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 Development of a Loop-Mediated Isothermal Amplification Assay for the Detection of Dickeya spp.

2017 ◽  
Vol 107 (11) ◽  
pp. 1339-1345 ◽  
Author(s):  
Jarred Yasuhara-Bell ◽  
Glorimar Marrero ◽  
Mohammad Arif ◽  
Asoka de Silva ◽  
Anne M. Alvarez
Keyword(s):  
Lamp Assay ◽  
Conventional Polymerase Chain Reaction ◽  
Isothermal Amplification ◽  
Northern Europe ◽  
Potato Seed ◽  
Chain Reaction ◽  
Bacterial Populations ◽  
Loop Mediated Isothermal Amplification ◽  
Amplification Assay ◽  
Polymerase Chain

Dickeya and Pectobacterium spp. are responsible for soft-rotting diseases of several plant species, some with overlapping host range. On potato, symptoms caused by these pathogens cannot be clearly differentiated. Disease results in the downgrading and rejection of potato seed, thus requiring additional phytosanitary restrictions across Northern Europe and other parts of the world. In an effort to provide a more timely and accurate diagnostic to distinguish these two groups of pathogens, a method for detecting Dickeya spp. using loop-mediated isothermal amplification (LAMP) was developed. The LAMP assay can be used to test crude extracts prepared directly from symptomatic lesions. The entire test can be completed in less than 30 min, making it faster than the current diagnostic standard, the pelADE conventional polymerase chain reaction. Additionally, the LAMP assay was able to detect Dickeya DNA in samples spiked with varying amounts of Pectobacterium DNA, thus demonstrating the highly specific and sensitive nature of the assay, which can be applied on survey samples with mixed soft-rotting bacterial populations.

scholarly journals Comparison of a TaqMan real-time polymerase chain reaction assay with a loop-mediated isothermal amplification assay for detection of Gallid herpesvirus 1

2011 ◽  
Vol 24 (1) ◽  
pp. 138-141 ◽  
Author(s):  
Shan-Chia Ou ◽  
Joseph J. Giambrone ◽  
Kenneth S. Macklin
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Chain Reaction ◽  
Loop Mediated Isothermal Amplification ◽  
Gallid Herpesvirus ◽  
Herpesvirus 1 ◽  
Polymerase Chain

A TaqMan real-time polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) assay were developed to detect Gallid herpesvirus 1 (GaHV-1, formerly Infectious laryngotracheitis virus). The standard curve of real-time PCR was established, and the sensitivity reached 10 copies/μl. In the current study, the conversion between viral titer and GaHV-1 genomic copy number was constructed. Six primers for LAMP assay amplified target gene at 65°C within 45 min, and the detection limit was 60 copies/μl. The 6 primers were highly specific, sensitive, and reproducible for detection of GaHV-1. Although the sensitivity of LAMP was lower than that of real-time PCR, LAMP was faster, less expensive, and did not require a thermocycler. The LAMP assay would be a viable alternative assay in diagnostic laboratories that do not employ real-time PCR technology.

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.

scholarly journals Validation of the loop-mediated isothermal amplification method for rapid and sensitive detection of Ureaplasma species in respiratory tracts of preterm infants

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0247618
Author(s):  
Yuta Mikami ◽  
Kazumasa Fuwa ◽  
Eriko Arima ◽  
Yasuo Suda ◽  
Itaru Yanagihara ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Preterm Infants ◽  
Sensitivity And Specificity ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Chain Reaction ◽  
Loop Mediated Isothermal Amplification ◽  
Polymerase Chain ◽  
Tracheal Aspirates

Introduction A simple and rapid diagnosis of Ureaplasma spp. is required for the choice of the appropriate antibiotic. However, an ideal detection method has not been available. This study examines the efficacy of the loop-mediated isothermal amplification (LAMP) assay, which provides rapid and sensitive results, to detect Ureaplasma spp. in respiratory tract samples of preterm infants. Methods The study included preterm infants born before 32 weeks of gestation admitted Kagoshima City Hospital from June 2018 to March 2020. Nasopharyngeal swabs and/or tracheal aspirates were obtained in the first seven postnatal days. One hundred sixty-seven nasopharyngeal swabs and 101 tracheal aspirates were analyzed by LAMP, culture, and quantitative real-time polymerase chain reaction. Results All 167 infants had a median (range) gestational age of 28.7 weeks (22.3–30.9) and birthweight 1030g (322–1828). One hundred sixty-seven nasopharyngeal swabs and 101 tracheal aspirates were obtained. In the results of nasopharyngeal swabs, the sensitivity and specificity of LAMP were 73.9% (17/23) and 97.2% (140/144), whereas those of quantitative real-time polymerase chain reaction were 73.9% (17/23) and 95.8% (138/144), compared to culture. In the results of tracheal aspirates, the sensitivity and specificity of LAMP were 89.5% (17/19) and 92.7% (76/82), whereas those of quantitative real-time polymerase chain reaction were 89.5% (17/19) and 93.9% (77/82), compared to culture. Conclusions The LAMP assay showed similar sensitivity and specificity with quantitative real-time polymerase chain reaction in the respiratory tracts of preterm infants including extremely preterm infants during the immediate postnatal period. Therefore, the LAMP is a practical alternative for the early detection so that appropriate antibiotics can be administered for preventing BPD.

scholarly journals Loop-Mediated Isothermal Amplification for the Detection of Tylenchulus semipenetrans in Soil

Plant Disease ◽  
2016 ◽  
Vol 100 (5) ◽  
pp. 877-883 ◽  
Author(s):  
Borong Lin ◽  
Honghong Wang ◽  
Kan Zhuo ◽  
Jinling Liao
Keyword(s):  
Restriction Analysis ◽  
Lamp Assay ◽  
Conventional Polymerase Chain Reaction ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Tylenchulus Semipenetrans ◽  
Slow Decline ◽  
Decline Disease ◽  
Individual Specimen ◽  
Polymerase Chain

Tylenchulus semipenetrans is an economically important plant-parasitic nematode occurring in all citrus-producing regions of the world and causing a disease called “slow decline”. For the rapid detection of this nematode, a loop-mediated isothermal amplification (LAMP) assay was developed, based on the ribosomal DNA internal transcribed spacer sequence. The optimal condition for the LAMP assay was 65°C for 50 min. The LAMP products were confirmed using conventional polymerase chain reaction (PCR) and restriction analysis with the BamHI enzyme, and by adding SYBR Green I to the LAMP products for visual inspection. The LAMP assay was highly specific for the detection of T. semipenetrans populations from different geographical origins. It was also sensitive, detecting a tenth of the DNA from an individual specimen of T. semipenetrans, which was 10 times more sensitive than conventional PCR. The LAMP protocol was applied to natural citrus rhizosphere soil samples from several orchards in China and the results were fast, sensitive, robust, and accurate. This study is the first to provide a diagnostic tool for T. semipenetrans using DNA extracted directly from citrus rhizosphere soils. This LAMP assay could be used as a practical molecular tool to identify T. semipenetrans and diagnose slow decline disease, even in remote locations.

scholarly journals Development of a Multiplex Loop-Mediated Isothermal Amplification Assay for Diagnosis of Plasmodium spp., Plasmodium falciparum and Plasmodium vivax

Diagnostics ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1950
Author(s):  
Woong Sik Jang ◽  
Da Hye Lim ◽  
YoungLan Choe ◽  
Hyunseul Jee ◽  
Kyung Chul Moon ◽  
...  
Keyword(s):  
Internal Control ◽  
Point Of Care ◽  
Lamp Assay ◽  
18S Rrna Gene ◽  
Isothermal Amplification ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Loop Mediated Isothermal Amplification

Malaria, caused by the parasite Plasmodium and transmitted by mosquitoes, is an epidemic that mainly occurs in tropical and subtropical regions. As treatments differ across species of malarial parasites, there is a need to develop rapid diagnostic methods to differentiate malarial species. Herein, we developed a multiplex malaria Pan/Pf/Pv/actin beta loop-mediated isothermal amplification (LAMP) to diagnose Plasmodium spp., P. falciparum, and P. vivax, as well as the internal control (IC), within 40 min. The detection limits of the multiplex malaria Pan/Pf/Pv/IC LAMP were 1 × 102, 1 × 102, 1 × 102, and 1 × 103 copies/µL for four vectors, including the 18S rRNA gene (Plasmodium spp.), lactate dehydrogenase gene (P. falciparum), 16S rRNA gene (P. vivax), and human actin beta gene (IC), respectively. The performance of the LAMP assay was compared and evaluated by evaluating 208 clinical samples (118 positive and 90 negative samples) with the commercial RealStar® Malaria S&T PCR Kit 1.0. The developed multiplex malaria Pan/Pf/Pv/IC LAMP assay showed comparable sensitivity (100%) and specificity (100%) with the commercial RealStar® Malaria S&T PCR Kit 1.0 (100%). These results suggest that the multiplex malaria Pan/Pf/Pv/IC LAMP could be used as a point-of-care molecular diagnostic test for malaria.

scholarly journals Development of Detection Equipment for a Polymerase Chain Reaction with a Loop-Mediated Isothermal Amplification Reaction

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Wei-Chien Weng ◽  
Yu-Cheng Lin
Keyword(s):  
Polymerase Chain Reaction ◽  
Temperature Control ◽  
Low Cost ◽  
Cost Effective ◽  
Isothermal Amplification ◽  
Raspberry Pi ◽  
Chain Reaction ◽  
Loop Mediated Isothermal Amplification ◽  
Processing Signal ◽  
Polymerase Chain

In this research, low-cost detection equipment intended to carry out a polymerase chain reaction (PCR) through a loop-mediated isothermal amplification (LAMP) reaction is presented. We designed the internal structure with SolidWorks and AutoCAD. The equipment comprised a Raspberry Pi development board, a temperature control module, and a fluorescent optical detection module. The main program, temperature control, florescent signal processing, signal analysis, and screen display were programmed with Java. We applied the digital temperature controller module to obtain precise temperature control of the equipment. The experimental results showed that the heating rate of the testing equipment could reach 65°C within 4 minutes and could be accurately controlled to within 1°C. The duration of the LAMP PCR experiment was found to be significantly shorter than that of the conventional PCR. The results also revealed that with LAMP PCR, the temperature could be accurately controlled within a specific range, and the designed heating tasks could be completed within 15 minutes to one hour, depending on the specimen. The equipment could also correctly read both the positive and negative reactions with fluorescent signals. Thus, the proposed LAMP PCR detection equipment is more sensitive, more stable, and more cost-effective than other conventional alternatives and can be used in numerous clinical applications.

scholarly journals Loop-Mediated Isothermal Amplification and Polymerase Chain Reaction Methods for Specific and Rapid Detection of Rhodococcus fascians

Plant Disease ◽  
2013 ◽  
Vol 97 (4) ◽  
pp. 517-529 ◽  
Author(s):  
M. Serdani ◽  
M. Curtis ◽  
M. L. Miller ◽  
J. Kraus ◽  
M. L. Putnam
Keyword(s):  
Polymerase Chain Reaction ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
In Planta ◽  
Bacterial Strains ◽  
Chain Reaction ◽  
Loop Mediated Isothermal Amplification ◽  
Rhodococcus Fascians ◽  
Polymerase Chain

Rhodococcus fascians is a phytopathogenic actinobacterium which causes leafy galls and other plant distortions that result in economically significant losses to nurseries producing ornamental plants. Traditional assays for detection and identification are time-consuming and laborious. We developed a rapid polymerase chain reaction (PCR) diagnostic assay based on two primer pairs, p450 and fas, which target the fasA and fasD genes, respectively, that are essential for pathogenicity. We also developed a faster, more convenient, loop-mediated isothermal amplification (LAMP) assay targeting the fasR gene, which regulates expression of virulence genes. Both assays were evaluated for sensitivity and specificity in vitro and in planta. The p450 and fas primers amplified DNA only from pure cultures of pathogenic reference isolates of R. fascians. Nonpathogenic isolates and 51 other plant-associated bacteria were not amplified. The PCR primers correctly detected pathogenic R. fascians from 73 of 75 (97%) bacterial strains isolated from naturally infected plants. The PCR assay correctly discriminated between pathogenic R. fascians and other bacteria in 132 of 139 (95%) naturally infected plants, and in 34 of 34 (100%) artificially inoculated plants. The fas primers were slightly more accurate than the p450 primers. The LAMP assay accurately detected pathogenic R. fascians in 26 of 28 (93%) naturally infected plants and did not react with 23 asymptomatic plants. The LAMP primers also amplified product for DNA extracts of 40 of 41 bacterial strains isolated from plants with leafy galls. The detection limit of both the PCR and LAMP assays was approximately 103 CFU/30-μl reaction. These new tools allow fast, reliable, and accurate detection of R. fascians in vitro and in planta. The LAMP assay in particular is a significant advancement in rapid R. fascians diagnostics, and enables those with limited laboratory facilities to confirm the presence of this pathogen in infected plants.

scholarly journals Species identification of felis and vulpes by a novel loop-mediated isothermal amplification assay in fur products

2019 ◽  
Vol 14 ◽  
pp. 155892501882072
Author(s):  
Shunji Yu ◽  
Wenjia Gu ◽  
Yi Yu ◽  
Qinfeng Qu ◽  
Yi’nan Zhang
Keyword(s):  
Polymerase Chain Reaction ◽  
Species Identification ◽  
Quantitative Polymerase Chain Reaction ◽  
Cost Effective ◽  
Isothermal Amplification ◽  
Chain Reaction ◽  
Species Identity ◽  
Loop Mediated Isothermal Amplification ◽  
Amplification Method ◽  
Polymerase Chain

In the chaotic market of fur goods, genetic distinction is increasingly important for identifying species. A vast diversity of species identification methods has been proposed, while little is developed, particularly those easy, fast, and cost-effective ones. In this study, a simple and reliable novel loop-mediated isothermal amplification method for identifying cytochrome c oxidase I of felis and vulpes was established. It saves laborious post–polymerase chain reaction procedures and shortens the time for high-fidelity gene amplification. The sensitivity of this method for felis and vulpes identification, which is well matched to quantitative polymerase chain reaction, could be 10 or 1.0 pg, respectively. Predominantly, the sensitivity of loop-mediated isothermal amplification is more tolerant to those polymerase chain reaction inhibitors such as pigments, dyes, or other fur ingredients, compared to quantitative polymerase chain reaction. Even without costly specialized equipment, a water bath is sufficient for genetic distinction. Our approach is a new technique with broad application perspective, such as on-site species identity tests, commercial fraud, and wildlife crimes.

Sign in / Sign up

Export Citation Format

Share Document