Species-specific Loop-mediated Isothermal Amplification Assays Research Articles

Loop-mediated isothermal amplification: Development, validation and application of simple and rapid assays for quantitative detection of species of Arcobacteraceae family- and species-specific Aliarcobacter faecis and Aliarcobacter lanthieri.

AimThe family Arcobacteraceae formerly genus Arcobacter has recently been reclassified into six genera. Among nine species of the genus Aliarcobacter, Aliarcobacter faecis and Aliarcobacter lanthieri have been identified as emerging pathogens potentially cause health risks to humans and animals. This study was designed to develop/optimize, validate and apply Arcobacteraceae family‐ and two species‐specific (A. faecis and A. lanthieri) loop‐mediated isothermal amplification (LAMP) assays to rapidly detect and quantify total number of cells in various environmental niches.Methods and ResultsThree sets of LAMP primers were designed from conserved and variable regions of 16S rRNA (family‐specific) and gyrB (species‐specific) genes. Optimized Arcobacteraceae family‐specific LAMP assay correctly amplified and detected 24 species, whereas species‐specific LAMP assays detected A. faecis and A. lanthieri reference strains as well as 91 pure and mixed culture isolates recovered from aquatic and faecal sources. The specificity of LAMP amplification of A. faecis and A. lanthieri was further confirmed by restriction fragment length polymorphism analysis. Assay sensitivities were tested using variable DNA concentrations extracted from simulated target species cells in an autoclaved agricultural water sample by achieving a minimum detection limit of 10 cells mL−1 (10 fg). Direct DNA‐based quantitative detection, from agricultural surface water, identified A. faecis (17%) and A. lanthieri (1%) at a low frequency compared to family‐level (93%) with the concentration ranging from 2·1 × 101 to 2·2 × 105 cells 100 mL−1.ConclusionsOverall, these three DNA‐based rapid and cost‐effective novel LAMP assays are sensitive and can be completed in less than 40 min. They have potential for on‐site quantitative detection of species of family Arcobacteraceae, A. faecis and A. lanthieri in food, environmental and clinical matrices.Significance and Impact of the StudyThe newly developed LAMP assays are specific, sensitive, accurate with higher reproducibility that have potential to facilitate in a less equipped lab setting and can help in early quantitative detection and rate of prevalence in environmental niches. The assays can be adopted in the diagnostic labs and epidemiological studies.

On-Site Detection of Tissues of Buffalo Origin by Loop-Mediated Isothermal Amplification (LAMP) Assay Targeting Mitochondrial Gene Sequences

Meat fraud and adulteration is a growing concern in the marketplace today. Species identification in food product is among primary duties of food office control. In this study, a one-step, species-specific, sensitive, and rapid loop-mediated isothermal amplification (LAMP) assay was developed for identification of tissues of buffalo origin. A set of six LAMP primers were designed by targeting mitochondrial cyt b gene. The reaction temperature of LAMP and component of reaction mixture were optimized as 64 °C amplification temperature, 6 U/μL concentration of Bst DNA polymerase, 3 mM concentration of MgSO4, 0.4 mM concentration of dNTP, 0.6 M concentration of betaine, and 1:6 ratio of outer and inner primer pair. Successful amplifications were confirmed using SYBR Green I dye and agarose gel electrophoresis. The specificity of assay was tested with DNA of cattle, goat, sheep, and pig where amplification was observed exclusively in buffalo. The analytical sensitivity of the LAMP assay for buffalo DNA detection was 0.1 pg. The applicability check of developed assay carried out on known/coded samples and binary meat admixture substantiated the accuracy of assay. Supernatant of tissue exposed to Phire Animal Tissue Direct PCR master mix treatment was observed rich enough with DNA of target species and successfully amplified under optimized LAMP reaction. The comparison of results on the samples undergone pre-standardized species-specific PCR taken as gold standard shown complete match with developed species-specific LAMP assay. Hence, the developed species-specific LAMP assays are effective in detection of tissue of buffalo origin.

Development of Three Loop-Mediated Isothermal Amplification (LAMP) Assays for the Rapid Detection of Calonectria ilicicola, Dactylonectria macrodidyma, and the Dactylonectria Genus in Avocado Roots.

Black root rot of avocado is a severe disease of nursery trees and young orchard transplants, causing tree death within a year after planting. In Australia, key pathogens include species complexes Calonectria ilicicola and Dactylonectria macrodidyma; however, several other Dactylonectria species also cause the disease. Rapid detection of these pathogens in planta is important to speed up implementation of disease management and reduce loss. The purpose of this study was to develop three loop-mediated isothermal amplification (LAMP) diagnostic assays to rapidly identify species within the C. ilicicola and D. macrodidyma complexes and species in the Dactylonectria genus in avocado roots. Primers were designed from β-tubulin sequence data of C. ilicicola and from histone H3 of D. macrodidyma and the Dactylonectria genus. The LAMP primers were tested for specificity and sensitivity with 82 fungal isolates, which included the target species complexes C. ilicicola and D. macrodidyma; species within the target Dactylonectria genus viz. D. macrodidyma, D. anthuriicola, D. novozelandica, D. pauciseptata, and D. vitis; and isolates of nontarget species, including Calonectria sp., Cylindrocladiella sp., Gliocladiopsis forsbergii, G. peggii, G. whileyi, Ilyonectria sp., Mariannaea sp., Fusarium sp., and Phytophthora cinnamomi. The species-specific LAMP assays were sensitive and specific at DNA concentrations of 1 pg/µl for C. ilicicola and 0.01 ng/µl for D. macrodidyma, whereas the Dactylonectria genus-wide assay was sensitive to 0.1 ng/µl. Detection of C. ilicicola occurred within 10 to 15 or 15 to 30 min when the template was pure DNA or crude extracts obtained from suspending fungal cultures in sterile water, respectively. Detection of D. macrodidyma was between 12 to 29 min with pure DNA and 16 to 30 min with crude extracts. Dactylonectria spp. were detected within 6 to 25 min with pure DNA and 7 to 23 min with crude extracts. The specificity of the assays was found to be dependent on time and isothermal amplification temperature, with optimal specificity occurring in reactions of <30 min and at temperatures of 67°C for C. ilicicola and D. macrodidyma assays and 69°C for Dactylonectria genus-wide assays. The assays were modified to accommodate a DNA extraction step and use of avocado roots as DNA templates. Detection in avocado roots ranged between 12 to 25 min for C. ilicicola, 12 to 26 min for D. macrodidyma, and 14 to 30 min for species in the Dactylonectria genus. The LAMP assays are applicable across multiple agricultural industries, because C. ilicicola, D. macrodidyma, and Dactylonectria spp. are also important pathogens of various crops and ornamental plants.

Species-specific loop-mediated isothermal amplification (LAMP) assay for identification of tissue of cattle origin by targeting mitochondrial gene sequences.

The present study was carried out with the objective of development of species-specific loop-mediated isothermal amplification (LAMP) assay for identification of tissue of cattle origin. The cattle-specific LAMP primer set was designed by targeting mitochondrial D-loop gene. The conditions for LAMP reaction for amplification of template DNA from cattle using designed cattle-specific primer set were optimized for the components of mixture and temperature of reaction. Amplified products were analysed using SYBR Green I dye and by agarose gel electrophoresis. The developed species-specific LAMP assay was evaluated for its specificity, sensitivity and validated in laboratory on samples from known, coded, binary meat admixture with other than cattle at relative percentage of 20%, 10%, 5% and 1%, Phire tissue direct PCR master mix treated tissues of cattle and on species-specific polymerase chain reaction assay positive samples. The developed LAMP assay using self-designed primer set was highly specific, amplifying the DNA template exclusively from cattle tissue under the optimized LAMP reaction conditions. The sensitivity assay using serially diluted DNA templates revealed lowest level of detection as 0.01ng of absolute DNA from target species. Laboratory validation substantiated the accuracy of assay in known/unknown (coded) samples and up to the 1% level of admixture in binary meat sample. DNA present in supernatant of Phire Animal tissue kit treated samples were also amplified successfully eliminating the extra step of extraction of genomic DNA. The developed assays exhibited comparable results with previously established species-specific PCR assay taken as gold standards. Thus, it was concluded that developed species-specific loop-mediated isothermal amplification assay was effective in identification of tissue of cattle origin.

Loop-Mediated Isothermal Amplification Assay for Identification of Five Human Plasmodium Species in Malaysia.

The lack of rapid, affordable, and accurate diagnostic tests represents the primary hurdle affecting malaria surveillance in resource- and expertise-limited areas. Loop-mediated isothermal amplification (LAMP) is a sensitive, rapid, and cheap diagnostic method. Five species-specific LAMP assays were developed based on 18S rRNA gene. Sensitivity and specificity of LAMP results were calculated as compared with microscopic examination and nested polymerase chain reaction. LAMP reactions were highly sensitive with the detection limit of one copy for Plasmodium vivax, Plasmodium falciparum, and Plasmodium malariae and 10 copies for Plasmodium knowlesi and Plasmodium ovale. LAMP positively detected all human malaria species in all positive samples (N = 134; sensitivity = 100%) within 35 minutes. All negative samples were not amplified by LAMP (N = 67; specificity = 100%). LAMP successfully detected two samples with very low parasitemia. LAMP may offer a rapid, simple, and reliable test for the diagnosis of malaria in areas where malaria is prevalent.

Genome Filtering for New DNA Biomarkers of Loa loa Infection Suitable for Loop-Mediated Isothermal Amplification.

Loa loa infections have emerged as a serious public health problem in patients co-infected with Onchocerca volvulus or Wuchereria bancrofti because of severe adverse neurological reactions after treatment with ivermectin. Accurate diagnostic tests are needed for careful mapping in regions where mass drug administration is underway. Loop-mediated isothermal amplification (LAMP) has become a widely adopted screening method because of its operational simplicity, rapidity and versatility of visual detection readout options. Here, we present a multi-step bioinformatic pipeline to generate diagnostic candidates suitable for LAMP and experimentally validate this approach using one of the identified candidates to develop a species-specific LAMP assay for L. loa. The pipeline identified ~140 new L. loa specific DNA repeat families as putative biomarkers of infection. The consensus sequence of one family, repeat family 4 (RF4), was compiled from ~ 350 sequences dispersed throughout the L. loa genome and maps to a L. loa-specific region of the long terminal repeats found at the boundaries of Bel/Pao retrotransposons. PCR and LAMP primer sets targeting RF4 specifically amplified L. loa but not W. bancrofti, O. volvulus, Brugia malayi, human or mosquito DNA. RF4 LAMP detects the DNA equivalent of one microfilaria (100 pg) in 25–30 minutes and as little as 0.060 pg of L. loa DNA (~1/1600th of a microfilaria) purified from spiked blood samples in approximately 50 minutes. In summary, we have successfully employed a bioinformatic approach to mine the L. loa genome for species-specific repeat families that can serve as new DNA biomarkers for LAMP. The RF4 LAMP assay shows promise as a field tool for the implementation and management of mass drug administration programs and warrants further testing on clinical samples as the next stage in development towards this goal.

Rapid identification and differentiation of Theileria sergenti and Theileria sinensis using a loop-mediated isothermal amplification (LAMP) assay

The present study developed and validated a species-specific loop-mediated isothermal amplification (LAMP) assay for the rapid detection and discrimination of two benign bovine Theileria species –T. sergenti and T. sinensis. The LAMP assay is inexpensive and easy to perform and involves a rapid reaction-the amplification can be performed in 55min or 50min under isothermal conditions of 61°C or 63°C, respectively, by employing a set of four species-specific primer mixtures. The results can be checked using agarose gels. The optimal assay conditions, under which the assay exhibited with no cross-reaction with other closely related tick-borne parasites (T. annulata, Babesia bovis, B. bigemina, B. major, B. ovata, B. U. sp., Anaplasma marginale) or between the two Theileria species of interest, was established. The assay is approximately 10-fold more sensitive than the conventional specific PCR assay. The LAMP assay was validated using DNA from 6 standard stocks in the laboratory and was evaluated for its diagnostic utility using blood samples collected from experimentally and naturally infection cattle or yaks in China. These findings indicate that this Theileria species-specific LAMP assay may have potential clinical applications for the detection and differentiation of two benign bovine Theileria species –T. sergenti and T. sinensis, especially in endemic countries.

Loop-mediated isothermal amplification (LAMP) method based on two species-specific primer sets for the rapid identification of Chinese Babesia bovis and B. bigemina

Bovine babesiosis is a tick-transmitted hemoprotozoan disease that is mainly caused by Babesia bovis and/or Babesia bigemina and is characterized by significant morbidity and mortality worldwide. This disease is widespread in most parts of China. However, it is difficult to rapidly discriminate between the B. bovis and B. bigemina species. To detect and distinguish these species, a loop-mediated isothermal amplification (LAMP) platform that targets specific sequences of the internal transcribed spacer (ITS) genes was developed. Specificity testing revealed that there was no cross-reaction with the other tick-borne parasites B. ovate, B. major, unnamed bovine Babesia, Theileria annulata, Theileria sinensis, Theileria sergenti, and Anaplasma marginale, or with bovine white blood cells. The sensitivity of the LAMP method was 0.1pg DNA for both B. bovis and B. bigemina, which was superior to that of the classical PCR methods. This assay was evaluated for its diagnostic utility using blood samples collected from experimentally and naturally infected cattle in China. These findings indicate that the Babesia species-specific LAMP assay may have potential clinical application in the detection and differentiation of Babesia species, particularly in countries in which babesiosis is endemic.

Loop-mediated isothermal amplification (LAMP) assays for the species-specific detection of Eimeria that infect chickens

BackgroundEimeria parasites can cause the disease coccidiosis in poultry and even subclinical infection can incur economic loss. Diagnosis of infection predominantly relies on traditional techniques including lesion scoring and faecal microscopy despite the availability of sensitive molecular assays, largely due to cost and the requirement for specialist equipment. Despite longstanding proven efficacy these traditional techniques demand time and expertise, can be highly subjective and may under-diagnose subclinical disease. Recognition of the tight economic margins prevailing in modern poultry production and the impact of avian coccidiosis on poverty in many parts of the world has highlighted a requirement for a panel of straightforward and sensitive, but cost-effective, Eimeria species-specific diagnostic assays.ResultsLoop-mediated isothermal amplification (LAMP) is an uncomplicated, quick and relatively inexpensive diagnostic tool. In this study we have developed a panel of species-specific LAMP assays targeting the seven Eimeria species that infect the chicken. Each assay has been shown to be genuinely species-specific with the capacity to detect between one and ten eimerian genomes, equivalent to less than a single mature schizont. Development of a simple protocol for template DNA preparation from tissue collected post mortem with no requirement for specialist laboratory equipment supports the use of these assays in routine diagnosis of eimerian infection. Preliminary field testing supports this hypothesis.ConclusionsDevelopment of a panel of sensitive species-specific LAMP assays introduces a valuable new cost-effective tool for use in poultry husbandry.

Rapid identification and differentiation of Fasciola hepatica and Fasciola gigantica by a loop-mediated isothermal amplification (LAMP) assay

The present study developed and validated a species-specific loop-mediated isothermal amplification (LAMP) assay for the rapid detection and discrimination of Fasciola hepatica and Fasciola gigantica. The LAMP assay is inexpensive, easy to perform and shows rapid reaction, wherein the amplification can be obtained in 45min under isothermal conditions of 61°C or 62°C by employing a set of four species-specific primer mixtures and results can be checked through naked-eye visualization. The optimal assay conditions with no cross-reaction with other closely related trematodes (Clonorchis sinensis, Opisthorchis viverrini, Orientobilharzia turkestanicum and Schistosoma japonicum) as well as within the two Fasciola species were established. The assay was validated by examining F. gigantica DNA in the intermediate host snails and in faecal samples. The results indicated that the LAMP assay is approximately 104 times more sensitive than the conventional specific PCR assays. These findings indicate that this Fasciola species-specific LAMP assay may have a potential clinical application for detection and differentiation of Fasciola species, especially in endemic countries.