from other major RKN species such as M. javanica, M. incognita and M. arenaria, and M. hapla. We also conducted a traditional morphology-based diagnostic assay and conventional polymerase chain reaction (PCR) assay to determine which of these techniques was less time consuming, more sensitive, and convenient to use in the field. The LAMP assay provided more rapid results, amplifying the target nematode species in less than 60 min at 70°C, with results 100 times more sensitive than conventional PCR (~2–3 hrs). Morphology-based, traditional diagnosis was highly time-consuming (2 days) and more laborious than conventional PCR and LAMP assays. These features greatly simplified the operating procedure and made the assay a powerful tool for rapid, on-site detection of pecan RKN, M. partityla. The developed LAMP assay will facilitate accurate pecan nematode diagnosis in the field and contribute to the management of the pathogen.

Background/aim Tuberculosis is still one of the most contagious diseases around the world. Key factors of tuberculosis control are rapid diagnostic, efficient treatment, and prevention of contamination by surveillance and monitoring. However, culture is the gold standard method for laboratory diagnosis of tuberculosis; the results are several weeks to obtain. In order to prevent contamination of tuberculosis, diagnosis must be made in short time and treatment should be started as soon as possible. The aim of this study is to optimize the loop-mediated isothermal amplification (LAMP) method, which provides a much faster and more sensitive result than the polymerase chain reaction (PCR) method and allows the replication of target nucleic acid sequences under isothermal conditions without the need for laboratory infrastructure.Materials and methods Sputum samples were homogenized with 5% trypsin solution in CaCl2 to obtain DNA. DNA was purified using QIAGEN QIAamp DNA mini kit. LAMP primers were design using Primer explorer V5 program according to IS6110 gene of Mycobacterium tuberculosis. NEB Bst 3.0 DNA polymerase kit was used for LAMP reactions. Besides, LAMP reactions were compared with TaqMan based RT-PCR method using NEB’s Taq polymerase kit. Finally, for visualization of LAMP products, lateral flow dipsticks that produced by Milenia Biotec, colorimetric 2X LAMP master mix that produced by NEB and 2% w/v agarose gel electrophoresis methods were used.ResultsOptimum amplification temperature for LAMP was found to be 71.4 °C. The detection limit of the method was 102 CFU/mL and sensitivity was determined 100% compared to five different Mycobacterium species. Conclusion The current study indicated that the LAMP-LFD and colorimetric LAMP protocol optimized with sputum samples can be reliable used as a rapid, sensitive and specific assay in the diagnosis of tuberculosis in the field.

Canine parvoviral enteritis remains one of the dreaded enteric viral diseases of dogs. Rapid detection of the etiological agent, Canine parvovirus-2 (CPV-2) is required for managemental intervention and initiating therapeutic and control measures. The present study describes Loop Mediated Isothermal Amplification (LAMP) using loop primers, validating the assay on all the circulating CPV-2 antigenic types and testing in 120 suspected clinical samples. The novel LAMP assay was completed within 30-45 min at 64°C in a water bath. Analytical sensitivity of the LAMP assay was 100 times more than conventional PCR and was comparable to real-time PCR. LAMP was able to detect 62 positive samples while conventional PCR could detect only in 51 samples. The developed LAMP provides a simple, rapid, cost-effective, sensitive, specific method of detection of CPV-2 infection in dogs in clinical settings.

Salmonella species are among the major pathogens that cause foodborne illness outbreaks. In this study, we aimed to develop a loop-mediated isothermal amplification (LAMP) assay for the rapid and sensitive detection of Salmonella species. We designed LAMP primers targeting the hilA gene as a universal marker of Salmonella species. A total of seven Salmonella species strains and 11 non-Salmonella pathogen strains from eight different genera were used in this study. All Salmonella strains showed positive amplification signals with the Salmonella LAMP assay; however, there was no non-specific amplification signal for the non-Salmonella strains. The detection limit was 100 femtograms (20 copies per reaction), which was ~1,000 times more sensitive than the detection limits of the conventional polymerase chain reaction (PCR) assay (100 pg). The reaction time for a positive amplification signal was less than 20 minutes, which was less than one-third the time taken while using conventional PCR. In conclusion, our Salmonella LAMP assay accurately detected Salmonella species with a higher degree of sensitivity and greater rapidity than the conventional PCR assay, and it may be suitable for point-of-care testing in the field.

A rapid, sensitive and visual loop‐mediated isothermal amplification (LAMP) method for detecting Acidovorax citrulli in cucurbit seed was developed in this study. The LAMP primers were designed to recognize the non‐ribosomal peptide synthetase (NRPS) gene (locus tag: Aave_4658) from A. citrulli. The LAMP assay was conducted at 64°C in 1 hr with calcein as an indicator. The sensitivity and specificity of the LAMP assay were further compared with those of a conventional polymerase chain reaction (PCR). The LAMP assay is highly specific to A. citrulli, and no cross‐reaction was observed with other bacterial pathogen. The sensitivity of the LAMP assay was 100‐fold higher than that of conventional PCR with a detection limit of 1 pg of genomic DNA. Using the LAMP assay, 7 of 12 cantaloupe seedlots collected from Xinjiang province were determined to be positive for A. citrulli. In contrast, only 2 of 12 seedlots showed positive for the pathogen with conventional PCR. Moreover, A. citrulli was detected in 100% of artificially infested seedlots with 0.01% infestation or greater. Our results demonstrated that the LAMP assay was simple, visual and sensitive for detecting A. citrulli, especially in seed health testing. Hence, this method has great potential application in routine detecting seed‐borne pathogens and reducing the risk of epidemics.

Infection of breeder flocks in China with subgroup J avian leukosis virus (ALV-J) has increased recently. In this study, we have developed a loop-mediated isothermal amplification (LAMP) assay for rapid detection of ALV-J from culture isolates and clinical samples. The ALV-J-specific LAMP assay efficiently amplified the target gene within 45 min at 63 degrees C using only a simple laboratory water bath. To determine the specificity of the LAMP assay, various subgroup ALVs and other related viruses were detected. A ladder pattern on gel electrophoresis was observed for ALV-J isolates but not for other viruses. To evaluate the sensitivities of the LAMP assay and conventional PCR, the NX0101 isolate plasmid DNA was amplified by them. The detection limit of the LAMP assay was 5 target gene copies/reaction, which was up to 20 times higher than that of conventional PCR. To evaluate the application of the LAMP assay for detection of ALV-J in clinical samples, 49 samples suspected of ALV infection from breeder flocks were tested by the LAMP assay and PCR. Moreover, virus isolation from these samples was also performed using cell culture. The positive-sample ratios were 21/49 (43%) by conventional PCR, 26/49 (53%) by the LAMP assay, and 19/46 (41%) by virus isolation. Additionally, a positive LAMP reaction can be visually ascertained by the observation of turbidity or a color change after addition of SYBR green I dye. Consequently, the LAMP assay is a simple, rapid, and sensitive diagnostic method and can potentially be developed for rapid detection of ALV-J infection in the field.