scholarly journals Dry loop mediated isothermal amplification assay for detection of SARS-CoV-2 from clinical specimens

2020 ◽  
Author(s):  
Yuki Higashimoto ◽  
Masaru Ihira ◽  
Yoshiki Kawamura ◽  
Masato Inaba ◽  
Kazuya Shirato ◽  
...  
Keyword(s):  
Developing Countries ◽  
Real Time ◽  
Predictive Value ◽  
Isothermal Amplification ◽  
Cold Chain ◽  
Pcr Analysis ◽  
Nasopharyngeal Swab ◽  
Lamp Method ◽  
Lamp Product ◽  
Loop Mediated Isothermal Amplification

Coronavirus disease 2019 (COVID-19) has had a major disease burden on many countries around the world. The spread of COVID-19 is anticipated to have a major impact on developing countries including African nations. To establish a point-of-care test for COVID-19, we developed a dry loop mediated isothermal amplification (LAMP) method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. We carried out reverse transcription (RT)-LAMP using the Loopamp SARS-CoV-2 Detection kit (Eiken Chemical, Tokyo, Japan). The entire mixture except for the primers is dried and immobilized inside the tube lid. To determine the specificity of the kit, 22 viral genomes associated with respiratory infections, including the SARS coronavirus, were tested. No LAMP product was detected in reactions performed with RNA from these pathogens. The sensitivity of this assay, determined by either a real-time turbidity assay or colorimetric change of the reaction mixture, as evaluated by the naked eye or under illumination with ultraviolet light, was 10 copies/reaction. After the initial validation analysis, we analyzed 24 nasopharyngeal swab specimens collected from patients suspected to have COVID-19. Nineteen (79.2%) of the 24 samples were positive for SARS-CoV-2 RNA, as determined by real-time RT-PCR analysis. Using the Loopamp SARS-CoV-2 Detection kit, we detected SARS-CoV-2 RNA in 15 (62.5%) of the 24 samples. Thus, the sensitivity, specificity, positive predictive value, and negative predictive value of the Loopamp 2019-CoV-2 detection reagent kit were 94.0%, 96.0%, 95.9%, and 94.1%, respectively. The dry LAMP method for detection of SARS-CoV-2 RNA was fast and easy to use, solves the cold chain problem, and therefore represents a promising tool for diagnosis of COVID-19 in developing countries.

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.

Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of Mycoplasma pneumoniae

2005 ◽  
Vol 54 (11) ◽  
pp. 1037-1041 ◽  
Author(s):  
Ryoichi Saito ◽  
Yoshiki Misawa ◽  
Kyoji Moriya ◽  
Kazuhiko Koike ◽  
Kimiko Ubukata ◽  
...  
Keyword(s):  
Real Time ◽  
Mycoplasma Pneumoniae ◽  
Rapid Detection ◽  
Clinical Laboratory ◽  
Direct Sequencing ◽  
Bacterial Species ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Nasopharyngeal Swab ◽  
Loop Mediated Isothermal Amplification

A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Mycoplasma pneumoniae was developed and evaluated. The assay specifically amplified only M. pneumoniae sequences, and no cross-reactivity was observed for other Mycoplasma species or respiratory bacterial species. The detection limit for this assay was found to be 2 × 102 copies, corresponding to 2–20 colour changing units of M. pneumoniae in 1 h, as observed in a real-time turbidimeter and electrophoretic analysis. The accuracy of the LAMP reaction was confirmed by restriction endonuclease analysis as well as direct sequencing of the amplified product. The assay was applied to 95 nasopharyngeal swab samples collected from patients or from healthy individuals, and compared to a real-time PCR assay in-house. A concordance of 100 % was observed between the two assays. The LAMP assay is easy to perform, shows a rapid reaction and is inexpensive. It may therefore be applied in the routine diagnosis of M. pneumoniae infection in the clinical laboratory.

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 of a loop-mediated isothermal amplification (LAMP) method for specific detection of Mycobacterium bovis

2021 ◽  
Vol 15 (1) ◽  
pp. e0008996
Author(s):  
Thoko Flav Kapalamula ◽  
Jeewan Thapa ◽  
Mwangala Lonah Akapelwa ◽  
Kyoko Hayashida ◽  
Stephen V. Gordon ◽  
...  
Keyword(s):  
Developing Countries ◽  
Mycobacterium Bovis ◽  
Color Change ◽  
Low Cost ◽  
Lamp Assay ◽  
Genomic Region ◽  
Isothermal Amplification ◽  
Diagnostic Methods ◽  
Lamp Method ◽  
Loop Mediated Isothermal Amplification

Bovine tuberculosis (TB) caused by Mycobacterium bovis is a significant health threat to cattle and a zoonotic threat for humans in many developing countries. Rapid and accurate detection of M. bovis is fundamental for controlling the disease in animals and humans, and for the proper treatment of patients as one of the first-line anti-TB drug, pyrazinamide, is ineffective against M. bovis. Currently, there are no rapid, simplified and low-cost diagnostic methods that can be easily integrated for use in many developing countries. Here, we report the development of a loop-mediated isothermal amplification (LAMP) assay for specific identification of M. bovis by targeting the region of difference 4 (RD4), a 12.7 kb genomic region that is deleted solely in M. bovis. The assay's specificity was evaluated using 139 isolates comprising 65 M. bovis isolates, 40 M. tuberculosis isolates, seven M. tuberculosis complex reference strains, 22 non-tuberculous mycobacteria and five other bacteria. The established LAMP detected only M. bovis isolates as positive and no false positives were observed using the other mycobacteria and non-mycobacteria tested. Our LAMP assay detected as low as 10 copies of M. bovis genomic DNA within 40 minutes. The procedure of LAMP is simple with an incubation at a constant temperature. Results are observed with the naked eye by a color change, and there is no need for expensive equipment. The established LAMP can be used for the detection of M. bovis infections in cattle and humans in resource-limited areas.

scholarly journals Assessment of a Loop-Mediated Isothermal Amplification (LAMP) Assay for the Rapid Detection of Pathogenic Bacteria from Respiratory Samples in Patients with Hospital-Acquired Pneumonia

2020 ◽  
Vol 8 (1) ◽  
pp. 103 ◽  
Author(s):  
Andrea Vergara ◽  
Hervé Boutal ◽  
Adrián Ceccato ◽  
Míriam López ◽  
Adrià Cruells ◽  
...  
Keyword(s):  
Positive Predictive Value ◽  
Negative Predictive Value ◽  
Predictive Value ◽  
Pathogenic Bacteria ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Loop Mediated Isothermal Amplification ◽  
Hospital Acquired Pneumonia ◽  
Hospital Acquired

Rapid identification of the causative agent of hospital-acquired pneumonia (HAP) will allow an earlier administration of a more appropriate antibiotic and could improve the outcome of these patients. The aim of this study was to develop a rapid protocol to identify the main microorganisms involved in HAP by loop-mediated isothermal amplification (LAMP) directly from respiratory samples. First of all, a rapid procedure (<30 min) to extract the DNA from bronchoalveolar lavage (BAL), endotracheal aspirate (EA) or bronchoaspirate (BAS) was set up. A specific LAMP for Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Acinetobacter baumannii was performed with the extracted solution at 65 °C for 30–40 min. Overall, 58 positive BAL and 83 EA/BAS samples were tested. The limits of detection varied according to the microorganism detected. Validation of the LAMP assay with BAL samples showed that the assay was 100% specific and 86.3% sensitive (positive predictive value of 100% and a negative predictive value of 50%) compared with culture. Meanwhile for BAS/EA samples, the assay rendered the following statistical parameters: 100% specificity, 94.6% sensitivity, 100% positive predictive value and 69.2% negative predictive value. The turnaround time including sample preparation and LAMP was circa 1 h. LAMP method may be used to detect the most frequent bacteria causing HAP. It is a simple, cheap, sensitive, specific and rapid assay.

scholarly journals Assessment of a loop-mediated isothermal amplification (LAMP) assay for the rapid detection of pathogenic bacteria from respiratory samples in patients with hospital-acquired pneumonia

2019 ◽  
Author(s):  
Andrea Vergara ◽  
Hervé Boutal ◽  
Adrián Ceccato ◽  
Míriam López ◽  
Adrià Cruells ◽  
...  
Keyword(s):  
Positive Predictive Value ◽  
Negative Predictive Value ◽  
Predictive Value ◽  
Pathogenic Bacteria ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Loop Mediated Isothermal Amplification ◽  
Hospital Acquired Pneumonia ◽  
Hospital Acquired

AbstractIntroductionHospital-acquired pneumonia (HAP) is the one that presents clinically two or more days after admission into the hospital. Rapid identification of the causative agent of HAP will allow an earlier administration of a more appropriate antibiotic therapy and could lead to an improved outcome of patients with HAP.MethodsFirst of all, a rapid procedure (< 30 min) to extract the DNA from bronchoalveolar lavage (BAL), endotracheal aspirate (EA) or bronchoaspirate (BAS) was set up. A loop-mediated isothermal amplification reaction (LAMP) specific for Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Acinetobacter baumannii was carried out with the extracted solution. The reaction was performed at 65ºC for 30-40 min. LAMP was compared with bacterial culture method.ResultsOverall, 58 positive BAL and 83 EA/BAS samples were tested. The limits of detection varied according to the microorganism detected and to the respiratory sample analyzed. Validation of the LAMP assay with BAL samples showed that the assay was 100% specific and 86.3% sensitive (positive predictive value of 100% and a negative predictive value of 50%). Meanwhile for BAS/EA samples, the assay rendered the following statistical parameters: 100% specificity, 94.6% sensitivity, 100% positive predictive value and 69.2% negative predictive value. These scores were obtained including minor errors as correct. The turnaround time including preparation of the sample and LAMP was circa 1 hour.ConclusionsLAMP method may be used to detect the most frequent bacteria causing HAP. It is a simple, cheap, sensitive, specific and rapid assay.

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.

Comparison of the detection of Toxocara spp. in the soils of public parks of Ahvaz (southwest of Iran) by PCR and Loop-Mediated Isothermal Amplification (LAMP)

2020 ◽  
Vol 20 ◽  
Author(s):  
Forough Kazemi ◽  
Reza Arjmand ◽  
Somayeh Fallahizadeh ◽  
Mehdi Tavalla
Keyword(s):  
Developing Countries ◽  
Isothermal Amplification ◽  
P Value ◽  
Lamp Method ◽  
Public Parks ◽  
Cross Sectional ◽  
The Public ◽  
Loop Mediated Isothermal Amplification ◽  
Southwest Of Iran ◽  
The City

Introduction: The infections caused by Toxocara spp. are considered as one of the most important zoonotic diseases in the world, especially in developing countries. Human toxocariasis, particularly in children is acquired by playing in public parks. Hence, the aim of the current study was to detect Toxocara spp. in the soils of public parks of the city of Ahvaz, southwest of Iran, using the PCR and loop-mediated isothermal amplification (LAMP) methods. Methods: In this descriptive cross-sectional study, 260 soil samples were randomly collected from the different public parks of the city of Ahvaz. After preforming zinc sulfate (ZnSO4) flotation technique, the DNA samples were extracted from the isolated Toxocara spp. egg. Lastly, the extracted DNA was used for PCR and LAMP-based molecular detection. Results: Of 260 specimens, 57 (21.9%) samples were found positive for Toxocara spp., using the PCR method, out of that 38 (14.6%) samples were positive for T. canis and 19 (7.3%) samples were positive for T. cati. Also, of 260, 81 (31.1%) cases were positive for Toxocara species, using the LAMP method, among them 51 (19.6%) samples were found positive for T. canis and 30 (11.5%) samples were positive for T. cati. Kappa (κ) coefficient between PCR and LAMP showed a strong agreement (0.766, P-value=0.002). Conclusion: Our data showed a relatively high outbreak of Toxocara spp. in the public parks’ soils of the city, using the PCR and LAMP methods. Since the parasite can cause human toxocariasis particularly in children; thus, the health authorities of the city of Ahvaz and other similar cities, particularly in developing countries must pay more attention to the hygiene of the public parks’ soils.

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