scholarly journals Fast, Precise, and Reliable Multiplex Detection of Potato Viruses by Loop-Mediated Isothermal Amplification

2020 ◽  
Vol 21 (22) ◽  
pp. 8741
Author(s):  
Güven Edgü ◽  
Lena Julie Freund ◽  
Stefanie Hartje ◽  
Eckhard Tacke ◽  
Hans-Reinhard Hofferbert ◽  
...  
Keyword(s):  
Disease Management ◽  
Management Practices ◽  
Tobacco Rattle Virus ◽  
Simultaneous Detection ◽  
Isothermal Amplification ◽  
Diagnostic Methods ◽  
Tuber Quality ◽  
Loop Mediated Isothermal Amplification ◽  
Tuber Necrosis ◽  
Corky Ringspot

Potato is an important staple food crop in both developed and developing countries. However, potato plants are susceptible to several economically important viruses that reduce yields by up to 50% and affect tuber quality. One of the major threats is corky ringspot, which is a tuber necrosis caused by tobacco rattle virus (TRV). The appearance of corky ringspot symptoms on tubers prior to commercialization results in ≈ 45% of the tubers being downgraded in quality and value, while ≈ 55% are declared unsaleable. To improve current disease management practices, we have developed simple diagnostic methods for the reliable detection of TRV without RNA purification, involving minimalized sample handling (mini), subsequent improved colorimetric loop-mediated isothermal amplification (LAMP), and final verification by lateral-flow dipstick (LFD) analysis. Having optimized the mini-LAMP-LFD approach for the sensitive and specific detection of TRV, we confirmed the reliability and robustness of this approach by the simultaneous detection of TRV and other harmful viruses in duplex LAMP reactions. Therefore, our new approach offers breeders, producers, and farmers an inexpensive and efficient new platform for disease management in potato breeding and cultivation.

scholarly journals Loop-mediated isothermal amplification assay forEnterococcus sp., E. coliandS. aureusin chicken

2018 ◽  
Author(s):  
HyeSoon Song ◽  
YouChan Bae ◽  
HyukMan Kwon ◽  
YongKuk Kwon ◽  
SeongJoon Joh
Keyword(s):  
Broiler Chicken ◽  
Simultaneous Detection ◽  
Isothermal Amplification ◽  
Diagnostic Methods ◽  
Economic Losses ◽  
Diagnostic Assays ◽  
E Coli ◽  
Loop Mediated Isothermal Amplification ◽  
Resource Limited ◽  
Amplification Assay

ABSTRACTBacterial chondronecrosis with osteomyelitis (BCO) is a major cause of lameness in broiler chicken, and results in serious economic losses worldwide. Although the pathogenesis mechanism leading to lameness is not entirely understood, some strains ofEnterococcus sp., avian pathogenicEscherichia coli, orStaphylococcus aureushave been long recognized as important causative pathogens. To prevent the progression ofEnterococcus sp., avian pathogenicE. coli, orS. aureusinfections, we developed rapid, sensitive, and convenient diagnostic assays using loop-mediated isothermal amplification (LAMP). Entero-Common-LAMP assays were developed for a simultaneous detection of eightEnterococcusspecies. To target specific microorganisms, seven Entero-Specific-LAMP assays forE. faecalis, E. faecium, E. hirae, E. gallinarum, E. avium, E. duransandE. cecorum, andE. coli-LAMP andS. aureus-LAMP assays, were developed. Considering the prevalence and economic impact ofEnterococcus sp., E. coli, andS. aureus, the developed ten different LAMP assays have a considerable potential as routine diagnostic methods in the field or in resource-limited environments.

scholarly journals A loop-mediated isothermal amplification (LAMP) assay to identify isotype 1 β-tubulin locus SNPs in synthetic double-stranded Haemonchus contortus DNA

2021 ◽  
Author(s):  
Livio M. Costa-Junior ◽  
Umer N. Chaudhry ◽  
Philip J. Skuce ◽  
Seamus Stack ◽  
Neil D. Sargison
Keyword(s):  
Genetic Susceptibility ◽  
Management Practices ◽  
Anthelmintic Resistance ◽  
Lamp Assay ◽  
Gastrointestinal Nematode ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Snp Typing ◽  
Field Samples ◽  
Β Tubulin

AbstractDevelopment of sustainable gastrointestinal nematode (GIN) control strategies depends on the ability to identify the frequencies of drug-susceptible and resistant genotypes in GIN populations arising from management practices undertaken on individual farms. Resistance to BZ drugs in GINs has been shown to be conferred by the presence of defined SNPs in the isotype 1 β-tubulin locus. Loop-mediated isothermal amplification (LAMP) assays are amenable to use on a range of DNA templates and are potentially adaptable to use in practical, cost-effective, pen-side diagnostic platforms that are needed to detect anthelmintic resistance in the field. In this study, we designed primers and examined LAMP assays to detect each of the three major isotype 1 β-tubulin SNPs conferring genetic susceptibility to BZ drugs. We used artificial pools of synthetic DNA, containing different proportions of susceptible and resistant SNPs to determine reproducibility of the assays. We demonstrated the detection of each of the isotype 1 β-tubulin SNPs conferring susceptibility to BZ drugs using the optimal LAMP assay. Isotype 1 β-tubulin SNP typing was effective in detecting BZ susceptibility, but the accuracy was reduced in samples with less than 60 % susceptible DNA. Our results show the potential for LAMP SNP typing to detect genetic susceptibility or resistance to anthelmintic drugs in livestock GINs, and some of the limitations in our approach that will need to be overcome in order to evaluate this assay using field samples.

scholarly journals Comparison of Loop-Mediated Isothermal Amplification and Real-Time PCR Assays for Detection ofStrongyloidesLarvae in Different Specimen Matrices

2019 ◽  
Vol 57 (4) ◽  
Author(s):  
Matthew R. Watts ◽  
Rady Kim ◽  
Vishal Ahuja ◽  
Gemma J. Robertson ◽  
Yasmin Sultana ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Chronic Infection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Diagnostic Methods ◽  
Percentage Agreement ◽  
Content Type ◽  
Loop Mediated Isothermal Amplification ◽  
Stool Specimens

ABSTRACTStrongyloides stercoraliscan cause disease that ranges from asymptomatic chronic infection to fatal hyperinfection. Diagnosis from stool can be challenging because the most sensitive conventional tests require live larvae to be effective and there can be low larval output in chronic infection. Nucleic acid amplification tests (NAAT) have been developed to complement existing diagnostic methods. We compared a recently developed loop-mediated isothermal amplification (LAMP) assay with a real-time PCR that has previously been validated with larval microscopy. The limits of detection—quantified using serial dilutions of DNA extracts from singleStrongyloides rattithird-stage (L3) larvae spiked into approximately 250 µl of 5 differentS. stercoralis-negative stool specimens—were 10−3(1/5 replicates) and 10−2(1/5 replicates) dilutions for PCR and LAMP, respectively. PCR was positive for 4/5 replicates at 10−2. LAMP was compared to PCR using extracts from 396 stool specimens collected in Bangladesh and Australia, of which 53 were positive and 343 were negative by PCR. The positive percentage agreement of LAMP was 77.3% (95% score confidence interval [CI], 64.5 to 86.6). The negative percentage agreement was 100% (95% CI, 98.9 to 100). In a preliminary investigation, PCR and LAMP assays were positive using DNA extracted from serum (PCR, 3/16 extracts; LAMP, 2/16 extracts) and bronchoalveolar lavage fluid (PCR and LAMP, 2/2 extracts), demonstrating proof of concept. Compared to PCR, the lower number of positive results using the LAMP assay may have been due to reaction inhibitors and DNA degradation, and strategies to improve the LAMP assay are discussed.

scholarly journals Enhancing colorimetric loop-mediated isothermal amplification speed and sensitivity with guanidine chloride

BioTechniques ◽  
2020 ◽  
Vol 69 (3) ◽  
pp. 178-185 ◽  
Author(s):  
Yinhua Zhang ◽  
Guoping Ren ◽  
Jackson Buss ◽  
Andrew J Barry ◽  
Gregory C Patton ◽  
...  
Keyword(s):  
Guanidine Hydrochloride ◽  
Isothermal Amplification ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Simple Method ◽  
Diagnostic Assays ◽  
Loop Mediated Isothermal Amplification ◽  
Dna And Rna ◽  
Versatile Technique ◽  
Primer Sets

Loop-mediated isothermal amplification (LAMP) is a versatile technique for detection of target DNA and RNA, enabling rapid molecular diagnostic assays with minimal equipment. The global SARS-CoV-2 pandemic has presented an urgent need for new and better diagnostic methods, with colorimetric LAMP utilized in numerous studies for SARS-CoV-2 detection. However, the sensitivity of colorimetric LAMP in early reports has been below that of the standard RT-qPCR tests, and we sought to improve performance. Here we report the use of guanidine hydrochloride and combined primer sets to increase speed and sensitivity in colorimetric LAMP, bringing this simple method up to the standards of sophisticated techniques and enabling accurate, high-throughput diagnostics.

scholarly journals Evaluation of Loop-Mediated Isothermal Amplification for Rapid Detection of Erwinia amylovora on Pear and Apple Fruit Flowers

Plant Disease ◽  
2011 ◽  
Vol 95 (4) ◽  
pp. 423-430 ◽  
Author(s):  
Todd N. Temple ◽  
Kenneth B. Johnson
Keyword(s):  
Rapid Detection ◽  
Pathogen Detection ◽  
Fire Blight ◽  
Management Practices ◽  
Erwinia Amylovora ◽  
Lamp Assay ◽  
Apple Fruit ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Forecasting Models

Fire blight of pear and apple is frequently an inoculum-limited disease but weather-based forecasting models commonly assume that the pathogen is omnipresent. To improve fire blight risk assessment during flowering, we developed a rapid pathogen detection protocol that uses loop-mediated isothermal amplification (LAMP) to detect DNA of epiphytic Erwinia amylovora on samples of pear and apple flowers. LAMP detected a single flower colonized epiphytically by E. amylovora in a sample of 100 flower clusters (approximately 600 flowers). Samples of 100 flower clusters from orchards (approximately one sample per hectare) were washed and subjected to LAMP, which was completed in 2 h. In three experimental orchards inoculated with E. amylovora, positive LAMP reactions were attained from nine of nine 100-flower cluster samples; pathogen populations in the floral washes averaged 5.2 × 103 CFU per flower as determined by dilution plating. Samples of pear and apple flowers obtained from 60 commercial orchards located in Oregon, Washington, California, and Utah resulted in detection of E. amylovora by LAMP assay from 34 sites, 20 of which developed fire blight. Of samples at early bloom, 10% were positive for epiphytic E. amylovora compared with 28% at petal fall; pathogen density in washes of positive samples averaged 3.2 × 102 CFU per flower. In another 26 orchards, all floral washes were negative for E. amylovora by LAMP and by dilution plating; a light severity of fire blight was observed in 8 of these orchards. Overall, positive detection of epiphytic E. amylovora in commercial orchards by LAMP-based scouting generally occurred at later stages of bloom after heat (risk) units had begun to accumulate, an indication that weather-based forecasting models may be an adequate measure of fire blight risk for many orchardists. Nonetheless, several orchardists communicated that information from the LAMP-based rapid detection protocol resulted in modification of their fire blight management practices.

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