scholarly journals Rapid detection of a novel B1-β-lactamase gene, blaAFM-1 using a loop-mediated isothermal amplification (LAMP) assay

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Yingcheng Qin ◽  
Xiaonv Duan ◽  
Yuan Peng ◽  
Yongyu Rui
Keyword(s):  
Recombinant Plasmid ◽  
Sanger Sequencing ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Conventional Pcr ◽  
Predictive Values ◽  
Clinical Strains ◽  
Loop Mediated Isothermal Amplification ◽  
Two Samples ◽  
Lactamase Gene

Abstract Background BlaAFM-1 (GenBank Accession No. 143105.1) is a new B1 subclass metallo-β-lactamase gene discovered by our group, and isolated from an Alcaligenes faecalis plasmid that renders carbapenem antibiotics ineffective. In this study, we generated a fast and reliable assay for blaAFM-1 detection. Methods We designed optimum loop-mediated isothermal amplification (LAMP) primers and constructed a recombinant plasmid AFM-1 to specifically detect blaAFM-1. Optimal LAMP primers were used to assess sensitivity of the recombinant plasmid AFM-1 and blaAFM-1-supplemented samples (simulated sputum and simulated feces). Fifty two samples, without blaAFM-1, were used to assess LAMP real-time assay specificity; these samples were verified by conventional PCR and sequencing for the absence of blaAFM-1. Three hundred clinical Gram-negative carbapenem-resistant strains were tested by LAMP assay for strains carrying blaAFM-1, which were confirmed by conventional PCR and Sanger sequencing. We calculated the sensitivity and its 95% confidence interval (95% CI), specificity and its 95% CI, and predictive values of the LAMP assay and conventional PCR/sequencing by investigating positive and negative clinical strains. Results The lowest limit of detection for the recombinant plasmid AFM-1 and blaAFM-1-supplemented samples (in both simulated sputum and simulated feces) was 101 copies/reaction. All amplification curves of the 52 blaAFM-1-free bacteria strains were negative, suggesting the LAMP assay had excellent specificity for detecting blaAFM-1. Among the 300 clinical strains, eight were positive for blaAFM-1 using LAMP. These LAMP results were consistent with conventional PCR and Sanger sequencing data. As with conventional PCR/sequencing, the LAMP method exhibits 100% sensitivity (95% CI 59.8–100%) and 100% specificity (95% CI 98.4–100%) for blaAFM-1 detection. The LAMP assay is also time-efficient (1 h) for blaAFM-1 detection. Conclusions We established a new LAMP assay with high sensitivity and specificity to detect the novel B1-β-lactamase gene, blaAFM-1.

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.

scholarly journals Rapid detection of cytochromecd1-containing nitrite reductase encoding genenirSwith loop-mediated isothermal amplification assay

2018 ◽  
Author(s):  
Qianqian Yang ◽  
Xuzhi Zhang ◽  
Xiaoyu Jiang ◽  
Xiaochun Wang ◽  
Yang Li ◽  
...  
Keyword(s):  
Nitrite Reductase ◽  
Genomic Dna ◽  
Limit Of Detection ◽  
Bacterial Species ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Target Sequence ◽  
Bacterial Cells ◽  
Conventional Pcr ◽  
Loop Mediated Isothermal Amplification

AbstractThe cytochromecd1-containing nitrite reductase,nirS, plays an important role in biological denitrification. Consequently, investigating the presence and abundance ofnirSis a commonly used approach to understand the distribution and potential activity of denitrifying bacteria, in addition to denitrifier communities. Herein, a new molecular biology technique termed loop-mediated isothermal amplification (LAMP) was developed to rapidly detectnirSgene using those ofPseudomonas aeruginosato optimize the assay. The LAMP assay relied on a set of four primers that were designed to recognize six target sequence sites, resulting in high target specificity. The specificity of the assay was confirmed by the lack of amplification when using DNA from 15 other bacterial species lackingnirSgene. The limit of detection for the LAMP assay under optimized conditions was 1.87 pg/reaction of genomic DNA, which was an order of magnitude lower than that required by conventional PCR assays. Moreover, a cell-template based LAMP assay was also developed for detectingnirSgene that directly used bacterial cells as template rather than genomic DNA. Only 1 h was needed from the addition of bacterial cells to the reaction to the verification of amplification success, and bulky and sophisticated equipment were not needed. Further, thenirSgene ofP. aeruginosain spiked seawater samples could be detected with both the DNA-template based LAMP assay and the cell-template based LAMP assay, thereby demonstrating the practicality of in-field use of them. In summary, the LAMP assays described here represent a rapid, user-friendly, and cost-effective alternative to conventional PCR.

scholarly journals Evaluation of Loop Mediated Isothermal Amplification (LAMP) for Diagnosis of Plasmodium falciparum in Wad Medani City, Gezira State, Sudan

2019 ◽  
pp. 1-7
Author(s):  
M. Y. Mohamed ◽  
A. D. Abakar ◽  
B. A. Talha ◽  
Salah Eldin G. Elzaki ◽  
Y. A. Mohammed ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Falciparum Malaria ◽  
Diagnostic Performance ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Teaching Hospitals ◽  
Molecular Technique ◽  
Rrna Gene ◽  
Predictive Values ◽  
Loop Mediated Isothermal Amplification

Plasmodium falciparum considered as the most serious form of species causes malaria compared with other species. Diagnosis of falciparum malaria in Sudan remain a major problem, the laboratory diagnosis depends solely on microscopy and RDTs. Loop mediated isothermal amplification (LAMP) assay is a molecular technique done in isothermal temperature using simple, inexpensive instruments for detection of falciparum malaria. The aim of the study is to evaluate the diagnostic performance of loop mediated isothermal amplification (LAMP) assay for detection of P. falciparum and compare with microscopic detection. A cross sectional hospital based study conducted on 220 blood samples collected from participants suspected to have falciparum malaria attending Wad Medani Teaching Hospitals and 26 healthy participants during the period November 2018 to January 2019. Thick blood films were done and used for P. falciparum detection. The extracted DNA by TE buffer was amplified by LAMP assay targeting 18S rRNA gene. Data were analyzed using Medical calculator (MedCalc) programs (V. 16). The results showed that the sensitivity, specificity, positive predictive value, negative predictive values were 99.1%, 84.6%, 53.2%, 99.8% respectively. Validation of LAMP diagnostic performance revealed that area under the curve is 0.919, while Weighted Kappa is 0.866. The study concluded that the LAMP assay had the identical diagnostic performance compared with microscopy in diagnosis of Plasmodium falciparum malaria. This gives a relative effortlessness application of LAMP assay in Sudan after availing the required logistics.

scholarly journals Evaluation of loop-mediated isothermal amplification assay for detection of scrub typhus in patients with acute febrile illness presenting to a Tertiary Care Center in Puducherry, India

2019 ◽  
Vol 11 (01) ◽  
pp. 082-086 ◽  
Author(s):  
Patricia Anitha Karthikeyan ◽  
Sugeerappa Laxmanappa Hoti ◽  
Reba Kanungo
Keyword(s):  
Diagnostic Test ◽  
Scrub Typhus ◽  
Tertiary Care ◽  
Lamp Assay ◽  
Febrile Illness ◽  
Isothermal Amplification ◽  
Acute Febrile Illness ◽  
Loop Mediated Isothermal Amplification ◽  
Igm Antibodies ◽  
Two Samples

Abstract PURPOSE: Scrub typhus an acute febrile illness has diverse clinical manifestations, which overlap with other febrile illnesses. Due to this reason, it is misdiagnosed, leading to inappropriate treatment, sometimes resulting in fatality. Thus, accurate diagnosis of scrub typhus is important for appropriate treatment. This study evaluated the loop-mediated isothermal amplification (LAMP) assay as a diagnostic test for scrub typhus among patients with fever. MATERIALS AND METHODS: A total of 50 cases of acute febrile illness clinically resembling scrub typhus, with or without an eschar, or cases of pyrexia of unknown origin were included in the study. Blood samples collected from these cases were subjected to detection of IgM antibodies to Orientia tsutsugamushi by ELISA, conventional groEL polymerase chain reaction (PCR), and the LAMP assay. RESULTS: Twelve cases had fever for less than a week, and two had fever for more than 3 weeks. IgM antibodies to O. tsutsugamushi were detected in 37 out of 50 samples (74%). LAMP assay was positive in 33 samples (66%). groEL gene-based PCR detected 35 (70%) samples as positive. Two samples negative by LAMP assay were positive by this PCR. Twenty samples collected from patients with dengue, typhoid, and malaria tested by the LAMP assay were negative, indicating its good specificity. LAMP assay and the conventional groEL-based PCR could detect 72.7% and 74.3% of the samples, respectively before the 10th day after onset of fever, whereas IgM ELISA could detect only 40.5% of the 37 samples. CONCLUSION: This study suggests that LAMP assay could be a useful diagnostic test for detecting scrub typhus in the acute phase of the illness and a cheaper alternative to other molecular methods in resource poor settings.

scholarly journals Development of Loop-Mediated Isothermal Amplification for Detection ofLeifsonia xylisubsp.xyliin Sugarcane

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Jing Liu ◽  
Liping Xu ◽  
Jinlong Guo ◽  
Rukai Chen ◽  
Michael Paul Grisham ◽  
...  
Keyword(s):  
Lamp Assay ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Conventional Pcr ◽  
Time Saving ◽  
Visual Judgment ◽  
Coryneform Bacterium ◽  
Loop Mediated Isothermal Amplification ◽  
Total Dna ◽  
Sensitive Method

Ratoon stunt, caused by the xylem-limited coryneform bacteriumLeifsonia xylisubsp.xyli(Lxx), is a deep bacteriosis and prevalent in most of sugarcane-producing countries. Based on loop-mediated isothermal amplification (LAMP), we developed a method for detectingLxx. The major advantages of the LAMP method are visual judgment by color and time saving with only 60 min for identification ofLxxand without the need for costly PCR apparatus and gel scanner. In the present study, positive and negative samples detected by the LAMP method were clearly distinguishable. When total DNA extracted from internode juice was used as the template, the sensitivity of LAMP was 10 times higher than that of the conventional PCR detection. The LAMP assay is a highly specific, rapid, and sensitive method for the diagnosis of ratoon stunt caused byLxxin sugarcane. This is the first report of LAMP-based assay for the detection ofLxxin sugarcane.

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 A loop-mediated isothermal amplification assay for Schistosoma mansoni detection in Biomphalaria spp. from schistosomiasis-endemic areas in Minas Gerais, Brazil

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Silvia Gonçalves Mesquita ◽  
Floria Gabriela dos Santos Neves ◽  
Ronaldo Guilherme Carvalho Scholte ◽  
Omar dos Santos Carvalho ◽  
Cristina Toscano Fonseca ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Lamp Assay ◽  
Minas Gerais ◽  
Cross Reactivity ◽  
Molecular Techniques ◽  
Isothermal Amplification ◽  
Conventional Pcr ◽  
Loop Mediated Isothermal Amplification ◽  
Field Samples ◽  
Collection Sites

Abstract Background Schistosomiasis a neglected tropical disease  endemic in Brazil. It is caused by the trematode Schistosoma mansoni, which is transmitted by snails of the genus Biomphalaria. Among measures used to control and eliminate schistosomiasis, accurate mapping and monitoring of snail breeding sites are recommended. Despite the limitations of parasitological methods, they are still used to identify infected snails. Loop-mediated isothermal amplification (LAMP) is a sensitive, rapid, and cost-effective diagnostic method for the identification of infected snails. In the work reported here, we aimed to validate the use of LAMP for the detection of S. mansoni in snails of the genus Biomphalaria. Methods Snails were collected in five municipalities of the Mucuri Valley and Jequitinhonha Valley regions in the state of Minas Gerais, Brazil. Snails were pooled according to collection site and then squeezed for the detection of S. mansoni and other trematode larvae. Pooled snails were subjected to pepsin digestion and DNA extraction. Molecular assays were performed for species-specific identification and characterization of the samples. A previously described LAMP assay was adapted, evaluated, and validated using laboratory and field samples. Results Using the parasitological method described here, S. mansoni cercariae were detected in snails from two collection sites, and cercariae of the family Spirorchiidae were found in snails from one site. The snails were identified by polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP). Biomphalaria glabrata, the main snail host of S. mansoni in Brazil, was detected in 72.2% of the collection sites. Biomphalaria kuhniana, which is resistant to S. mansoni infection, was found in the remaining sites. Multiplex, low stringency (LS), and conventional PCR allowed the detection of positive snails in four additional sites. Trematodes belonging to the families Strigeidae and Echinostomatidae were detected by multiplex PCR in two sites. The LAMP assay was effective in detecting the presence of S. mansoni infection in laboratory (7 days post-infection) and field samples with no cross-reactivity for other trematodes. When compared to LS and conventional PCR, LAMP showed 100% specificity, 85.7% sensitivity, and a κ index of 0.88. Conclusions Our findings suggest that LAMP is a good alternative method for the detection and monitoring of transmission foci of S. mansoni, as it was three times as effective as the parasitological examination used here for the detection of infection, and is more directly applicable in the field than other molecular techniques. Graphical abstract

scholarly journals Optimization of a Loop-Mediated Isothermal Amplification (LAMP) Assay for Schistosoma Mansoni (Trematoda: Digenea) Detection in Biomphalaria spp. from Endemic Areas for Schistosomiasis in Brazil

2021 ◽  
Author(s):  
Silvia Gonçalves Mesquita ◽  
Floria Gabriela dos Santos Neves ◽  
Ronaldo Guilherme Carvalho Scholte ◽  
Omar dos Santos Carvalho ◽  
Cristina Toscano Fonseca ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
False Negative ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Conventional Pcr ◽  
Promising Alternative ◽  
Loop Mediated Isothermal Amplification ◽  
Field Samples ◽  
Endemic Areas ◽  
Collection Sites

Abstract Background: Schistosomiasis mansoni is a neglected tropical disease endemic in Brazil caused by Schistosoma mansoni, which is transmitted by Biomphalaria snails. Among all measures to control and eliminate the disease, accurate mapping and monitoring of snail breeding sites for susceptible and/or infected hosts in endemic areas are recommended. Parasitological methods are frequently used to identify infected snails, although they have many limitations, often providing false-negative results. Loop-mediated isothermal amplification (LAMP) is a promising alternative method for a more sensitive, rapid, and cost-effective diagnosis. However, standardization of LAMP assays is challenging due to the variety of parasites that are co-endemic with S. mansoni, and their varying prevalence rates in different areas. In this work, we aimed to optimize a LAMP assay for the detection of S. mansoni in Biomphalaria snails from endemic areas in the state of Minas Gerais, Brazil. Methods: A total of 1,001 snails were collected in five municipalities of the Mucuri and Jequitinhonha Valleys. Snails were pooled and squeezed according to the collection site to detect the presence of the larval forms of S. mansoni and other trematodes. Pooled snails were submitted to pepsin digestion and DNA extraction. Then molecular assays were performed for the species-specific identification and characterization of the samples. A LAMP assay was optimized and validated using laboratory and field samples. Results: Using the parasitological method, S. mansoni cercariae were detected in snails from two collection sites. Biomphalaria glabrata, the main snail host of S. mansoni in Brazil, was detected in 72.2% of the collection sites by PCR-RFLP. Multiplex PCR, LS-PCR, and conventional PCR allowed the detection of positive snails in four additional sites. The optimized LAMP assay was effective in detecting the presence of S. mansoni infection with 100% sensitivity, 91.66% specificity, and a Kappa index of 0.88, when compared to LS-PCR and conventional PCR. Conclusions: Our findings suggest that LAMP is a good alternative for the detection and monitoring of transmission foci of S. mansoni, as it enabled the detection of infection three times more than the parasitological examination and is more applicable directly in the field when compared to other molecular approaches.

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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