Simple protocol for population (Sanger) sequencing for Zika virus genomic regions

Cabral, Gabriela Bastos; Ferreira, João Leandro de Paula; Souza, Renato Pereira de; Cunha, Mariana Sequetin; Luchs, Adriana; Figueiredo, Cristina; Brígido, Luis Fernando de Macedo

doi:10.1590/0074-02760170248

Cited by 5 publications

(3 citation statements)

References 22 publications

(36 reference statements)

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“…Patients living in an endemic area with suspected clinical manifestations of early “Lyme disease”, such as unexplained sudden headache, low-grade fever, chills, muscle aches, and lymphadenopathy, with or without a skin rash, should be tested for spirochetemia at a local hospital laboratory, rather than having the blood sample sent to geographically distant commercial laboratories for a possible diagnosis of bacteremia. In the past 40 years, Lyme disease has not been seriously studied as an emerging infectious disease among patients as for other newly emerging infectious diseases, such as Ebola [67] and Zika [68] which were diagnosed by nucleic acid amplification tests from the start [67,68]. The laboratory criteria of Ebola virus disease case definition for reporting in the European Union is “Detection of Ebola virus nucleic acid in a clinical specimen and confirmation by sequencing or a second assay on different genomic targets; or isolation of Ebola virus from a clinical specimen” [69].…”

Section: Discussionmentioning

confidence: 99%

Single Core Genome Sequencing for Detection of both Borrelia burgdorferi Sensu Lato and Relapsing Fever Borrelia Species

Lee

Healy

Lambert

2019

IJERPH

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Lyme disease, initially described as Lyme arthritis, was reported before nucleic-acid based detection technologies were available. The most widely used diagnostic tests for Lyme disease are based on the serologic detection of antibodies produced against antigens derived from a single strain of Borrelia burgdorferi. The poor diagnostic accuracy of serological tests early in the infection process has been noted most recently in the 2018 Report to Congress issued by the U.S. Department of Health and Human Services Tick-Borne Disease Working Group. Clinical Lyme disease may be caused by a diversity of borreliae, including those classified as relapsing fever species, in the United States and in Europe. It is widely accepted that antibiotic treatment of Lyme disease is most successful during this critical early stage of infection. While genomic sequencing is recognized as an irrefutable direct detection method for laboratory diagnosis of Lyme borreliosis, development of a molecular diagnostic tool for all clinical forms of borreliosis is challenging because a “core genome” shared by all pathogenic borreliae has not yet been identified. After a diligent search of the GenBank database, we identified two highly conserved segments of DNA sequence among the borrelial 16S rRNA genes. We further developed a pair of Borrelia genus-specific PCR primers for amplification of a segment of borrelial 16S rRNA gene as a “core genome” to be used as the template for routine Sanger sequencing-based metagenomic direct detection test. This study presented examples of base-calling DNA sequencing electropherograms routinely generated in a clinical diagnostic laboratory on DNA extracts of human blood specimens and ticks collected from human skin bites and from the environment. Since some of the tick samples tested were collected in Ireland, borrelial species or strains not known to exist in the United States were also detected by analysis of this 16S rRNA “core genome”. We recommend that hospital laboratories located in Lyme disease endemic areas begin to use a “core genome” sequencing test to routinely diagnose spirochetemia caused by various species of borreliae for timely management of patients at the early stage of infection.

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Section: Discussionmentioning

confidence: 99%

Single Core Genome Sequencing for Detection of both Borrelia burgdorferi Sensu Lato and Relapsing Fever Borrelia Species

Lee

Healy

Lambert

2019

IJERPH

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“…As control of species and internal control, complementary primers were used to a sequence of a mitochondrial gene mtDNA from bats [21], as a positive control of the lyophilized vaccine prepared with the attenuated virus of equine encephalomyelitis TC83 was used; molecular water grade as a negative control was used. The positive samples were reamplified, and the amplicons were sequenced using the Sanger method [22]. For the analysis of pathological anatomy and immunohistochemistry, positive and negative samples were included using molecular biology.…”

Section: Molecular Methodsmentioning

confidence: 99%

Molecular and cellular evidence of natural Venezuelan equine encephalitis virus infection in frugivorous bats in Colombia

et al. 2020

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Background and Aim: Venezuelan equine encephalitis virus (VEEV) is an alphavirus that causes encephalitis with a high impact on public health in Latin America. However, only in Guatemala, Trinidad and Tobago, and Mexico have found antibodies in VEEV in bats, using immunohistochemistry, the sensitivity and specificity are improved; thus, it is better for demonstrating natural infection in bats as potential hosts. This study aimed to determine the presence of VEEV in tissues of frugivorous bats. Materials and Methods: A prospective descriptive cross-sectional study with a non-probabilistic sampling was carried out in 12 localities of Córdoba and Sucre area of the Colombian Caribbean. Two hundred and eighty-six bats were captured using fog nets, and the specimens according to taxonomic keys were classified. According to the Ethics Committee of the University of Córdoba, the bats were treated with analgesics and anesthetics. Blood samples were taken and then euthanized to obtain tissues and organs which were preserved in liquid N2 at –196°C. A portion of each organ was fixed in 10% buffered formalin for the detection of antigens by immunohistochemistry. Several pathological anatomy analyses were performed to determine the histological characteristics of tissue lesions of frugivorous bats naturally infected with the VEEV. Results: Of the 286 bats captured, 23 species were identified. In samples of the brain, spleen, and lung of two frugivorous bats (2/286=0.70%) Artibeus planirostris and Sturnira lilium, the presence of VEEV was confirmed by immunohistochemistry. Conclusion: A fragment of the nsP4 non-structural protein gene corresponding to the alphavirus was amplified. Two samples were positive (2/286=0.70%) in frugivorous bats; A. planirostris (code GenBank: MG820274) and S. lilium (code GenBank: MG820275). The present study showed the first molecular evidence and cellular evidence (histopathology and immunohistochemistry) of natural VEEV infection in frugivorous bats in Colombia; these bats could be a host of this zoonosis.

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“…Nevertheless, Sanger sequencing continues to be used for whole genome sequencing of pathogens with small genomes, such as humanin fluenza A viruses, and Zika virus, as well as for bacteria 16S rRNA gene sequencing and fungi 18S rRNA gene sequencing. [7][8][9][10] It is also used to verify the authenticity of microorganisms detected by next-generation sequencing (NGS). For example, in 2017, Chinese scientists detected Pseudorabies Virus in vitreous humor for the first time using NGS technology, while Sanger sequencing was used to further verify their findings.…”

Section: First-generation Sequencing Technologiesmentioning

confidence: 99%

High‐Throughput Metagenomics for Identification of Pathogens in the Clinical Settings

et al. 2020

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The application of sequencing technology is shifting from research to clinical laboratories owing to rapid technological developments and substantially reduced costs. However, although thousands of microorganisms are known to infect humans, identification of the etiological agents for many diseases remains challenging as only a small proportion of pathogens are identifiable by the current diagnostic methods. These challenges are compounded by the emergence of new pathogens. Hence, metagenomic next‐generation sequencing (mNGS), an agnostic, unbiased, and comprehensive method for detection, and taxonomic characterization of microorganisms, has become an attractive strategy. Although many studies, and cases reports, have confirmed the success of mNGS in improving the diagnosis, treatment, and tracking of infectious diseases, several hurdles must still be overcome. It is, therefore, imperative that practitioners and clinicians understand both the benefits and limitations of mNGS when applying it to clinical practice. Interestingly, the emerging third‐generation sequencing technologies may partially offset the disadvantages of mNGS. In this review, mainly: a) the history of sequencing technology; b) various NGS technologies, common platforms, and workflows for clinical applications; c) the application of NGS in pathogen identification; d) the global expert consensus on NGS‐related methods in clinical applications; and e) challenges associated with diagnostic metagenomics are described.

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Simple protocol for population (Sanger) sequencing for Zika virus genomic regions

Cited by 5 publications

References 22 publications

Single Core Genome Sequencing for Detection of both Borrelia burgdorferi Sensu Lato and Relapsing Fever Borrelia Species

Single Core Genome Sequencing for Detection of both Borrelia burgdorferi Sensu Lato and Relapsing Fever Borrelia Species

Molecular and cellular evidence of natural Venezuelan equine encephalitis virus infection in frugivorous bats in Colombia

High‐Throughput Metagenomics for Identification of Pathogens in the Clinical Settings

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