Design and evaluation of loop-mediated isothermal amplification for rapid detection of Enterocytozoon bieneusi
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ABSTRACT:
SUBMITTER: Mahdavi F
PROVIDER: S-EPMC10958217 | biostudies-literature | 2024 Mar
REPOSITORIES: biostudies-literature
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Project description:BackgroundEnterocytozoon hepatopenaei (EHP) is a newly emerged microsporidian parasite that causes retarded shrimp growth in many countries. But there are no effective approaches to control this disease to date. The EHP could be an immune risk factor for increased dissemination of other diseases. Further, EHP infection involves the absence of obvious clinical signs and it is difficult to identify the pathogen through visual examination, increasing the risk of disease dissemination. It is urgent and necessary to develop a specific, rapid and sensitive EHP-infected shrimp diagnostic method to detect this parasite. In the present study, we developed and evaluated a rapid real-time loop-mediated isothermal amplification (real-time LAMP) for detection of EHP.MethodsA rapid and efficient real-time LAMP method for the detection of EHP has been developed. Newly emerged EHP pathogens in China were collected and used as the sample, and three sets of specificity and sensitivity primers were designed. Three other aquatic pathogens were used as templates to test the specificity of the real-time LAMP assay. Also, we compared the real-time LAMP with the conventional LAMP by the serial dilutions of EHP DNA and their amplification curves. Application of real-time LAMP was carried out with clinical samples.ResultsPositive products were amplified only from EHP, but not from other tested species, EHP was detected from the clinical samples, suggesting a high specificity of this method. The final results of this assay were available within less than 45 min, and the initial amplification curve was observed at about 6 min. We found that the amplification with an exponential of sixfold dilutions of EHP DNA demonstrated a specific positive signal by the real-time LAMP, but not for the LAMP amplicons from the visual inspection. The real-time LAMP amplification curves demonstrated a higher slope than the conventional LAMP.DiscussionIn this study, pathogen virulence impacts have been increased in aquaculture and continuous observation was predominantly focused on EHP. The present study confirmed that the real-time LAMP assay is a promising and convenient method for the rapid identification of EHP in less time and cost. Its application greatly aids in the detection, surveillance, and prevention of EHP.
| S-EPMC6284447 | biostudies-literature
Project description:AimThe present study was undertaken to develop a nucleic acid-based diagnostic assay loop-mediated isothermal amplification assay (LAMP) targeting highly conserved genomic regions of Capripoxvirus (CaPVs) and its comparative evaluation with real-time polymerase chain reaction (PCR).Material and methodsLyophilized vaccine strain of sheeppox virus (SPPV) was used for optimization of LAMP assay. The LAMP assay was designed using envelope immunogenic protein (P32) coding gene targeting highly conserved genomic regions of CaPV responsible for causing sheep pox, goat pox, and lumpy skin disease in sheep, goat and cattle respectively. Serial tenfold dilution of SPPV recombinant plasmid DNA was used for a calculating limit of detection. Analytical sensitivity and specificity were performed.ResultsThe test described is quick (30 min), sensitive and specific for detection of CaPVs. The described assay did not show any cross-reactivity to other related viruses that cause apparently similar clinical signs. It was found to be ten times more sensitive than conventional PCR however, 100 times less sensitive than quantitative PCR (qPCR). LAMP assay results were monitored by color change method using picogreen dye and agarose gel electrophoresis.ConclusionLAMP assay can be a very good alternative for CaPV detection to other molecular techniques requiring sophisticated equipments.
| S-EPMC4774739 | biostudies-literature
Project description:Mycoplasma synoviae (MS) remains a serious concern in production of poultry and affects world production of chickens and turkeys. Loop-mediated isothermal amplification (LAMP) of DNA has been recently used for the identification of different economically important avian pathogens. The aim of this study was to develop LAMP for simple and inexpensive detection of MS strains in poultry using specifically designed primers targeting hemagglutin A (vlh) gene. The assay was conducted in a water bath for 1 h at 63 °C. The results were visualized after addition of SYBR Green(®) fluorescent dye. LAMP was specific exclusively for MS without cross-reactivity with other Mycoplasma species. The sensitivity of LAMP was determined as 10(-1) CFU/ml and was 1,000 times higher than MS-specific polymerase chain reaction. LAMP assay was conducted on 18 MS field strains to ensure its reliability and usefulness. This is the first report on LAMP development and application for the rapid detection of MS isolated from chickens. This simple method may be applied by diagnostic laboratories without access to expensive equipment.
| S-EPMC4326666 | biostudies-literature
Project description:The main strategy for response and control of COVID-19 demands the use of rapid, accurate diagnostic tests aimed at the first point of health care. During the emergency, an increase in asymptomatic and symptomatic cases results in a great demand for molecular tests, which is promoting the development and application of rapid diagnostic technologies. In this study, we describe the development and evaluation of RT-LAMP to detect SARS-CoV-2 based on three genes (ORF1ab, M and N genes) in monoplex and triplex format. RT-LAMP assays were compared with the gold standard method RT-qPCR. The triplex format (RdRp, M and N genes) allowed obtaining comparable results with de RT-qPCR (RdRp and E genes), presented a sensitivity of 98.9% and a specificity of 97.9%, opening the opportunity to apply this method to detect SARS-CoV-2 at primary health-care centers.
| S-EPMC8688429 | biostudies-literature
Project description:To improve the diagnosis and treatment of Mycoplasma pneumoniae (Mp) infection and reduce the misuse of antibiotics, we sought to establish a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Mp.Six primers specific for the Mp P1 gene were designed, and the LAMP method was used to rapidly detect Mp. The sensitivity of the LAMP method was determined by serial dilution of the standard Mp strain FH (standard strains of Mycoplasma pneumoniae). Specificity was assessed with 17 common pathogenic microorganisms in the respiratory tract. Patient samples were collected from the Department of Respiratory and Critical Care Medicine at the 307th Hospital of Chinese People's Liberation Army from March 2016 to May 2017, examined prospectively, and compared with diagnosis by quantitative real-time polymerase chain reaction (qRT-PCR).The LAMP assay for Mp detection can be completed within 60 minutes. The minimum detection limit was 39 pg/μL, and no cross-reaction was observed with 17 common respiratory tract pathogens. Of the 125 clinical specimens tested, 43 cases were positive by LAMP assay, and 40 cases were positive by qRT-PCR (P = .162). All 43 samples determined as positive by LAMP test were confirmed to be Mp by Mp P1 protein sequencing.The LAMP assay is suitable for rapid detection of Mp. It has high sensitivity and specificity, and the detection results are not inferior to those of qRT-PCR.
| S-EPMC6023700 | biostudies-literature
Project description:Anaplasma ovis is an intraerythrocytic rickettsial pathogen of small ruminants. Loop-mediated isothermal amplification (LAMP) is a nucleic acid detection method in which the target DNA can be efficiently amplified with high specificity and sensitivity under isothermal conditions. In this study, a LAMP method was developed for the specific detection of A. ovis, using LAMP primers designed on the basis of the major surface protein 4 gene. LAMP was performed at 65 °C for 30 min. Its specificity was confirmed by successful amplification of several A. ovis isolates and through EcoRI restriction analysis of LAMP products. No cross-reactivity with the A. marginale Lushi isolate, Mycoplasma mycoides subsp. capri, Chlamydophila psittaci, Theileria ovis, T. luwenshuni, T. uilenbergi, or the Babesia sp. Xinjiang isolate was observed. Detection using the LAMP method was compared with that using conventional PCR in 227 field samples; LAMP demonstrated a sensitivity of 95.45%. In summary, LAMP is a specific, sensitive, and rapid test for the diagnosis of A. ovis infection, with the potential to be standardized as a detection method for A. ovis in areas of endemicity.
| S-EPMC3122716 | biostudies-literature
Project description:Hepatitis C virus (HCV) is a major public health problem and a leading cause of chronic liver disease. An estimated 180 million people are infected worldwide. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay for rapid detection of HCV genomic RNA and compared the sensitivity of LAMP with nested-PCR. A total of 30 blood samples from HCV-infected patients were analyzed with six primers targeting conserved sequences of the HCV 5'UTR within 70 min, under isothermal conditions at 62 °C. Then, visualized by gel electrophoresis with ethidium bromide staining and detected by the naked-eye after adding SYBR Green I. All samples positive for HCV by nested PCR were confirmed by LAMP method. When visualized by gel electrophoresis and ethidium bromide staining, the HCV LAMP assay products appeared in a ladder pattern, with many bands of different sizes. The HCV LAMP product could also be detected by the naked-eye after adding SYBR Green I to the reaction tube and observing a color change from orange to green in positive samples. The HCV LAMP had the same sensitivity as a nested-PCR assay, the detection limit for the both systems were found to be 10 copies/mL of HCV RNA. The LAMP assay reported here is superior for rapid amplification, simple operation, and easy detection and will be useful for rapid and reliable clinical diagnosis of HCV in areas with limited resources, such as developing countries.
| S-EPMC3550813 | biostudies-literature
Project description:A method of loop-mediated isothermal amplification (LAMP) was employed to develop a rapid and simple detection system for porcine circovirus type 2 (PCV2). The amplification could be finished in 60 min under isothermal condition at 64 degrees C by employing a set of four primers targeting the cap gene of PCV2. The LAMP assay showed higher sensitivity than the conventional PCR, with a detection limit of five copies per tube of purified PCV2 genomic DNA. No cross-reactivity was observed from the samples of other related viruses including porcine circovirus type 1 (PCV1), porcine parvovirus (PPV), porcine pseudorabies virus (PRV) and porcine reproductive and respiratory syndrome virus (PRRSV). The detection rate of PCV2 LAMP for 86 clinical samples was 96.5% and appeared greater than that of the PCR method. The LAMP assay reported can provide a rapid yet simple test of PCV2 suitable for laboratory diagnosis and pen-side detection due to ease of operation and the requirement of only a regular water bath or heat block for the reaction.
| S-EPMC7112855 | biostudies-literature
Project description:A method of loop-mediated isothermal amplification (LAMP) was employed to develop a rapid and simple detection system for porcine parvovirus (PPV) DNA. The amplification could be finished in 45 min under isothermal condition at 62 degrees C by employing a set of four primers targeting VP2 gene of PPV. LAMP assay showed higher sensitivity than PCR, with a detection limit of 5 copies of PPV genomic DNA per reaction. No cross reactivity was observed from the samples of other related viruses including canine parvovirus, parvovirus B19, porcine circovirus type 1, porcine circovirus type 2 and porcine peudorabies virus. The detection rate of PPV LAMP for 125 clinical samples was 97.6% and appeared higher than that of PCR method. The result indicated the potential usefulness of the technique as a simple, rapid procedure for the detection of PPV.
| S-EPMC7112806 | biostudies-literature
Project description:Zika virus (ZIKV) is a mosquito-borne flavivirus, which is a pathogen affecting humans in Africa, Asia, and America. It is necessary to detect ZIKV with a rapid and sensitive molecular method to guide timely treatment. In this study, a loop-mediated isothermal amplification (LAMP) assay was described, which is an attractive option as a fast, sensitive, and specific method for ZIKV detection using the NS5 protein coding region and the envelope protein (EP) coding region as target sequences. Two different techniques, a calcein/Mn2+ complex chromogenic method and real-time turbidity monitoring, were employed. The specificity and sensitivity of the LAMP assay were determined. The assay's detection limit was 0.5?×?10-9 pmol/µl DNA for NS5 protein coding region and 1.12?×?10-11 pmol/µl DNA for E coding region, respectively, which is a 100-fold increase in sensitivity compared with real-time reverse transcription-polymerase chain reaction (RT-PCR) and conventional PCR. All 12 non-ZIKA respiratory pathogens tested were negative for LAMP detection, indicating the high specificity of the primers for ZIKV. In conclusion, a visual detection LAMP assay was developed, which could be a useful tool for primary quarantine purposes and clinical screening, especially in situations where resources are poor and in point-of-care tests.
| S-EPMC7089109 | biostudies-literature