scholarly journals Rapid And Sensitive Detection Of Lawsonia Intracellularis In Pigs By Real-Time Loop-Mediated Isothermal Amplification

2015 ◽  
Vol 65 (1) ◽  
pp. 20-29 ◽  
Author(s):  
PARK Byung-Yong ◽  
SHIM Kwan-Seob ◽  
KIM Won-Il ◽  
HOSSAIN Md Mukter ◽  
KIM Bumseok ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Sensitive Detection ◽  
Detection Methods ◽  
Conventional Pcr ◽  
Fecal Samples ◽  
Loop Mediated Isothermal Amplification ◽  
Time Loop

Abstract A simple and rapid real-time loop-mediated isothermal amplification (LAMP) assay designed to detect Lawsonia (L.) intracellularis, an important bacteria causing proliferative enteropathy in pigs. A set of four primers targeting the ubiquinone/menaquinone biosynthesis methylase (ubiE) gene was designed for the LAMP reaction. Additionally, serial 10-fold dilutions of cultured L. intracellularis and spiked feces were also used for the optimization of real-time LAMP. The lower limit of the linear range of the assay in L. intracellularis was 1.0 × 100 L. intracellularis. Real-time LAMP was 10 and 100 times more sensitive than real-time PCR and conventional PCR detection methods, respectively. Based on testing of 213 porcine fecal samples using real-time LAMP, realtime PCR and PCR, the agreement quotients of real-time LAMP with conventional PCR and with real-time PCR were 0.77 and 0.95, respectively. This study demonstrated that real-time LAMP was a powerful tool for the rapid and sensitive detection of L. intracellularis in porcine fecal samples.

scholarly journals Rapid identification of transgenic cotton (Gossypium hirsutum L.) plants by loop-mediated isothermal amplification

2011 ◽  
Vol 47 (No. 4) ◽  
pp. 140-148 ◽  
Author(s):  
N. Rostamkhani ◽  
A. Haghnazari ◽  
M. Tohidfar ◽  
A. Moradi
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Lamp Assay ◽  
Transgenic Cotton ◽  
Rapid Identification ◽  
Isothermal Amplification ◽  
Detection Sensitivity ◽  
Lamp Method ◽  
Loop Mediated Isothermal Amplification ◽  
Leaf Tissues

In an attempt to speed up the process of screening of transgenic cotton (G. hirsutum L.) plants, a visual and rapid loop-mediated isothermal amplification (LAMP) assay was adopted. Genomic DNA was extracted from fresh leaf tissues of T<sub>2</sub> transgenic cotton containing chitinase (chi) and cry1A(b) genes. Detection of genes of interest was performed by polymerase chain reaction (PCR), LAMP and real-time PCR methods. In LAMP assay the amplification was performed after 30 min at 65&deg;C when loop primers were involved in the reaction. The involvement of loop primers decreased the time needed for amplification. By testing serial tenfold dilutions (10<sup>&ndash;1</sup> to 10<sup>&ndash;8</sup>) of the genes of interest, the detection sensitivity of LAMP was found to be 100-fold higher than that of PCR. The rapid DNA extraction method and LAMP assay can be performed within 30 min and the derived LAMP products can be directly observed as visually detectable based on turbidity in the reaction tube. The accuracy of LAMP method in the screening of transgenes was confirmed by PCR and real-time PCR. The developed method was efficient, rapid and sensitive in the screening of cotton transgenic plants. This method can be applied to any other crops.

scholarly journals Comparison of Loop-Mediated Isothermal Amplification and Real-Time PCR Assays for Detection ofStrongyloidesLarvae in Different Specimen Matrices

2019 ◽  
Vol 57 (4) ◽  
Author(s):  
Matthew R. Watts ◽  
Rady Kim ◽  
Vishal Ahuja ◽  
Gemma J. Robertson ◽  
Yasmin Sultana ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Chronic Infection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Diagnostic Methods ◽  
Percentage Agreement ◽  
Content Type ◽  
Loop Mediated Isothermal Amplification ◽  
Stool Specimens

ABSTRACTStrongyloides stercoraliscan cause disease that ranges from asymptomatic chronic infection to fatal hyperinfection. Diagnosis from stool can be challenging because the most sensitive conventional tests require live larvae to be effective and there can be low larval output in chronic infection. Nucleic acid amplification tests (NAAT) have been developed to complement existing diagnostic methods. We compared a recently developed loop-mediated isothermal amplification (LAMP) assay with a real-time PCR that has previously been validated with larval microscopy. The limits of detection—quantified using serial dilutions of DNA extracts from singleStrongyloides rattithird-stage (L3) larvae spiked into approximately 250 µl of 5 differentS. stercoralis-negative stool specimens—were 10−3(1/5 replicates) and 10−2(1/5 replicates) dilutions for PCR and LAMP, respectively. PCR was positive for 4/5 replicates at 10−2. LAMP was compared to PCR using extracts from 396 stool specimens collected in Bangladesh and Australia, of which 53 were positive and 343 were negative by PCR. The positive percentage agreement of LAMP was 77.3% (95% score confidence interval [CI], 64.5 to 86.6). The negative percentage agreement was 100% (95% CI, 98.9 to 100). In a preliminary investigation, PCR and LAMP assays were positive using DNA extracted from serum (PCR, 3/16 extracts; LAMP, 2/16 extracts) and bronchoalveolar lavage fluid (PCR and LAMP, 2/2 extracts), demonstrating proof of concept. Compared to PCR, the lower number of positive results using the LAMP assay may have been due to reaction inhibitors and DNA degradation, and strategies to improve the LAMP assay are discussed.

Visual and Real-Time Event-Specific Loop-Mediated Isothermal Amplification Based Detection Assays for Bt Cotton Events MON531 and MON15985

2015 ◽  
Vol 98 (5) ◽  
pp. 1207-1214 ◽  
Author(s):  
Gurinder Jit Randhawa ◽  
Rashmi Chhabra ◽  
Rajesh K Bhoge ◽  
Monika Singh
Keyword(s):  
Real Time ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Bt Cotton ◽  
Loop Mediated Isothermal Amplification ◽  
Commercial Cultivation ◽  
Detection And Identification ◽  
Cotton Seeds ◽  
Gm Detection ◽  
Time Loop

Abstract Bt cotton events MON531 and MON15985 are authorized for commercial cultivation in more than 18 countries. In India, four Bt cotton events have been commercialized; more than 95% of total area under genetically modified (GM) cotton cultivation comprises events MON531 and MON15985. The present study reports on the development of efficient event-specific visual and real-time loop-mediated isothermal amplification (LAMP) assays for detection and identification of cotton events MON531 and MON15985. Efficiency of LAMP assays was compared with conventional and real-time PCR assays. Real-time LAMP assay was found time-efficient and most sensitive, detecting up to two target copies within 35 min. The developed real-time LAMP assays, when combined with efficient DNA extraction kit/protocol, may facilitate onsite GM detection to check authenticity of Bt cotton seeds.

Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for the Rapid Detection of Alternaria alternata

2017 ◽  
Vol 100 (1) ◽  
pp. 99-103 ◽  
Author(s):  
Xinyue Zhang ◽  
Guojie Xu ◽  
Huaqi Tang ◽  
Yanpeng Li ◽  
Chunsheng Liu
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Alternaria Alternata ◽  
Lamp Assay ◽  
High Specificity ◽  
Etiologic Agent ◽  
Isothermal Amplification ◽  
Pcr Assay ◽  
The Real ◽  
Loop Mediated Isothermal Amplification

Abstract Fungi of the Alternaria genus are associated with allergic diseases, with Alternaria alternata being one of the most prevalent species. A. alternata has been frequently reported as the etiologic agent of hypersensitivity pneumonitis, allergic rhinosinusitis, bronchial asthma,and other diseases. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay and a real-time PCR assay to detect low levels of A. alternata in herbal tea samples. The LAMP assay can detect as little as 3 pg/μL of A. alternata genomic DNA with high specificity. In addition, both the LAMP assay and the real-time PCR assay can be used for quantification of A. alternata. Although the newly developed LAMP assay is more rapid and specific in A. alternata identification, the real-time PCR assay is more precise in quantitation analysis.

scholarly journals Comparison of a TaqMan real-time polymerase chain reaction assay with a loop-mediated isothermal amplification assay for detection of Gallid herpesvirus 1

2011 ◽  
Vol 24 (1) ◽  
pp. 138-141 ◽  
Author(s):  
Shan-Chia Ou ◽  
Joseph J. Giambrone ◽  
Kenneth S. Macklin
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Chain Reaction ◽  
Loop Mediated Isothermal Amplification ◽  
Gallid Herpesvirus ◽  
Herpesvirus 1 ◽  
Polymerase Chain

A TaqMan real-time polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) assay were developed to detect Gallid herpesvirus 1 (GaHV-1, formerly Infectious laryngotracheitis virus). The standard curve of real-time PCR was established, and the sensitivity reached 10 copies/μl. In the current study, the conversion between viral titer and GaHV-1 genomic copy number was constructed. Six primers for LAMP assay amplified target gene at 65°C within 45 min, and the detection limit was 60 copies/μl. The 6 primers were highly specific, sensitive, and reproducible for detection of GaHV-1. Although the sensitivity of LAMP was lower than that of real-time PCR, LAMP was faster, less expensive, and did not require a thermocycler. The LAMP assay would be a viable alternative assay in diagnostic laboratories that do not employ real-time PCR technology.

scholarly journals Real-time loop-mediated isothermal amplification for rapid detection of Enterocytozoon hepatopenaei

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5993 ◽  
Author(s):  
Shao-Xin Cai ◽  
Fan-De Kong ◽  
Shu-Fei Xu ◽  
Cui-Luan Yao
Keyword(s):  
Real Time ◽  
Clinical Signs ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Lamp Method ◽  
The Real ◽  
Loop Mediated Isothermal Amplification ◽  
Enterocytozoon Hepatopenaei ◽  
Time Loop

Background Enterocytozoon 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. Methods A 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. Results Positive 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. Discussion In 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.

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