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 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 Development of a loop-mediated isothermal amplification assay for rapid detection of African swine fever

2020 ◽  
Vol 19 (1) ◽  
pp. 87-100
Author(s):  
Arphaphorn Dokphut ◽  
◽  
Prakit Boonpornprasert ◽  
Tapanut Songkasupa ◽  
Supansa Tangdee ◽  
...  
Keyword(s):  
Early Detection ◽  
Real Time ◽  
Sensitivity And Specificity ◽  
Real Time Pcr ◽  
African Swine Fever ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Analytical Sensitivity ◽  
Blue Color ◽  
Loop Mediated Isothermal Amplification

Since the first African swine fever (ASF) outbreak was reported in China in 2018, the disease has spread rapidly to several countries in Asia. The early detection of this disease is essential for the ASF control strategy to be effective. Loop-mediated isothermal amplification (LAMP) is a nucleic acid detection assay that is rapid, simple, cost-effective and field-friendly. In this study, we have developed a colorimetric assay of LAMP to detect ASF virus (ASFV). A set of LAMP primers was designed to target the conserved region of the VP72 gene. The conditions of LAMP were optimized. The amplification products were easily detected by the naked eye using hydroxynaphthol blue (HNB). The positive LAMP reaction generated a violet to sky blue color change. The sensitivity and specificity of LAMP assay were demonstrated in comparison with the OIE-recommended real-time PCR. A total of 211 samples including 121 confiscated pork products and 90 spiked clinical specimens were tested. The optimal amplification of ASFV DNA by LAMP was incubation at 60 °C for 90 min. The analytical sensitivity of ASFV LAMP assay was at least 368 plasmid DNA copies/µL without cross-reactivity with other swine pathogens. The diagnostic sensitivity and specificity of LAMP were 88% and 100%, respectively. There was almost perfect agreement between LAMP and real-time PCR assays (Kappa value=0.84). This novel LAMP assay is deemed to be a rapid, simple, sensitive, specific diagnostic tool and suitable for early detection of ASF to minimize the likelihood of ASF spread nationwide.

scholarly journals Loop-Mediated Isothermal Amplification for Detection ofStaphylococcus aureusin Dairy Cow Suffering from Mastitis

2012 ◽  
Vol 2012 ◽  
pp. 1-5 ◽  
Author(s):  
Zhang Tie ◽  
Wang Chunguang ◽  
Wei Xiaoyuan ◽  
Zhao Xinghua ◽  
Zhong Xiuhui
Keyword(s):  
Staphylococcus Aureus ◽  
High Sensitivity ◽  
High Specificity ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Specific Primers ◽  
Loop Mediated Isothermal Amplification ◽  
Reaction Tube ◽  
Specificity And Sensitivity ◽  
Detectable Concentration

To develop a rapid detection method ofStaphylococcus aureususing loop-mediated isothermal amplification (LAMP), four specific primers were designed according to six distinct sequences of thenucgene. In addition, the specificity and sensitivity of LAMP were verified and compared with those of PCR. Results showed that the LAMP reaction was completed within 45 min at 62.5°C, and ladder bands were appeared in LAMP products analyzed by gel electrophoresis. After adding 1x SYBR Green l, the positive reaction tube showed green color and the negative reaction tube remained orange, indicating that the LAMP has high specificity. The minimal detectable concentration of LAMP was1×102 CFU/mL and that of PCR was1×104 CFU/mL, indicating that the LAMP was 100 times more sensitive than the PCR. The LAMP method for detection ofStaphylococcus aureushas many advantages, such as simple operation, high sensitivity, high specificity, and rapid analysis. Therefore, this method is more suitable for the rapid on-site detection ofStaphylococcus aureus.

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 Development of an in-house loop-mediated isothermal amplification (LAMP) assay for detection of Mycobacterium tuberculosis and evaluation in sputum samples of Nepalese patients

2008 ◽  
Vol 57 (4) ◽  
pp. 439-443 ◽  
Author(s):  
Basu Dev Pandey ◽  
Ajay Poudel ◽  
Tomoko Yoda ◽  
Aki Tamaru ◽  
Naozumi Oda ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Lamp Assay ◽  
High Sensitivity ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Lamp Method ◽  
Rrna Gene ◽  
Predictive Values ◽  
Loop Mediated Isothermal Amplification ◽  
System A

A number of nucleic acid amplification assays (NAAs) have been employed to detect tubercle bacilli in clinical specimens for tuberculosis (TB) diagnosis. Among these, loop-mediated isothermal amplification (LAMP) is an NAA possessing superior isothermal reaction characteristics. In the present study, a set of six specific primers targeting the Mycobacterium tuberculosis 16S rRNA gene with high sensitivity was selected and a LAMP system (MTB-LAMP) was developed. Using this system, a total of 200 sputum samples from Nepalese patients were investigated. The sensitivity of MTB-LAMP in culture-positive samples was 100 % (96/96), and the specificity in culture-negative samples was 94.2 % (98/104, 95 % confidence interval 90.5–97.9 %). The positive and negative predictive values of MTB-LAMP were 94.1 and 100 %, respectively. These results indicate that this MTB-LAMP method may prove to be a powerful tool for the early diagnosis of TB.

HIGHLY SENSITIVE H7N9 AVIAN INFLUENZA VIRUS DETECTION USING REVERSE TRANSCRIPTION LOOP-MEDIATED ISOTHERMAL AMPLIFICATION AND OLIGONUCLEOTIDE MICROARRAY

2015 ◽  
Vol 41 (04) ◽  
pp. 251-255
Author(s):  
Lih-Chiann Wang ◽  
Dean Huang
Keyword(s):  
Avian Influenza ◽  
Sensitivity And Specificity ◽  
Reverse Transcription ◽  
High Sensitivity ◽  
Oligonucleotide Microarray ◽  
Influenza Viruses ◽  
Isothermal Amplification ◽  
Detection Methods ◽  
Loop Mediated Isothermal Amplification ◽  
Viral Copy Number

H7N9 avian influenza viruses have circulated in the human population and poultry flocks in China since 2013. H7N9 virus monitoring is imperative in Taiwan due to the frequent contact between China and Taiwan. Traditional viral molecular detection methods using RT-PCR and sequencing are time- and labor-intensive, thus a simpler and cheaper method with high sensitivity and specificity is worth developing. We successfully detected human and wild bird H7N9 viruses in this study using reverse transcription loop-mediated isothermal amplification (RT-LAMP) and oligonucleotide microarray. The detection limit was as low as one viral copy number. The specific matching reaction between the templates and microarray probes during hybridization ensured the high detection effectiveness. The excellent sensitivity and specificity of the RT-LAMP-microarray makes it a powerful H7N9 surveillance approach in Taiwan.

scholarly journals Loop-Mediated Isothermal Amplification (LAMP) Assay to De-tect Toxoplasmosis in Schizophrenia Patients

2020 ◽  
Author(s):  
Hadi MIRAHMADI ◽  
Raheleh HASANZADEH ◽  
Hamid MALEK RAEESI ◽  
Shirzad FALLAHI ◽  
Mahdi KHOSHSIMA SHAHRAKI ◽  
...  
Keyword(s):  
Nested Pcr ◽  
High Efficiency ◽  
Parasitic Infection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Intermediate Hosts ◽  
Cross Sectional ◽  
Loop Mediated Isothermal Amplification ◽  
Schizophrenic Patients

Background: Toxoplasma gondii (T. gondii) causes an important parasitic infection known as toxoplasmosis, which is a globally distributed important zoonosis. One of the major serious characteristics of T. gondii is its ability to manipulate the behavior of intermediate hosts. We performed a cross-sectional study to determine toxoplasmosis in schizophrenic patients, as one of the major neuropsychiatric disorders, using loop-mediated isothermal amplification (LAMP) technic by targeting parasite B1 gene. Methods: Blood samples were taken from 118 schizophrenic patients hospitalized in tow hospitals including Baharan, Clinic of Psychiatric Ali-ibn-Abi-Talib Hospital (in Zahedan City), and Amir-al Momenin Psychiatric Hospital (in Zabol City), Sistan and Baluchestan Province, southeast Iran in 2016. They were analyzed using LAMP, and compared with the previous data of nested-PCR and serology. Results: Out of the 118 schizophrenic individuals, 56 patients (47.4%) were found to be infected with T. gondii. The diagnosis of toxoplasmosis was confirmed in 41 patients (34.7%) via the nested-PCR. The seroprevalence of toxoplasmosis in schizophrenic patients was 55.9% (66/118). Conclusion: We found a high efficiency of LAMP method in identifying toxoplasmosis and its high prevalence among schizophrenic patients. Our findings could provide viable offer implications for the prevention of schizophrenia.

scholarly journals LOOP-MEDIATED ISOTHERMAL AMPLIFICATION FOR DETECTION OF C. BURNETII IN BULGARIA

2021 ◽  
Vol 19 (2) ◽  
pp. 147-151
Author(s):  
M. Kunchev ◽  
V. Belcheva ◽  
E. Grigorov
Keyword(s):  
Polymerase Chain Reaction ◽  
Sensitivity And Specificity ◽  
Q Fever ◽  
High Sensitivity ◽  
Isothermal Amplification ◽  
Chain Reaction ◽  
Retrospective Diagnosis ◽  
Loop Mediated Isothermal Amplification ◽  
The World ◽  
Polymerase Chain

Q fever, which is caused by Coxiella burnetii, a small, pleomorphic intracellular bacterium, is the most widespread zoonosis in the world. The chronic form of the disease can lead to disability and death. Rapid diagnosis of Q fever is needed in order that effective treatment can be initiated. The conventional retrospective diagnosis of Q fever, based on serology, is useless for the treatment of afflicted patients. Thus, molecular methods have been created to close the diagnostic gap between the onset of the disease and the presence of specific antibodies in serum. A polymerase chain reaction is a suitable and reliable method with high sensitivity and specificity, but it requires expensive equipment and post-amplification protocol. Loop-mediated isothermal amplification (LAMP) is an isothermal technique, conducted at constant temperature that can amplify a negligible amount of DNA to more than 109 copies within one hour, using special primers and polymerase. We have tested the sensitivity and specificity of LAMP in the detection of C. burnetii. The mean positive rate of LAMP and polymerase chain reaction in patients was 100% and 74%, respectively. LAMP reacted negatively with non-C. burnetii pathogens and non-infected blood samples. We conclude that LAMP is a sensitive and specific technique for the detection of C. burnetii and has advantages over serological methods and PCR that make it attractive for diagnosing Q fever in countries around the world.

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.

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.

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