一种快速检测微囊藻毒素mcyG基因的新技术——环介导恒温扩增

African swine fever (ASF) is a contagious viral disease, which can cause up to 100% mortality among domestic pigs. Pig production is growing rapidly in Uganda among East African countries and is not only a source of food but also an important income for many people living in the rural areas. Field diagnosis of ASF depends only on clinical signs and has to be confirmed in the laboratory since the clinical signs are not pathognomonic. Diagnostic techniques for ASF are focused on serological tests for detection of antigen and antibody, genomic DNA detection by polymerase chain reaction (PCR), and on virus isolation and localization in clinical samples. There have been many recent reports of ASF outbreaks in Uganda yet laboratory diagnosis is limited due to the high cost and expertise required. This work reports the evaluation and application of a loop-mediated isothermal amplification (LAMP) test for detecting African swine fever virus (ASFV) DNA based on the topoisomerase II gene. Thirty (30) tissue samples obtained from suspected ASF outbreaks were collected from different regions of Uganda. The tissue samples were found to have lesions consistent with ASF. One hundred and eighty eight (188) additional blood samples were obtained from the abattoir and field surveillance. Six primers targeting the topoisomerase II gene were used. The sensitivity and specificity of LAMP and OIE recommended diagnostic PCR were compared. The LAMP assay is rapid with results obtained within 1 h (45-60 min). The sensitivity of LAMP for the detection of ASFV was 100% (95% CI: 91.78-100) while the specificity was 44% (95% CI: 36.52-51.69). The Kappa statistic for level of agreement between PCR and LAMP test in the detection of ASFV was 23.7% (95% CI: 16.42-30.91). This Kappa value indicated a fair agreement between the two assays. No cross reaction was observed with Porcine circovirus type 2 virus and E. coli isolated from pigs in Uganda. This is the first study evaluating and applying the LAMP assay in the detection of ASF in domestic pigs in Uganda. The LAMP assay was found to be more sensitive than PCR. Due to its simplicity, sensitivity and specificity, the LAMP assay has the potential for use in the diagnosis and routine surveillance of ASF in Uganda.   Key words: African swine fever virus, loop-mediated isothermal amplification (LAMP), polymerase chain reaction (PCR), sensitivity, specificity, topoisomerase II gene.

Haemonchus contortus is one of the most significant strongylid nematodes infecting small ruminants, and it causes great economic losses to the livestock industry worldwide. Accurate diagnosis of H. contortus is crucial to control strategies. Traditional microscopic examinations are the most common methods for the diagnosis of H. contortus , but they are time-consuming and inaccurate. Molecular methods based on PCR are more accurate, but need expensive machines usually only used in the laboratory. Loop-mediated isothermal amplification (LAMP) is a rapid, simple, specific, and sensitive method that has been widely used to detect viruses, bacteria, and parasites. In the present study, a LAMP method targeting ribosomal ITS-2 gene for detection of the H. contortus in goat fecal samples has been established. The established LAMP method was H. contortus specific, and the sensitivity of LAMP was the same as that of the H. contortus species-specific PCR, with the lowest DNA level detected as being 1 pg. Examination of the clinical samples indicated that the positive rate of LAMP was higher than that of PCR, but no statistical difference was observed between LAMP and PCR (χ2 = 17.991, P = 0.053). In conclusion, a LAMP assay with a high specificity and a good sensitivity has been developed to detect H. contortus infection in goats. The established LAMP assay is useful for clinical diagnosis of H. contortus .

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.

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.

Introduction: Community-acquired pneumonia (CAP) caused by M. pneumoniae affect 3%-10% of children. It is not possible to spot M. pneumoniae infection depending entirely on clinical signs and symptoms. Correct identification of M. pneumoniae infections is crucial to initiate proper antibiotic therapy. The role of culture is questionable as this organism is fastidious. Diagnostic accuracy of serology depends on the specimen collection time. IgM can usually be tracked within one week after the onset of clinical illness, followed by IgG two weeks later. Nucleic Acid Amplification Techniques (NAATs) are proved to be the “new gold standard”. Loop-Mediated Isothermal Amplification (LAMP) amplifies DNA of mycoplasma in less than an hour under isothermal conditions with the use of six primers in a single tube. The amplified products can be visualized by naked eye as turbidity or fluorescence. Aim: To assess the diagnostic value of LAMP assay compared to serum Mycoplasma IgM for rapid detection of M. pneumoniae. Materials and methods: A 6-months study was conducted on hospitalized children diagnosed with community acquired pneumonia (CAP) admitted to Cairo University specialized pediatric hospital (CUSPH). M. pneumoniae IgM was done on serum samples by RD-Ratio Diagnostics ELISA kit, Germany. LAMP assay was done on nasopharyngeal swabs using Loopampᵀᴹ RNA/DNA, Eiken Chemical, Japan. Kappa measure of agreement was calculated to assess the concordance between LAMP and Mycoplasma IgM. Results: 90 hospitalized children with CAP were included in the study. Serum IgM was positive in 27(30%) of cases. LAMP assay was positive in 33 cases (36.7%). The sensitivity of LAMP was 85.2%, the specificity was 84.1%, PPV was 69.7%, NPV 93.0% with AUC=0.847. The agreement between serology and LAMP was good K=0.652 with 95% CI (0.487-0.816). Conclusion: LAMP technique is a rapid, sensitive and specific method for diagnosis of M. pneumoniae. Combination of LAMP assay and serology may be optimal for the diagnosis of M. pneumoniae infection.

Rapid diagnosis of lymph node metastasis in lung cancer with loop-mediated isothermal amplification assay using carcinoembryonic antigen–mRNA

Accurately identifying and targeting the human reservoir of malaria parasitemia is critical for malaria control, and requires a reliable and sensitive diagnostic method. Loop-mediated isothermal amplification (LAMP) is increasingly used to diagnose submicroscopic parasitemia. Although most published studies report the sensitivity of LAMP compared with nested polymerase chain reaction (PCR) as ≥ 80%, they have failed to use a consistent, sensitive diagnostic as a comparator. We used cross-sectional samples from children and adults in Tororo, Uganda, a region with high but declining transmission due to indoor residual spraying, to characterize the sensitivity and specificity of pan-Plasmodium LAMP for detecting submicroscopic infections. We compared LAMP results targeting a mitochondrial DNA sequence conserved in all Plasmodium species, performed on DNA extracted from dried blood spots, to those of a gold standard quantitative PCR assay targeting the var gene acidic terminal sequence of Plasmodium falciparum (varATS qPCR), performed on DNA extracted from 200 µL of whole blood. Using LAMP and varATS qPCR increased the detection of parasitemia 2- to 5-fold, compared with microscopy. Among microscopy-negative samples, the sensitivity of LAMP was 81.5% for detecting infection ≥ 1 parasites/µL. However, low density infections were common, and LAMP failed to identify more than half of all infections diagnosed by varATS qPCR, performing with an overall sensitivity of 44.7% for detecting submicroscopic infections ≥ 0.01 parasites/µL. Thus, although the LAMP assay is more sensitive than microscopy, it missed a significant portion of the submicroscopic reservoir. These findings have important implications for malaria control, particularly in settings where low-density infections predominate.

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.

Acanthamoeba, one of the free-living amoeba, has been detected in many environmental samples, mainly in water, soil and air. Acanthamoeba keratitis and granulomatous amoebic encephalitis are among the most important clinical manifestations caused by Acanthamoeba. In this study, it was aimed to determine the sensitivity of the rapid loop mediated isothermal amplification (LAMP) test designed with primers specific to Acanthamoeba 18S rRNA gene to detect more rapidly the presence of Acanthamoeba in clinical and environmental samples. Acanthamoeba strain grown in culture was diluted in 200 μL as 1x106 trophozoites and DNA was isolated, and the amount of DNA was determined by Nano-Drop Spectrophotometer. The purified DNAs were diluted from 1000 pg to 0.001 pg and used in colorimetric and fluorescence-based LAMP reactions. The LAMP reaction mixture was incubated for 60 minutes at 63 °C in a total volume of 25 μL. To determine the sensitivity of the test, positivity of Acanthamoeba genomic DNA was observed at 1, 10, 100 and 1000 pg/reaction in both colorimetric and fluorescence-based LAMP tests. The lowest analytical sensitivity of both calorimetric and fluorescent LAMP assay was determined as 1 pg/reaction. In addition, as a result of LAMP reaction applied with other parasite DNAs to evaluate the specificity of the test, no LAMP product was detected in calorimetric and 1% agarose gel electrophoresis, except for positive control, and the specificity of the test was determined as 100%. It has been demonstrated that the LAMP assay designed by targeting 18S rRNA gene of Acanthamoeba has a detection limit of 1 pg of genomic DNA. It is promising that LAMP test is more sensitive and faster than culture method, as well as simple, inexpensive and highly sensitive. For this reason, it is thought that developed test can be applied in the diagnosis of Acanthamoeba in environmental and clinical samples.

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.

Ortleppascaris sinensis is the dominant nematode species infecting the gastrointestinal tract of the captive Chinese alligator, a critically endangered species. Gastrointestinal nematode infection may cause a loss of appetite, growth, a development disorder, and even mortality in alligators, especially young ones. This research first establishment a loop-mediated isothermal amplification (LAMP) assay in rapidly identifying O. sinensis, upon the basis of the complete internal transcribed spacers (ITS) gene. Eight sets of primers were designed for recognition of the unique conserved ITS gene sequences, and one set was selected to be the most suitable primer for rapid detection. The specific as well as the sensitive features of the most appropriate primer in LAMP reactions for O. sinensis, and feces specimens of Chinese alligators suffering from O. sinensis were determined. Turbidity monitoring and Te Visual Reagent methods were used for determining negative and positive consequences. According to this study, amplification and visualization of the target DNA could be realized through two detection approaches during 50 min at 65 °C isothermal temperature. The sensitivity of LAMP was a detecting limitation of 3.46 pg/µl DNA. No cross-reactions were found between O. sinensis and any other of the nine heterologous nematode parasites, which shows the outstanding specific features of the primers. The LAMP assay could also perform a detection of target DNA of O. sinensis in the feces samples of Chinese alligators. This LAMP assay is useful for directly detecting O. sinensis in the Chinese alligator breeding centers, particularly due to its rapidity, simplicity and low cost.

Development of a real-time loop-mediated isothermal amplification (LAMP) assay and visual LAMP assay for detection of African swine fever virus (ASFV)

The purpose of this study was to establish a loop-mediated isothermal amplification (LAMP) method for the rapid, sensitive detection of Vibrio harveyi in mariculture shellfish. A set of four primers, two outer and two inner primers, were designed from the toxR gene sequence of V. harveyi. The LAMP reaction was conducted at 65 degrees C for 60 min. There were no cross-reactions with other bacterial strains indicating a high specificity of the LAMP. The detection sensitivity of the LAMP assay for V. harveyi with both of pure cultures and added shellfish cultures is about 10(-5) dilution level (equivalent to 17.2 cells per reaction). The amplification products were detected by visual inspection using SYBR Green I. The detection sensitivity using the LAMP method was 10 times higher than that of conventional PCR. The LAMP assay established in this study is an extremely specific, sensitive and rapid for identification of V. harveyi in mariculture shellfish. This LAMP technique provides an important detecting tool for the detection of V. harveyi infection both in the laboratory and field. This technique is recommended as an applied protocol for health management programme and disease surveillance of in hatcheries as well as in grow-out pond, to prevent the disease outbreak.

Rapid and sensitive detection of Banana bunchy top virus by loop-mediated isothermal amplification

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.