Loop-mediated isothermal amplification assay for the detection of Ehrlichia canis DNA in blood samples from dogs

Faggion, SA; Salvador, Alessia; Jacobino, KL; Bortolotto, L.F.B.; Lopes, M.B.; Silva, M.; Santos, EV; Fachin, AL; França, SC; Marins, Mozart

doi:10.4067/s0301-732x2013000200012

Cited by 12 publications

(14 citation statements)

References 22 publications

(19 reference statements)

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“…The assay used dilutions of plasmid DNA containing a fragment of the p30 gene, and had a detection limit of 1 copy per reaction. This limit is comparable to that of other LAMP assays developed for Anaplasmataceae (Nakao et al, 2010;Ma et al, 2011;Pan et al, 2011;Faggion et al, 2013;Li et al, 2014). The performance of the p30-based LAMP assay for detection of E. canis DNA in 137 field samples was superior to that of the PCR assay, which was based on the amplification of the p30 gene.…”

Section: Discussionmentioning

confidence: 64%

“…We have previously developed a LAMP assay for the detection of E. canis DNA using the groESL operon gene; though this showed a similar sensitivity to the p30-based LAMP assay, its performance in the field samples used in this study were not satisfactory (Faggion et al, 2013). The exact reason for these differences between the PCR and LAMP assays must be further clarified; however, despite many articles in literature claiming the superiority of the LAMP assay over PCR, our results and those of others must be considered with caution when applying this technique to field samples.…”

Section: Discussionmentioning

confidence: 93%

“…In contrast, LAMP is performed in a water bath and the results, which can be observed with the naked eye without the need for a specialized gel electrophoresis unit, are obtained within 1 h (Mori and Notomi, 2009). Since its introduction, LAMP has been used in a wide range of applications, including the detection of canine pathogens (Adaszek et al, 2013;Chaouch et al, 2013;Faggion et al, 2013). In this respect, LAMP may be a useful tool for monitoring the treatment of CME in dogs in countries like Brazil, where the disease is endemic and highly prevalent, and veterinary laboratories often have limited access to sophisticated equipment.…”

Section: Discussionmentioning

confidence: 99%

“…The sensitivity of the assay was evaluated as described in a previous study (Faggion et al, 2013). Briefly, the fragment of the p30 gene encompassing the region amplified by the LAMP reaction was amplified by PCR using the P30-1SPF and P30-1SPR primers, using the ReadyMix™ Taq PCR Reaction Mix (Sigma-Aldrich) according to the manufacturer protocols; the following reaction condition were employed: 40 cycles of denaturation at 95°C for 45 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. The amplicons were cloned using the pGEM-T easy vector system (Promega, Madison, WI, USA).…”

Section: Detection Threshold and Specificity Of The Lamp Assaymentioning

confidence: 99%

“…However, despite the high specificity and sensitivity of nested PCR, they are often criticized because of the time-consuming steps and high risk of cross-contamination, which cause false-positive results (Labruna et al, 2007). Recently, loop-mediated isothermal DNA amplification (LAMP) has been successfully applied to the detection of different pathogens, including E. canis and other related rickettsial agents (Nakao et al, 2010;Ma et al, 2011;Faggion et al, 2013). LAMP assays are highly sensitive and specific, and permit rapid generation and visualization of results (Notomi et al, 2000;Francois et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

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Development and evaluation of a loop-mediated isothermal amplification assay for detection of Ehrlichia canis DNA in naturally infected dogs using the p30 gene

et al. 2015

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ABSTRACT. Canine monocytic ehrlichiosis (CME) is a common tick-borne disease caused by the rickettsial bacterium Ehrlichia canis (Rickettsiales: Anaplasmataceae). In view of the different stages and variable clinical signs of CME, which can overlap with those of other infections, a conclusive diagnosis can more readily be obtained by combining clinical and hematological evaluations with molecular diagnostic methods. In this study, a loop-mediated isothermal amplification (LAMP) assay targeting the p30 gene of E. canis was developed. The assay was developed using DNA extracted from E. canis-infected cultures of the macrophage cell line DH82 and samples from dogs testing positive for E. canis DNA by PCR. The LAMP assay was compared to a p30-based PCR assay, using DNA extracted from EDTA-anticoagulated blood samples of 137 dogs from an endemic region in Brazil. The LAMP assay was sensitive enough to detect a single copy of the target gene, and identified 74 (54.0%) E. canis DNA- (2015) positive samples, while the p30 PCR assay detected 50 positive samples (36.5%) among the field samples. Agreement between the two assays was observed in 42 positive and 55 negative samples. However, 32 positive samples that were not detected by the PCR assay were identified by the LAMP assay, while eight samples identified as E. canis-positive by PCR showed negative results in LAMP. The developed E. canis LAMP assay showed the potential to maximize the use of nucleic acid tests in a veterinary clinical laboratory, and to improve the diagnosis of CME.

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Section: Discussionmentioning

confidence: 64%

Section: Discussionmentioning

confidence: 93%

Section: Discussionmentioning

confidence: 99%

Section: Detection Threshold and Specificity Of The Lamp Assaymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Development and evaluation of a loop-mediated isothermal amplification assay for detection of Ehrlichia canis DNA in naturally infected dogs using the p30 gene

et al. 2015

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Tick-borne zoonoses and commonly used diagnostic methods in human and veterinary medicine

et al. 2021

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Around the world, human health and animal health are closely linked in terms of the One Health concept by ticks acting as vectors for zoonotic pathogens. Animals do not only maintain tick cycles but can either be clinically affected by the same tick-borne pathogens as humans and/or play a role as reservoirs or sentinel pathogen hosts. However, the relevance of different tick-borne diseases (TBDs) may vary in human vs. veterinary medicine, which is consequently reflected by the availability of human vs. veterinary diagnostic tests. Yet, as TBDs gain importance in both fields and rare zoonotic pathogens, such as Babesia spp., are increasingly identified as causes of human disease, a One Health approach regarding development of new diagnostic tools may lead to synergistic benefits. This review gives an overview on zoonotic protozoan, bacterial and viral tick-borne pathogens worldwide, discusses commonly used diagnostic techniques for TBDs, and compares commercial availability of diagnostic tests for humans vs. domestic animals, using Germany as an example, with the aim of highlighting existing gaps and opportunities for collaboration in a One Health framework.

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An innovative and user-friendly smartphone-assisted molecular diagnostic approach for rapid detection of canine vector-borne diseases

et al. 2021

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Present-day diagnostic tools and technologies for canine diseases and other vector-borne parasitic diseases hardly meet the requirements of an efficient and rapid diagnostic tool, which can be suitable for use at the point-of-care in resource-limited settings. Loop-mediated isothermal amplification (LAMP) technique has been always a method of choice in the development and validation of quick, precise, and sensitive diagnostic assays for pathogen detection and to reorganize point-of-care (POC) molecular diagnostics. In this study, we have demonstrated an efficient detection system for parasitic vector-borne pathogens like Ehrlichia canis and Hepatozoon canis by linking the LAMP assay to a smartphone via a simple, inexpensive, and a portable “LAMP box,” All the components of the LAMP box were connected to each other wirelessly. This LAMP box was made up of an isothermal heating pad mounted below an aluminum base which served as a platform for the reaction tubes and LAMP assay. The entire setup could be connected to a smartphone via an inbuilt Wi-Fi that allowed the user to establish the connection to control the LAMP box. A 5 V USB power source was used as a power supply. The sensitivity of the LAMP assay was estimated to be up to 10 −6 dilution limit using the amplified, purified, and quantified specific DNA templates. It can also serve as an efficient diagnostic platform for many other veterinary infectious or parasitic diseases of zoonotic origin majorly towards field-based diagnostics. Supplementary Information The online version contains supplementary material available at 10.1007/s00436-021-07077-z.

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Loop-mediated isothermal amplification assay for the detection of Ehrlichia canis DNA in blood samples from dogs

Cited by 12 publications

References 22 publications

Development and evaluation of a loop-mediated isothermal amplification assay for detection of Ehrlichia canis DNA in naturally infected dogs using the p30 gene

Development and evaluation of a loop-mediated isothermal amplification assay for detection of Ehrlichia canis DNA in naturally infected dogs using the p30 gene

Tick-borne zoonoses and commonly used diagnostic methods in human and veterinary medicine

An innovative and user-friendly smartphone-assisted molecular diagnostic approach for rapid detection of canine vector-borne diseases

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