scholarly journals Identification of Trueperella bernardiae isolated from peking ducks (Anas platyrhynchos domesticus) by phenotypical and genotypical investigations and by a newly developed loop-mediated isothermal amplification (LAMP) assay

2021 ◽  
Author(s):  
Marwa F. E. Ahmed ◽  
Mazen Alssahen ◽  
Christoph Lämmler ◽  
Bernd Köhler ◽  
Martin Metzner ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Lamp Assay ◽  
High Sensitivity ◽  
Housekeeping Gene ◽  
Isothermal Amplification ◽  
Specific Gene ◽  
Rrna Gene ◽  
Phenotypic Properties ◽  
Loop Mediated Isothermal Amplification ◽  
Encoding Gene

AbstractTrueperella (T.) bernardiae is a well-known bacterial pathogen in infections of humans, rarely in animals. In the present study, five T. bernardiae isolates, isolated from five Peking ducks of four different farms, were identified by phenotypic properties, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, and genotypically by sequencing the 16S ribosomal RNA (rRNA) gene, the superoxide dismutase A encoding gene sodA, and the glyceraldehyde-3-phosphate dehydrogenase encoding gene gap. In addition, the T. bernardiae isolates could be identified with a newly developed loop-mediated isothermal amplification (LAMP) assay based on the gyrase encoding housekeeping gene gyrA. All these tests clearly identified the T. bernardiae isolates to the species level. However, the detection of the specific gene gyrA with the newly designed LAMP assay appeared with a high sensitivity and specificity, and could help to identify this bacterial species in human and animal infections in future. The importance of the T. bernardiae isolates for the clinical condition of the ducks and for the problems at farm level remains unclear.

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.

Bench-scale experiments for the development of a unified loop-mediated isothermal amplification (LAMP) assay for the in vitro diagnosis of Leishmania species' promastigotes

2013 ◽  
Vol 142 (8) ◽  
pp. 1671-1677 ◽  
Author(s):  
M. KARANI ◽  
I. SOTIRIADOU ◽  
J. PLUTZER ◽  
P. KARANIS
Keyword(s):  
Lamp Assay ◽  
High Sensitivity ◽  
Leishmania Species ◽  
18S Rrna Gene ◽  
Isothermal Amplification ◽  
Rrna Gene ◽  
Bench Scale ◽  
Loop Mediated Isothermal Amplification ◽  
Wide Range

SUMMARYWe developed, in bench-scale experiments, a unified loop-mediated isothermal amplification (LAMP) assay for the detection of cutaneous, mucocutaneous and visceral leishmaniasis using DNA of cultivated promastigotes. Two primer sets for the LAMP assay were designed based on the 18S rRNA gene, and their sensitivity and specificity were tested and compared. Both of them were specific for Leishmania as the DNA of all ten Leishmania species tested was amplified, whereas the DNA of other parasites, including that of Trypanosoma, was not. The detection limit for primer set 1 ranged between 30 pg and 3·6 fg, depending on which Leishmania species tested. Primer set 2 showed high sensitivity, but was less sensitive than primer set 1. Our findings lead to the conclusion that the LAMP assay with primer set 1 is a promising and effective assay for the successful detection of a wide range of Leishmania infections using only a unified multiplex LAMP test.

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 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.

First report on optimization of loop-mediated isothermal amplification (LAMP) for the diagnosis of Babesia felis

2017 ◽  
Author(s):  
Muhammad Awais Salim ◽  
Raheela Akhtar ◽  
Muhammad Lateef ◽  
Imran Rashid ◽  
Harron Akbar ◽  
...  
Keyword(s):  
Lamp Assay ◽  
Cost Effective ◽  
Conventional Polymerase Chain Reaction ◽  
18S Rrna Gene ◽  
Isothermal Amplification ◽  
Rrna Gene ◽  
Chain Reaction ◽  
Loop Mediated Isothermal Amplification ◽  
Polymerase Chain ◽  
Effective Diagnosis

The objective of present study was to optimize loop mediated isothermal amplification (LAMP) assay for the diagnosis of Babesia felis in cats. LAMP primers were designed recognizing four sections of 18SribosomalRNA (18S rRNA) gene of B. felis. The blood samples of cats microscopically positive for Babesia felis were further used to extract deoxyribo neuclic acid (DNA) and the reaction mixture of 25 µL was standardized at 63°C temperature for 1 hour. LAMP assay provided more positive samples than conventional polymerase chain reaction (PCR). The prevalence of B. felis was also determined in cats using this optimized LAMP assay and it was found that the prevalence was more in younger cats as compare to adults. The application of LAMP can be helpful in rapid, reliable and cost effective diagnosis of B. felis in field.

scholarly journals Evaluation of Loop-Mediated Isothermal Amplification Assay for the Detection of Pneumocystis jirovecii in Immunocompromised Patients

2015 ◽  
Vol 2015 ◽  
pp. 1-6 ◽  
Author(s):  
Preeti Singh ◽  
Sundeep Singh ◽  
Bijay Ranjan Mirdha ◽  
Randeep Guleria ◽  
Sanjay Kumar Agarwal ◽  
...  
Keyword(s):  
Diagnostic Test ◽  
Nested Pcr ◽  
Lamp Assay ◽  
High Sensitivity ◽  
Single Step ◽  
Isothermal Amplification ◽  
Pneumocystis Jirovecii ◽  
Immunocompromised Patients ◽  
Loop Mediated Isothermal Amplification ◽  
Common Opportunistic Infection

Pneumocystis pneumonia (PCP) is one of the common opportunistic infection among HIV and non-HIV immunocompromised patients. The lack of a rapid and specific diagnostic test necessitates a more reliable laboratory diagnostic test for PCP. In the present study, the loop-mediated isothermal amplification (LAMP) assay was evaluated for the detection of Pneumocystis jirovecii. 185 clinical respiratory samples, including both BALF and IS, were subjected to GMS staining, nested PCR, and LAMP assay. Of 185 respiratory samples, 12/185 (6.5%), 41/185 (22.2%), and 49/185 (26.5%) samples were positive by GMS staining, nested PCR, and LAMP assay, respectively. As compared to nested PCR, additional 8 samples were positive by LAMP assay and found to be statistically significant (p<0.05) with the detection limit of 1 pg. Thus, the LAMP assay may serve as a better diagnostic tool for the detection of P. jirovecii with high sensitivity and specificity, less turn-around time, operational simplicity, single-step amplification, and immediate visual detection.

scholarly journals First Report of Crown Gall of Kiwifruit (Actinidia deliciosa) Caused by Agrobacterium fabacearum in China and the Establishment of Loop-Mediated Isothermal Amplification Technique

2021 ◽  
Vol 23 (1) ◽  
pp. 207
Author(s):  
Linan He ◽  
Jinqiao Shi ◽  
Zhibo Zhao ◽  
Fei Ran ◽  
Feixu Mo ◽  
...  
Keyword(s):  
Crown Gall ◽  
High Sensitivity ◽  
Isothermal Amplification ◽  
Guizhou Province ◽  
Specific Gene ◽  
Lamp Method ◽  
Crown Gall Disease ◽  
Loop Mediated Isothermal Amplification ◽  
Detection Technology ◽  
Amplification Technique

Kiwifruit is moderately sweet and sour and quite popular among consumers; it has been widely planted in some areas of the world. In 2019, the crown gall disease of kiwifruit was discovered in the main kiwifruit-producing area of Guizhou Province, China. This disease can weaken and eventually cause the death of the tree. The phylogeny, morphological and biological characteristics of the bacteria were described, and were related to diseases. The pathogenicity of this species follows the Koch hypothesis, confirming that A. fabacearum is the pathogen of crown gall disease of kiwifruit in China. In this study, Loop-mediated isothermal amplification (LAMP) analysis for genome-specific gene sequences was developed for the specific detection of A. fabacearum. The detection limit of the LAMP method is 5 × 10−7 ng/μL, which has high sensitivity. At the same time, the amplified product is stained with SYBR Green I after the reaction is completed, so that the amplification can be detected with the naked eye. LAMP analysis detected the presence of A. fabacearum in the roots and soil samples of the infected kiwifruit plant. The proposed LAMP detection technology in this study offers the advantages of ease of operation, visibility of results, rapidity, accuracy and high sensitivity, making it suitable for the early diagnosis of crown gall disease of kiwifruit.

Rapid detection of Helicobacter pylori using cytotoxin-associated gene A based on loop-mediated isothermal amplification assay and magnetic nanoparticles

2020 ◽  
Vol 10 (2) ◽  
pp. 283-289
Author(s):  
Junwang Zhang ◽  
Meixia Wang ◽  
Ying Shi ◽  
Qi Wang ◽  
Wubo Zhao
Keyword(s):  
Helicobacter Pylori ◽  
Magnetic Nanoparticles ◽  
Lamp Assay ◽  
High Sensitivity ◽  
Isothermal Amplification ◽  
Bacterial Strains ◽  
Loop Mediated Isothermal Amplification ◽  
Cytotoxin Associated Gene A ◽  
H Pylori ◽  
Sensitive Method

The current methods for detecting Helicobacter pylori infection are time-consuming and have relatively low sensitivity. More appropriate tests are needed. A rapid, specific, and sensitive method was presently developed to detect the cytotoxin-associated gene A (cagA) of H. pylori. Genomic DNA was extracted using magnetic nanoparticles and then amplified by the loop-mediated isothermal amplification (LAMP) reaction using primers we designed. To assess the diagnostic value of the LAMP assay in detecting H. pylori cagA, agarose gel electrophoresis as well as detection of fluorescence intensity after adding fluorescent dye were done. Specificity analysis showed that 11 pathogenic bacterial strains common in human gut were negative for cagA, with a positive result obtained only for H. pylori. Sensitivity analysis demonstrated a cagA detection limit of 100 fg. The results were consistent with that of the 3C-urea breath test. The novel LAMP assay can directly identify H. pylori cagA in the gastric juice of clinical patients with high sensitivity and specificity. The comparatively more rapid and more sensitive method may be valuable for clinical applications.

scholarly journals Field Evaluation of a Loop-Mediated Isothermal Amplification (LAMP) Platform for the Detection of Schistosoma japonicum Infection in Oncomelania hupemsis Snails

2018 ◽  
Author(s):  
Zhi-Qiang Qin ◽  
Jing Xu ◽  
Ting Feng ◽  
Shan Lv ◽  
Yin-Jun Qian ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Point Of Care ◽  
Infected Snail ◽  
Lamp Assay ◽  
High Sensitivity ◽  
Field Evaluation ◽  
Isothermal Amplification ◽  
Pcr Analysis ◽  
Schistosomiasis Control ◽  
Loop Mediated Isothermal Amplification

Schistosoma infection in snails can be monitored by microscopy or indirectly by sentinel mice. As both these approaches sometimes miss infections, more sensitive tests are needed, particularly in low-level transmission settings. In this study, the loop-mediated isothermal amplification (LAMP) technique, designed to detect a specific 28S ribosomal S. japonicum gene with high sensitivity, was compared to microscopy using snail samples from 51 areas endemic for schistosomiasis in five Chinese provinces. The results were compared with those by polymerase chain reaction (PCR) adding DNA sequencing as a reference when needed. The testing of pooled snail samples showed that a dilution factor of 1/50, i.e., one infected snail plus 49 non-infected ones, would still result in a positive reaction after the recommended number of amplification cycles. Testing a total of 232 pooled samples, emanating from 4,006 snail specimens, with the LAMP assay showed a 6.5% rate of infection, while traditional microscopy found only 0.04% positive samples in the same materials. Parallel PCR analysis confirmed the diagnostic accuracy of the LAMP assay, with DNA sequencing even giving LAMP a slight lead. Microscopy and the LAMP test were carried out at local schistosomiasis-control stations demonstrating that the potential of the latter assay to serve as a point-of-care (POC) test with results available within 60&ndash;90 minutes, while the more complicated PCR test had to be carried out at the National Institute of Parasitic Diseases (NIPD) in Shanghai, China. In conclusion, LAMP was found to be clearly superior to microscopy and as good as, or better, than PCR. Application of LAMP testing would be useful for surveillance and risk prediction as it requires less time than other techniques and can be used under field conditions, which improves and accelerates schistosomiasis control.

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