scholarly journals Development of a New LAMP Assay for the Detection of Ancylostoma caninum DNA (Copro-LAMPAc) in Dog Fecal Samples

2021 ◽  
Vol 8 ◽  
Author(s):  
Héctor Gabriel Avila ◽  
Marikena Guadalupe Risso ◽  
Marta Cabrera ◽  
Paula Ruybal ◽  
Silvia Analía Repetto ◽  
...  
Keyword(s):  
Gold Standard ◽  
Genomic Dna ◽  
Low Cost ◽  
Lamp Assay ◽  
Epidemiological Studies ◽  
Lamp Method ◽  
Analytical Sensitivity ◽  
Ancylostoma Caninum ◽  
Fecal Samples ◽  
Screening And Identification

Ancylostoma caninum is a zoonotic nematode which is able to affect animals and humans. Diagnosis in the definitive host and environmental detection are key to prevent its dissemination and achieve control. Herein, a new coprological LAMP method for the detection of A. caninum (Copro-LAMPAc) DNA was developed. DNA extraction was performed using a low-cost method and a fragment of the cox-1 gene was used for primer design. The analytical sensitivity, evaluated with serial dilutions of genomic DNA from A. caninum adult worms, was 100 fg. A specificity of 100% was obtained using genomic DNA from the host and other pathogens. The Copro-LAMPAc was evaluated using environmental canine fecal samples. When compared with gold standard optical microscopy in epidemiological studies, it proved to be more sensitive. This new LAMP assay can provide an alternative protocol for screening and identification of A. caninum for epidemiological studies in endemic areas.

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.

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.

scholarly journals Development of a LAMP Method for Detecting SDHI Fungicide Resistance in Botrytis cinerea

Plant Disease ◽  
2018 ◽  
Vol 102 (8) ◽  
pp. 1612-1618 ◽  
Author(s):  
F. Fan ◽  
W. X. Yin ◽  
G. Q. Li ◽  
Y. Lin ◽  
C. X. Luo
Keyword(s):  
Succinate Dehydrogenase ◽  
Botrytis Cinerea ◽  
High Efficiency ◽  
Color Change ◽  
Low Cost ◽  
Point Mutations ◽  
Lamp Assay ◽  
High Specificity ◽  
Lamp Method ◽  
Development Of Resistance

Resistance to succinate dehydrogenase inhibitors (SDHI) in Botrytis cinerea is associated with point mutations in the target gene succinate dehydrogenase subunit B (SdhB). The substitution from histidine to arginine at codon 272 (H272R) is currently the predominant mutation in SDHI-resistant populations in B. cinerea worldwide. In order to monitor the development of resistance to SDHI, a rapid, simple, and efficient method with high specificity to the H272R point mutation was developed based on loop-mediated isothermal amplification (LAMP). To specifically detect the H272R mutation, a set of four primers was designed based on the sequence of SdhB, and the LAMP reaction was optimized. When SYBR Green I was added after reaction, only samples with the H272R mutation showed the color change (from brown to fluorescent yellow), indicating that this set of primers could successfully discriminate the H272R genotype from other genotypes. Specificity and accuracy tests showed that this LAMP assay had high specificity and accuracy. Moreover, the LAMP method was further simplified with fungal mycelia and conidia as the amplification template which could be prepared within 5 min. Due to the low cost, simplicity, high efficiency, and specificity, the developed LAMP assay may contribute to the monitoring of resistance development to SDHI in B. cinerea, especially in field and high-throughput experiments.

scholarly journals Rapid Detection of Pityophthorus juglandis (Blackman) (Coleoptera, Curculionidae) with the Loop-Mediated Isothermal Amplification (LAMP) Method

Plants ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1048
Author(s):  
Domenico Rizzo ◽  
Salvatore Moricca ◽  
Matteo Bracalini ◽  
Alessandra Benigno ◽  
Umberto Bernardo ◽  
...  
Keyword(s):  
Early Detection ◽  
Low Cost ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Limit Values ◽  
Loop Mediated Isothermal Amplification ◽  
Wood Processing ◽  
Pityophthorus Juglandis ◽  
Specificity And Sensitivity

The walnut twig beetle Pityophthorus juglandis is a phloem-boring bark beetle responsible, in association with the ascomycete Geosmithia morbida, for the Thousand Cankers Disease (TCD) of walnut trees. The recent finding of TCD in Europe prompted the development of effective diagnostic protocols for the early detection of members of this insect/fungus complex. Here we report the development of a highly efficient, low-cost, and rapid method for detecting the beetle, or even just its biological traces, from environmental samples: the loop-mediated isothermal amplification (LAMP) assay. The method, designed on the 28S ribosomal RNA gene, showed high specificity and sensitivity, with no cross reactivity to other bark beetles and wood-boring insects. The test was successful even with very small amounts of the target insect’s nucleic acid, with limit values of 0.64 pg/µL and 3.2 pg/µL for WTB adults and frass, respectively. A comparison of the method (both in real time and visual) with conventional PCR did not display significant differences in terms of LoD. This LAMP protocol will enable quick, low-cost, and early detection of P. juglandis in areas with new infestations and for phytosanitary inspections at vulnerable sites (e.g., seaports, airports, loading stations, storage facilities, and wood processing companies).

scholarly journals Rapid and Visual Identification of Chlorophyllum molybdites With Loop-Mediated Isothermal Amplification Method

2021 ◽  
Vol 12 ◽  
Author(s):  
Nan Wang ◽  
Zhiyong Zhao ◽  
Jie Gao ◽  
Enjing Tian ◽  
Wenjie Yu ◽  
...  
Keyword(s):  
Color Change ◽  
Low Cost ◽  
Food Poisoning ◽  
Lamp Assay ◽  
Forensic Analysis ◽  
Isothermal Amplification ◽  
Mushroom Poisoning ◽  
Lamp Method ◽  
Mushroom Species ◽  
Loop Mediated Isothermal Amplification

Chlorophyllum molybdites is a kind of common poisonous mushroom in China that is widely distributed in different areas. Food poisoning caused by accidentally eating C. molybdites has become more frequent in recent years. In 2019, there were 55 food poisoning incidents caused by eating this mushroom in China. Mushroom poisoning continues to be a common health issue of global concern. When mushroom poisoning occurs, an effective, simple, and rapid detection method is required for accurate clinical treatment or forensic analysis. For the first time, we established a loop-mediated isothermal amplification (LAMP) assay for the visual detection of C. molybdites. A set of specific LAMP primers was designed, and the specificity was confirmed against 43 different mushroom species. The LAMP method could detect as low as 1 pg of genomic DNA. Boiled mushrooms and artificial gastric-digested mushroom samples were prepared to test the applicability of the method, and the results showed that as low as 1% C. molybdites in boiled and digested samples could be successfully detected. The LAMP method can also be completed within 45 min, and the reaction results could be directly observed based on a color change under daylight by the naked eye. Therefore, the LAMP assay established in this study provides an accurate, sensitive, rapid, and low-cost method for the detection of C. molybdites.

scholarly journals A new hybrid record linkage process to make epidemiological databases interoperable: application to the GEMO and GENEPSO studies involving BRCA1 and BRCA2 mutation carriers

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Yue Jiao ◽  
Fabienne Lesueur ◽  
Chloé-Agathe Azencott ◽  
Maïté Laurent ◽  
Noura Mebirouk ◽  
...  
Keyword(s):  
Record Linkage ◽  
Gold Standard ◽  
Brca2 Mutation ◽  
Epidemiological Studies ◽  
Supervised Machine Learning ◽  
Training Dataset ◽  
Support Vector ◽  
Genetic Modifiers ◽  
Brca1 And Brca2 ◽  
Mutation Carriers

Abstract Background Linking independent sources of data describing the same individuals enable innovative epidemiological and health studies but require a robust record linkage approach. We describe a hybrid record linkage process to link databases from two independent ongoing French national studies, GEMO (Genetic Modifiers of BRCA1 and BRCA2), which focuses on the identification of genetic factors modifying cancer risk of BRCA1 and BRCA2 mutation carriers, and GENEPSO (prospective cohort of BRCAx mutation carriers), which focuses on environmental and lifestyle risk factors. Methods To identify as many as possible of the individuals participating in the two studies but not registered by a shared identifier, we combined probabilistic record linkage (PRL) and supervised machine learning (ML). This approach (named “PRL + ML”) combined together the candidate matches identified by both approaches. We built the ML model using the gold standard on a first version of the two databases as a training dataset. This gold standard was obtained from PRL-derived matches verified by an exhaustive manual review. Results The Random Forest (RF) algorithm showed a highest recall (0.985) among six widely used ML algorithms: RF, Bagged trees, AdaBoost, Support Vector Machine, Neural Network. Therefore, RF was selected to build the ML model since our goal was to identify the maximum number of true matches. Our combined linkage PRL + ML showed a higher recall (range 0.988–0.992) than either PRL (range 0.916–0.991) or ML (0.981) alone. It identified 1995 individuals participating in both GEMO (6375 participants) and GENEPSO (4925 participants). Conclusions Our hybrid linkage process represents an efficient tool for linking GEMO and GENEPSO. It may be generalizable to other epidemiological studies involving other databases and registries.

Development of a low-cost copro-LAMP assay for simultaneous copro-detection of Toxocara canis and Toxocara cati

Parasitology ◽  
2021 ◽  
pp. 1-8
Author(s):  
Héctor Gabriel Avila ◽  
Marikena Guadalupe Risso ◽  
Paula Ruybal ◽  
Silvia Analía Repetto ◽  
Marcos Javier Butti ◽  
...  
Keyword(s):  
Low Cost ◽  
Lamp Assay ◽  
Toxocara Canis ◽  
Toxocara Cati

Abstract

scholarly journals Development of a loop-mediated isothermal amplification assay for the detection of Tilletia controversa based on genome comparison

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Somayyeh Sedaghatjoo ◽  
Monika K. Forster ◽  
Ludwig Niessen ◽  
Petr Karlovsky ◽  
Berta Killermann ◽  
...  
Keyword(s):  
Test Performance ◽  
Genomic Dna ◽  
Lamp Assay ◽  
Fungal Species ◽  
Isothermal Amplification ◽  
Genome Comparison ◽  
Performance Study ◽  
Loop Mediated Isothermal Amplification ◽  
Tilletia Controversa ◽  
Genomic Regions

AbstractTilletia controversa causing dwarf bunt of wheat is a quarantine pathogen in several countries. Therefore, its specific detection is of great phytosanitary importance. Genomic regions routinely used for phylogenetic inferences lack suitable polymorphisms for the development of species-specific markers. We therefore compared 21 genomes of six Tilletia species to identify DNA regions that were unique and conserved in all T. controversa isolates and had no or limited homology to other Tilletia species. A loop-mediated isothermal amplification (LAMP) assay for T. controversa was developed based on one of these DNA regions. The specificity of the assay was verified using 223 fungal samples comprising 43 fungal species including 11 Tilletia species, in particular 39 specimens of T. controversa, 92 of T. caries and 40 of T. laevis, respectively. The assay specifically amplified genomic DNA of T. controversa from pure cultures and teliospores. Only Tilletia trabutii generated false positive signals. The detection limit of the LAMP assay was 5 pg of genomic DNA per reaction. A test performance study that included five laboratories in Germany resulted in 100% sensitivity and 97.7% specificity of the assay. Genomic regions, specific to common bunt (Tilletia caries and Tilletia laevis together) are also provided.

scholarly journals A set of simple methods for detection and extraction of laminarinase

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ananthamurthy Koteshwara ◽  
Nancy V. Philip ◽  
Jesil Mathew Aranjani ◽  
Raghu Chandrashekhar Hariharapura ◽  
Subrahmanyam Volety Mallikarjuna
Keyword(s):  
Ammonium Sulfate ◽  
Ammonium Sulphate ◽  
Gold Standard ◽  
Direct Detection ◽  
Low Cost ◽  
Direct Analysis ◽  
Reducing Sugar ◽  
Ammonium Sulfate Precipitation ◽  
Simple Method ◽  
Functional Step

AbstractA carefully designed ammonium sulfate precipitation will simplify extraction of proteins and is considered to be a gold standard among various precipitation methods. Therefore, optimization of ammonium sulfate precipitation can be an important functional step in protein purification. The presence of high amounts of ammonium sulphate precludes direct detection of many enzymatically active proteins including reducing sugar assays (e.g. Nelson-Somogyi, Reissig and 3,5-dinitrosalicylic acid methods) for assessing carbohydrases (e.g. laminarinase (β (1–3)-glucanohydrolase), cellulases and chitinases). In this study, a simple method was developed using laminarin infused agarose plate for the direct analysis of the ammonium sulphate precipitates from Streptomyces rimosus AFM-1. The developed method is simple and convenient that can give accurate results even in presence of ammonium sulfate in the crude precipitates. Laminarin is a translucent substrate requiring the use of a stain to visualize the zones of hydrolysis in a plate assay. A very low-cost and locally available fluorescent optical fabric brightener Tinopal CBS-X has been used as a stain to detect the zones of hydrolysis. We also report simple methods to prepare colloidal chitin and cell free supernatant in this manuscript.

scholarly journals A20 DIAGNOSTIC ACCURACY OF A LOW COST, WIDELY AVAILABLE TEST STRIP FOR PREDICTING A POSITIVE FECAL CALPROTECTIN TEST

2021 ◽  
Vol 4 (Supplement_1) ◽  
pp. 210-212
Author(s):  
R Trasolini ◽  
S Wong ◽  
B Salh
Keyword(s):  
Sample Size ◽  
Likelihood Ratio ◽  
Gold Standard ◽  
Low Cost ◽  
Positive Likelihood Ratio ◽  
Negative Likelihood Ratio ◽  
Rapid Test ◽  
Test Strip ◽  
Fecal Calprotectin ◽  
Type I

Abstract Background Fecal calprotectin is a non-invasive test of colonic inflammation used for monitoring inflammatory bowel disease activity and for risk stratifying non-specific colonic symptoms. Calprotectin is a leukocyte specific enzyme. A similar test, leukocyte esterase is used to detect leukocytes in urine and is widely available as a low-cost point-of-care test strip. We hypothesize that an unmodified version of the urine test strip would be highly accurate in predicting a positive fecal calprotectin test in a real world sample of patients. Aims To explore a low cost, rapid alternative to the fecal calprotectin test Methods All inpatient and outpatient stool samples tested for calprotectin by the Vancouver General Hospital laboratory from February 2020 to November 2020 were included prospectively. Samples were simultaneously tested for fecal leukocyte esterase using an unmodified Roche Cobas Chemstrip urinalysis test strip by central lab personnel. An identical aliquot was sent to LifeLabs for calprotectin as per standard protocol. All samples were suspended in buffer using established laboratory protocols prior to testing. Fecal leukocyte esterase results were reported as 0–4+ based on visual interpretation, calprotectin results were reported as mcg/g of stool. REB review and approval was obtained prior to data collection. Sensitivity, Specificity and AUROC were calculated using Microsoft Excel and JROCFIT. Results 26 samples were collected. Using a fecal calprotectin greater than 120 mcg/g as a gold standard an AUROC of 0.89 (SE= .06) was calculated. A leukocyte esterase reading of 2+ or greater had the best test characteristics based on ROC curve analysis. Using this cutoff, 21/26 samples were concordant, giving an accuracy of 80.8%, sensitivity of 90.9% and specificity of 73.3%. Positive likelihood ratio was 8.07 and negative likelihood ratio was 0.29. Assuming an AUROC of 0.8, the sample size N=26 is 90% powered (β=0.9) to predict the true AUROC within 0.1 with a type I error rate of .05 (α<.05). Conclusions This study suggests application of a prepared stool sample to a urinalysis test strip gives a result highly predictive of a positive fecal calprotectin test. Further results are being collected prospectively to improve the robustness of these preliminary data. Secondary outcomes including comparison to endoscopy and biopsy results where available are planned if an adequate sample size can be accrued. Future studies justifying independent clinical use of leukocyte esterase would require a common gold standard comparator such as endoscopy. Fecal calprotectin testing is not universally insured and is not available as a rapid test strip. Use of fecal leukocyte esterase may reduce costs and shorten time to results if proven to be independently reliable. Funding Agencies None

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