Fast multiplex real-time PCR assay for simultaneous detection of dog and human blood and Leishmania parasites in sand flies

Sales, Kamila Gaudêncio da Silva; Miranda, Débora Elienai de Oliveira; Paiva, Marcelo Henrique Santos; Figueredo, Luciana Aguiar; Otranto, Domenico; Dantas‐Torres, Filipe

doi:10.1186/s13071-020-3994-6

Cited by 12 publications

(10 citation statements)

References 34 publications

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“…The extracted RNA samples were subjected to cDNA synthesis using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, California, USA). To confirm the presence and integrity of the extracted RNA, dog-specific cytochrome oxidase subunit 1 (COX-1) amplification was performed (Sales et al, 2020). In the case of cats, after cDNA synthesis, to check the RNA quality, PCR was performed for the endogenous control, COX-1, by conventional PCR (cPCR) using the primers and conditions of Hafner et al (1994).…”

mentioning

confidence: 99%

No molecular evidence of SARS‐CoV‐2 infection in companion animals from Veracruz, Mexico

Sánchez‐Montes

Ballados‐González

Gamboa-Prieto

et al. 2021

Transbounding Emerging Dis

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Active epidemiological surveillance of infectious agents represents a fundamental tool for understanding the transmission dynamics of pathogens and establishing public policies that can reduce or limit their expansion. Epidemiological surveillance of emerging agents, such as the recently recognized severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), the cause of COVID‐19, is essential to establish the risk of transmission between species. Recent studies reveal that companion animals are organisms susceptible to being infected by this pathogen due to the close contact they have with their owners. For this reason, the aim of the present work was to detect the presence of SARS‐CoV‐2 in dogs and cats in the state of Veracruz, Mexico, where there is active transmission of this microorganism in human populations. Oral and nasopharyngeal swab samples were collected from dogs and cats with a history of exposure to patients with COVID‐19. Total RNA was extracted and detection of viral genes N1 and N2 was performed by reverse transcription polymerase chain reaction (RT‐qPCR). All 130 samples of companion animals tested by RT‐qPCR for SARS‐CoV‐2 were negative at the time they were collected. This study represents the second active surveillance of SARS‐CoV‐2 in populations of domestic dogs and cats in Latin America and the first approach in Mexico. Given that coronaviruses have shown a high capacity to be transmitted between species, it is imperative to establish measures to prevent this agent from entering and establishing in populations of companion animals.

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mentioning

confidence: 99%

No molecular evidence of SARS‐CoV‐2 infection in companion animals from Veracruz, Mexico

Sánchez‐Montes

Ballados‐González

Gamboa-Prieto

et al. 2021

Transbounding Emerging Dis

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“…In addition, the integrity of the extracted DNA at all storage times was confirmed by the amplification of CAC and cox 1 genes by PCR in 99.6% of the cases; only one out of 250 DNA extracts failed to amplify the CAC gene (Table 1 ), which suggests that DNA extraction was not successful for this sample. More recently, a fast multiplex real-time PCR assay for simultaneous detection of blood meals and Leishmania parasites in female phlebotomine sand flies demonstrated promising results using specimens that were stored in 70% ethanol for approximately two years [ 20 , 33 ]. Interestingly, female phlebotomine sand flies stored at − 20 °C for approximately four years were successfully used to detect host DNA through real-time PCR assays [ 34 ].…”

Section: Discussionmentioning

confidence: 99%

Evaluation of different storage times and preservation methods on phlebotomine sand fly DNA concentration and purity

et al. 2020

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Background: Different methods have been used to preserve phlebotomine sand flies for research purposes, including for taxonomic studies and detection of Leishmania spp. Here, we evaluated the effect of various preservation methods at different storage times on phlebotomine sand fly DNA concentration and purity. Methods: Field-collected phlebotomine sand flies were individually stored in 70% ethanol (G1) and 95% ethanol (G2) at room temperature, 70% ethanol (G3) and 95% ethanol (G4) at 8 °C or frozen dry (i.e. no preservation solution) at − 20 °C (G5). DNA concentration and purity were assessed at various storage times (T1, ≤ 12 h; T2, 3 months; T3, 6 months; T4, 9 months; and T5, 12 months). Fragments of the cytochrome c oxidase subunit 1 (cox1) and cacophony (CAC) genes of phlebotomine sand flies were also amplified. Results: Mean DNA concentration (P = 0.178) and 260/280 purity ratios (P = 0.584) did not vary significantly among various preservation methods and storage times. Within each group, DNA concentration varied in G1 (Kruskal-Wallis H-test, P = 0.009) for T3 vs T4 (Dunn's post-hoc, P < 0.05), and in G2 (Kruskal-Wallis H-test, P = 0.004) for T1 vs T2 and T1 vs T4 (Dunn's post-hoc, P < 0.05). For 260/280 purity ratios, the only statistically significant difference was found for G5 (Kruskal-Wallis H-test, P = 0.020) between T1 vs T4 (Dunn's post-hoc test, P < 0.05). The cox1 and CAC genes were successfully amplified, regardless of the preservation method and storage time; except in one sample from G2 at T1, for which the CAC gene failed to amplify. Conclusions: The preservation methods and storage times herein evaluated did not affect the concentration and purity of DNA samples obtained from field-collected phlebotomine sand flies, for up to 12 months. Furthermore, these preservation methods did not interfere with PCR amplification of CAC and cox1 genes, being suitable for molecular analyses under the conditions studied herein.

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“…These assays are limited by primer sets selected in advance to amplify the DNA of known hosts; they are not useful for detection of blood from uncommon or unexpected hosts. Regardless of this limitation, recent studies utilizing real-time PCR technology were able to easily identify multi-host bloodmeals from mosquitoes and sand flies [ 94 , 149 , 150 ], and assays proved to be extremely specific and had limits of detection below one picogram of DNA for flea bloodmeals [ 151 ]. Interestingly, the bloodmeal hosts of ticks were detectable for up to 8 months after feeding, though in a low percentage of samples [ 14 ].…”

Section: Molecular Advancesmentioning

confidence: 99%

Modernizing the Toolkit for Arthropod Bloodmeal Identification

Borland

Kading

2021

Insects

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Understanding vertebrate–vector interactions is vitally important for understanding the transmission dynamics of arthropod-vectored pathogens and depends on the ability to accurately identify the vertebrate source of blood-engorged arthropods in field collections using molecular methods. A decade ago, molecular techniques being applied to arthropod blood meal identification were thoroughly reviewed, but there have been significant advancements in the techniques and technologies available since that time. This review highlights the available diagnostic markers in mitochondrial and nuclear DNA and discusses their benefits and shortcomings for use in molecular identification assays. Advances in real-time PCR, high resolution melting analysis, digital PCR, next generation sequencing, microsphere assays, mass spectrometry, and stable isotope analysis each offer novel approaches and advantages to bloodmeal analysis that have gained traction in the field. New, field-forward technologies and platforms have also come into use that offer promising solutions for point-of-care and remote field deployment for rapid bloodmeal source identification. Some of the lessons learned over the last decade, particularly in the fields of DNA barcoding and sequence analysis, are discussed. Though many advancements have been made, technical challenges remain concerning the prevention of sample degradation both by the arthropod before the sample has been obtained and during storage. This review provides a roadmap and guide for those considering modern techniques for arthropod bloodmeal identification and reviews how advances in molecular technology over the past decade have been applied in this unique biomedical context.

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Fast multiplex real-time PCR assay for simultaneous detection of dog and human blood and Leishmania parasites in sand flies

Cited by 12 publications

References 34 publications

No molecular evidence of SARS‐CoV‐2 infection in companion animals from Veracruz, Mexico

No molecular evidence of SARS‐CoV‐2 infection in companion animals from Veracruz, Mexico

Evaluation of different storage times and preservation methods on phlebotomine sand fly DNA concentration and purity

Modernizing the Toolkit for Arthropod Bloodmeal Identification

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