scholarly journals EFFECTIVENESS OF THE LOOP-MEDIATED ISOTHERMAL AMPLIFICATION WITH FLUORESCENT DETECTION IN THE DIAGNOSIS OF PARVOVIRUS ENTERITIS IN CARNIVORES

2019 ◽  
Vol 1 (1) ◽  
pp. 90-95 ◽  
Author(s):  
O. A. Petrusha ◽  
T. L. Chernichenko ◽  
I. A. Kofiadi ◽  
V. V. Zverev ◽  
E. B. Faizuloev
Keyword(s):  
Real Time ◽  
Point Of Care ◽  
Reference Method ◽  
Diagnostic Value ◽  
Isothermal Amplification ◽  
Fluorescent Detection ◽  
Lamp Method ◽  
Analytical Sensitivity ◽  
Loop Mediated Isothermal Amplification ◽  
Syto 9

The aim of the study was to evaluate the diagnostic value of the method of loop-mediated isothermal amplification of DNA with real-time fluorescent detection (real-time LAMP, or RT-LAMP) on the model of carnivore parvoviruses. Materials and methods. Samples of feces, blood and swabs from the rectum of different species of predatory animals with parvovirus enteritis (n = 39) and healthy animals (n = 31), as well as laboratory strains of mink enteritis virus, were analyzed by RT-LAMP using SYTO-9 and SYTO-82 dyes. Real-time PCR was used as a reference method. Results. In our study, the LAMP method with real-time fluorescence detection (RT-LAMP) in the carnivore parvovirus enteritis model provides high analytical sensitivity (1.5×103 copies of DNA/ml), diagnostic sensitivity and specificity (up to 100% under optimal conditions). Comparison of the two intercalating dyes showed that the SYTO-82 dye provides a higher signal-to-background ratio (22.6 ± 2.1) than the SYTO-9 dye (6.3 ± 1.5) (p <0.0000001 ). At the same time, SYTO-9 dye at the sensitivity limit (10 copies of DNA) provides an increase in fluorescence in the reaction mixture 13 minutes earlier than for SYTO-82 (23 and 36 minutes, respectively). Conclusion. RT-LAMP is a promising method for rapid and highly sensitive «point-of-care» diagnosis of infectious diseases, as well as in conditions of livestock farms or in field conditions.

scholarly journals Rapid diagnostics of genital herpes by loop-mediated isothermal amplification method with fluorescent detection

2019 ◽  
pp. 40-46
Author(s):  
D. I. Smirnova ◽  
O. A. Petrusha ◽  
A. V. Gracheva ◽  
E. A. Volynskaya ◽  
V. V. Zverev ◽  
...  
Keyword(s):  
Real Time ◽  
Sensitivity And Specificity ◽  
High Efficiency ◽  
High Sensitivity ◽  
Isothermal Amplification ◽  
Fluorescent Detection ◽  
Lamp Method ◽  
Analytical Sensitivity ◽  
Loop Mediated Isothermal Amplification

Introduction. Due to the high clinical significance of herpesvirus diseases, the searching of fast and effective methods for their diagnosis remains relevant.The aim of the study was to evaluate the diagnostic efficiency of the loop-mediated isothermal amplification of DNA with real-time fluorescent detection (RT-LAMP) with SYTO-82 dye on a model of herpes simplex virus (HSV) infection.Materials and methods. A total of 44 urogenital swabs containing type 1 and type 2 HSV DNA and 43 swabs without HSV DNA, including 33 samples containing the DNA of cytomegalovirus, Epstein-Barr virus and herpesvirus type 6, were studied. For RT-LAMP, Bst 2.0 WarmStart DNA polymerase, SYTO-82 dye, LAMP primers were used.Results. The high efficiency of HSV DNA detection in the RT-LAMP reaction with SYTO-82 dye was shown. RT-LAMP in optimal conditions allowed to reduce reaction time for 2-3 times compared to real-time PCR (to 35 minutes). Analytical sensitivity of HSV type 1 and 2 detection in RT-LAMP was 103 copies of DNA/ml. The diagnostic sensitivity and specificity of the RT-LAMP diagnosis of HSV infection were 96% and 100%, respectively.Discussion. RT-LAMP method has a high sensitivity and specificity comparable to RTPCR, while the risk of false positive results obtaining is minimal.Conclusion. Thus, the reaction of RT-LAMP with SYTO-82 dye allows quickly, with high sensitivity and specificity to detect HSV DNA in clinical material and can be considered as a promising point-of-care testing method.

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 Comparative analysis of loop-mediated isothermal amplification (LAMP)-based assays for rapid detection of SARS-CoV-2 genes

2020 ◽  
Author(s):  
Daniel Urrutia-Cabrera ◽  
Roxanne Hsiang-Chi Liou ◽  
Jianxiong Chan ◽  
Sandy Shen-Chi Hung ◽  
Alex W Hewitt ◽  
...  
Keyword(s):  
Comparative Analysis ◽  
Reaction Time ◽  
False Positive ◽  
Point Of Care ◽  
Low Cost ◽  
Isothermal Amplification ◽  
Fluorescent Detection ◽  
Experimental Conditions ◽  
Diagnostic Laboratory ◽  
Loop Mediated Isothermal Amplification

AbstractThe COVID-19 pandemic caused by SARS-CoV-2 has infected millions worldwide and there is an urgent need to increase our diagnostic capacity to identify infected cases. Although RT-qPCR remains the gold standard for SARS-CoV-2 detection, this method requires specialised equipment in a diagnostic laboratory and has a long turn-around time to process the samples. To address this, several groups have recently reported development of loop-mediated isothermal amplification (LAMP) as a simple, low cost and rapid method for SARS-CoV-2 detection. Herein we present a comparative analysis of three LAMP-based assays that target different regions of the SARS-CoV-2: ORF1ab RdRP, ORF1ab nsp3 and Gene N. We perform a detailed assessment of their sensitivity, kinetics and false positive rates for SARS-CoV-2 diagnostics in LAMP or RT-LAMP reactions, using colorimetric or fluorescent detection. Our results independently validate that all three assays can detect SARS-CoV-2 in 30 minutes, with robust accuracy at detecting as little as 1000 RNA copies and the results can be visualised simply by color changes. We also note the shortcomings of these LAMP-based assays, including variable results with shorter reaction time or lower load of SARS-CoV-2, and false positive results in some experimental conditions. Overall for RT-LAMP detection, the ORF1ab RdRP and ORF1ab nsp3 assays have higher sensitivity and faster kinetics for detection, whereas the Gene N assay exhibits no false positives in 30 minutes reaction time. This study provides validation of the performance of LAMP-based assays for SARS-CoV-2 detection, which have important implications in development of point-of-care diagnostic for SARS-CoV-2.

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.

Development and application of a real-time loop-mediated isothermal amplification method for quantification of Acetobacter aceti in red wine

2020 ◽  
Vol 367 (19) ◽  
Author(s):  
Jingfeng Zhang ◽  
Li Wang ◽  
Lei Shi ◽  
Xun Chen ◽  
Meidan Liang ◽  
...  
Keyword(s):  
Real Time ◽  
Red Wine ◽  
Reference Method ◽  
Acetic Acid Bacteria ◽  
Accurate Method ◽  
Isothermal Amplification ◽  
Quantitative Detection ◽  
Acetobacter Aceti ◽  
Loop Mediated Isothermal Amplification ◽  
Time Loop

ABSTRACT This study reports the development and optimization of a real-time loop-mediated isothermal amplification (qLAMP) method for rapid detection of Acetobacter aceti strain in red wine samples. Our results showed that the primers and probes designed for 16S rRNA were effective for A. aceti detection. The quantification limit of real-time polymerase chain reaction (qPCR) and qLAMP in pure culture was 2.05 × 101 colony forming units (CFU) mL−1. qLAMP had a sensitivity of 6.88 × 101 CFU mL−1 in artificially contaminated Changyu dry red wine (CDRW) and Changyu red wine (CRW), and 6.88 × 102 CFU mL−1 in artificially contaminated Greatwall dry red wine (GDRW), which was 10 times higher than that of qPCR. In conclusion, this newly developed qLAMP is a reliable, rapid and accurate method for the detection and quantification of A. aceti species in red wine samples. Furthermore, our work provides a standard reference method for the quantitative detection of A. aceti and other acetic acid bacteria during the fermentation and storage of red wine samples.

Development and Evaluation of a Loop-Mediated Isothermal Amplification (LAMP) Method for Detecting Foodborn Salmonella in Raw Milk

2013 ◽  
Vol 647 ◽  
pp. 577-582 ◽  
Author(s):  
Yong Zhen Wang ◽  
De Guo Wang
Keyword(s):  
Reference Method ◽  
Lamp Assay ◽  
Raw Milk ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Conventional Pcr ◽  
Detection Level ◽  
Loop Mediated Isothermal Amplification ◽  
Milk Samples ◽  
Food Borne

In present study, we reported the performance of a Loop-mediated isothermal amplification (LAMP) assay detecting food-borne pathogen Salmonella. Three pairs of primers were specially designed for recognizing eight distinct sequences on the target invA gene. Time and temperature conditions for amplification of Salmonella were optimized to be 40 min at 61°C. The LAMP assay gave with artificially contaminated raw milk samples detection limit level of 142 CFU/ml which corresponds to 6-9 cells per reaction tube, while the detection level of conventional PCR was 103 CFU/ml. Data on naturally contaminated raw milk samples indicated that the LAMP method was highly specific and sensitive, giving 89.58% concordance with the ISO 6579 reference method for the samples without enrichment and 100% concordance for the samples after enrichment.

scholarly journals Development and evaluation of a loop-mediated isothermal amplification assay (LAMP) for the diagnosis of campylobacteriosis

2021 ◽  
Author(s):  
Anna Shuryaeva ◽  
Tatyana Malova ◽  
Anna Tolokonceva ◽  
Sofia Karseka ◽  
Ekaterina Davydova ◽  
...  
Keyword(s):  
Russian Federation ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Analytical Sensitivity ◽  
Central Research ◽  
Gastrointestinal Infections ◽  
Loop Mediated Isothermal Amplification ◽  
Diagnostic Characteristics ◽  
Amplification Assay ◽  
Campylobacter Spp

Background: Different species of Campylobacter are the most common causes of bacterial gastroenteritis. There are many methods to detect the presence of Campylobacter, including PCR, but it takes about 5-6 hours. Using loop-mediated amplification assay allowed reducing the time of detection and simplifying the procedure at all. Aims: To develop a loop-mediated isothermal amplification assay (LAMP) with fluorescent probe for the diagnosis of campylobacteriosis. Methods: Stool suspensions were prepared and bacterial fractions were separated as in methodological recommendation of Central Research Institute of Epidemiology described. DNA was extracted using AmpliTest RIBO-prep (FSBI SPC FMBA, Russian Federation) according to the manufacturers instruction and detected with AmpliSens OKI-screen-FL" (FBIS CRIE, Russian Federation). Primers and probes were selected in 16S rDNA gene region. Analytical specificity was confirmed on bacterial cultures, analytical sensitivity was assessed using a recombinant plasmid containing the target Campylobacter DNA sequence fragment. LAMP amplification was performed at 65 C for 30 min. Results: An assay for the detection of Campylobacter spp. based on loop-mediated isothermal amplification is developed, the reaction time does not exceed 30 minutes. The analytical sensitivity of the developed technique is comparable to the real-time PCR and is equal to 103 copies / ml, the analytical specificity is 100%. The evaluation of 127 clinical samples, previously characterized by the commercial kit "AmpliSens OKI-screen-FL" (FBIS CRIE, Russian Federation), showed high diagnostic specificity and sensitivity of the developed LAMP-method. No false positive results were found, 108 samples were negative by LAMP and PCR. Campylobacter spp. DNA was detected by the LAMP method in 18 out of 19 PCR-positive samples. One discordant LAMP negative sample can be attributed to the low bacterial load of Campylobacter spp. for a given sample. Conclusions: A method for the rapid detection of Campylobacter spp. loop-mediated isothermal amplification is developed, and its high analytical and diagnostic characteristics have been shown experimentally. Keywords: Gastrointestinal infections, molecular diagnostics, rapid diagnostics, Loop-Mediated Isothermal Amplification (LAMP), Campylobacter spp.

scholarly journals Development and evaluation of a multiplex loop-mediated isothermal amplification (LAMP) assay for differentiation of Mycobacterium tuberculosis and non-tuberculosis mycobacterium in clinical samples

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0244753
Author(s):  
Jeeyong Kim ◽  
Borae G. Park ◽  
Da Hye Lim ◽  
Woong Sik Jang ◽  
Jeonghun Nam ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Real Time ◽  
Positive Reaction ◽  
Real Time Pcr ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Published Data ◽  
Analytical Sensitivity ◽  
Loop Mediated Isothermal Amplification

Introduction The rapid and accurate diagnosis of tuberculosis (TB) is important to reduce morbidity and mortality rates and risk of transmission. Therefore, molecular detection methods such as a real-time PCR–based assay for Mycobacterium tuberculosis (MTB) have been commonly used for diagnosis of TB. Loop-mediated isothermal amplification (LAMP) assay was believed to be a simple, quick, and cost-effective isothermal nucleic acid amplification diagnostic test for infectious diseases. In this study, we designed an in-house multiplex LAMP assay for the differential detection of MTB and non-tuberculosis mycobacterium (NTM), and evaluated the assay using clinical samples. Material and methods For the multiplex LAMP assay, two sets of specific primers were designed: the first one was specific for IS6110 genes of MTB, and the second one was universal for rpoB genes of mycobacterium species including NTM. MTB was confirmed with a positive reaction with both primer sets, and NTM was identified with a positive reaction by only the second primer set without a MTB-specific reaction. Total 333 clinical samples were analyzed to evaluate the multiplex LAMP assay. Clinical samples were composed of 195 positive samples (72 MTB and 123NTM) and 138 negative samples. All samples were confirmed positivity or negativity by real-time PCR for MTB and NTM. Analytical sensitivity and specificity were evaluated for the multiplex LAMP assay in comparison with acid fast bacilli staining and the culture method. Results Of 123 NTM samples, 121 were identified as NTM and 72/72 MTB were identified as MTB by the multiplex LAMP assay. False negative reactions were seen only in two NTM positive samples with co-infection of Candida spp. All 138 negative samples were identified as negative for MTB and NTM. Analytical sensitivity of the multiplex LAMP assay was 100% (72/72) for MTB, and 98.4% (121/123) for NTM. And the specificity of assay was 100% (138/138) for all. Conclusions Our newly designed multiplex LAMP assay for MTB and NTM showed relatively good sensitivity in comparison with previously published data to detect isolated MTB. This multiplex LAMP assay is expected to become a useful tool for detecting and differentiating MTB from NTM rapidly at an acceptable sensitivity.

A rapid, sensitive, specific and visual detection of Salmonella in retail meats using the loop-mediated isothermal amplification (LAMP), targeting the invA gene

2021 ◽  
Author(s):  
Can Wang ◽  
Ziheng Xu ◽  
Xuejiao Hou ◽  
Min Wang ◽  
Chenyu Zhou ◽  
...  
Keyword(s):  
Food Safety ◽  
Visual Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
National Standard ◽  
Lamp Method ◽  
Analytical Sensitivity ◽  
Conventional Pcr ◽  
Inva Gene ◽  
Loop Mediated Isothermal Amplification

Salmonella is one of the major pathogenic bacteria causing food-borne diseases. The rapid detection of Salmonella in food is of great significance to food safety. In this study, the loop-mediated isothermal amplification (LAMP) method was developed and the primers were designed targeting the invA gene of Salmonella. Then, the standard samples of recombinant invA-plasmid and 100 retail meat samples were tested by LAMP and compared with the results tested by the conventional PCR and the routine China National Food Safety Standard Methods for Food Microbiological Examination-Salmonella (GB/T4789.4-2016), respectively. The results showed that Salmonella strains of 8 different serotypes were amplified successfully by the developed LAMP assay. And, it was 1000-fold more sensitive than the conventional PCR with the analytical sensitivity of 8×102 copies/μL of the standard sample of invA-plasmid. The results were visualized directly by adding Calcein/MnCl2 into the LAMP reaction tube and the positively amplified products turned green after an incubation of 2 min. In the parallel detection, the positive rate of Salmonella by the LAMP assay was highly correlated with the routine China national standard method. The results of the study demonstrated that the developed LAMP assay is a simple, rapid, strongly specific, highly sensitive and visual detection method for Salmonella.

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