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.

The loop-mediated isothermal amplification assay for rapid diagnosis of Babesia canis canis infections in dogs

2013 ◽  
Vol 16 (1) ◽  
pp. 131-133 ◽  
Author(s):  
Ł. Adaszek ◽  
M. Jankowska ◽  
M. Kalinowski ◽  
T. Banach ◽  
D. Wułupek ◽  
...  
Keyword(s):  
High Specificity ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Babesia Canis ◽  
Canine Babesiosis ◽  
Loop Mediated Isothermal Amplification ◽  
Specificity And Sensitivity ◽  
Amplification Assay ◽  
Analytical Laboratories ◽  
Babesia Canis Canis

Abstract The aim of this study was to use a rapid and easy DNA-based test, the loop-mediated isothermal amplification (LAMP), for diagnosis of Babesia canis canis infections in dogs. 10 DNA samples of 18S RNA-A and 10 DNA samples of 18S RNA-B of B. canis canis were used in the study. LAMP method could successfully detect DNA in all examined samples down to 0.1 pg dilution. Obtained results suggest that this method has high specificity and sensitivity and can be applied in analytical laboratories in diagnosis of canine babesiosis.

scholarly journals Suitability of scienciometric analysis targeting Loop Mediated Isothermal Amplification Assay applied to farm animals / Adequação da análise cienciométrica direcionada ao ensaio de Amplificação Isotérmica Mediada por Alça aplicada à animais de produção

2021 ◽  
Vol 7 (12) ◽  
pp. 115333-115354
Author(s):  
Sarah Amado Ribeiro ◽  
Calebe Bertolino Marins De Campos ◽  
Hérida Samaya Gonçalves De Sousa ◽  
Alex Silva Da Cruz ◽  
Aparecido Divino Da Cruz
Keyword(s):  
Isothermal Amplification ◽  
Farm Animals ◽  
Scientometric Analysis ◽  
Lamp Method ◽  
Bibliometric Data ◽  
Web Interface ◽  
Microsoft Excel ◽  
Loop Mediated Isothermal Amplification ◽  
Amplification Assay ◽  
Number Of Publications

The objective of the study was to carry out, with the aid of Scopus®️, a scientometric analysis of Loop Mediated Isothermal Amplification Assay (LAMP) applied to farm animals. The research has considered articles from January 2000 to December 2019 and only open or closed access articles published in English. The bibliometric matrices were run through RStudio, applying Biblioshiny as a web interface to Bibliometrix resources for R environment. Later, several bibliometric data were collected with the aid of Bibliometrix, most of which were converted into graphs using Microsoft Excel®️. The scientometric analysis base of the current study was composed by 438 articles from 504 researched in the Scopus®️. Of the 438 articles analyzed, it stands out as results: 1) the years of 2015 (11,4%) and 2019 (11,4%) had equally the highest number of publications in the area; 2) Journal of Virological Methods (12,5%) ranked first in the ranking of journals according to total articles published; 3) China (49,8%), Japan (12,7%) and India (7,1%) have been countries of more published articles; 4) most articles applied the assay to detect microorganisms affecting the farm animals; and, 5) together, the animal groups fish, bovine, poultry, and swine corresponded to 2/3 (71,1%) of the animals used in scientific research using the LAMP method. With all these results, it is concluded that the scientometric analysis showed an overview of the information in the articles about LAMP applied to farm animals.

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.

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 Establishment and Evaluation of a Loop-Mediated Isothermal Amplification Assay for Detection of Raccoon Dog in Meat Mixtures

2017 ◽  
Vol 2017 ◽  
pp. 1-8
Author(s):  
Jinhua Liu ◽  
Yanyu Shi ◽  
Siying Teng ◽  
Lianpeng Wu ◽  
Xinmin Zhang
Keyword(s):  
Lamp Assay ◽  
Cross Reactivity ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Nyctereutes Procyonoides ◽  
Raccoon Dog ◽  
Loop Mediated Isothermal Amplification ◽  
Different Temperatures ◽  
Amplification Assay ◽  
Minced Meat

Raccoon dog (Nyctereutes procyonoides) is an economically important animal used for fur production, but consuming its meat is injurious to human health. Currently, no rapid and sensitive method for detecting raccoon dog meat in meat mixtures is available. In this study, we developed an easily applicable, rapid, and economically feasible method for identifying the presence of raccoon dog in meat mixtures based on loop-mediated isothermal amplification (LAMP). Four sets of LAMP primers were tested at different temperatures, and the primers that worked best at 62°C (set 2) were determined. In the LAMP assay, there was no cross-reactivity with the meat procured from other species of animals and the detection limit of DNA concentration was 0.1 pg·μL−1, slightly higher than TaqMan real-time PCR (0.01 pg·μL−1), but sensitivity of 0.1 pg·μL−1 complies with most requirements of routine analysis. Moreover, by the LAMP method, the meat mixtures containing more than 0.5% of the raccoon dog component were directly detected (without DNA extraction) in the supernatant isolated from the meat mixtures after performing repeated cycles of thawing and freezing of minced meat mixtures. Our results show that LAMP assay is a valuable, straightforward, and sensitive detection tool for identification of raccoon dog meat in mixtures.

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.

Rapid detection of Betacoronavirus 1 from clinical fecal specimens by a novel reverse transcription loop-mediated isothermal amplification assay

2011 ◽  
Vol 24 (1) ◽  
pp. 174-177 ◽  
Author(s):  
Jun Qiao ◽  
Qingling Meng ◽  
Xuepeng Cai ◽  
Chuangfu Chen ◽  
Zaichao Zhang ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Rapid Detection ◽  
Lamp Assay ◽  
High Specificity ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Lamp Method ◽  
Nucleocapsid Gene ◽  
Loop Mediated Isothermal Amplification ◽  
Amplification Assay

Betacoronavirus 1 (BCoV-1) is an important pathogen causing diarrhea in calves. In the current study, a novel reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid detection of BCoV-1 was successfully developed. The primers were designed to target the highly conserved fragment of BCoV-1 nucleocapsid gene. The assay displayed high specificity detecting only BCoV-1 with no cross reaction with other viruses. When 418 clinical samples from 6 different geographical areas of Xinjiang province were tested by the RT-LAMP method, the results indicated that this test is a simple, rapid, accurate, and sensitive method for the detection of BCoV-1.

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