Dataset Information

Sensitive detection of Borrelia burgdorferi sensu lato DNA and differentiation of Borrelia species by LightCycler PCR.

ABSTRACT: In order to differentiate species within the Borrelia burgdorferi sensu lato complex, LightCyler PCR and melting-curve analysis of the amplicons of two genes with intraspecies variability, the p66 gene and the recA gene, were performed. It was demonstrated that nested LightCycler PCR amplification of p66 is more sensitive in the detection of borrelia DNA than amplification of the recA gene. B. burgdorferi sensu stricto could be differentiated from Borrelia garinii and Borrelia afzelii by melting-curve analysis of the p66 gene amplicon. B. garinii could be differentiated from B. afzelii and B. burgdorferi sensu stricto by melting-curve analysis of the recA gene amplicon. Therefore, the PCRs complement each other in subtyping different Borrelia species, and combined LightCycler PCR and melting-curve analysis of both target genes is a rapid method to distinguish the three species of B. burgdorferi sensu lato.

SUBMITTER: Mommert S

PROVIDER: S-EPMC88206 | biostudies-literature | 2001 Jul

REPOSITORIES: biostudies-literature

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Sensitive detection of Borrelia burgdorferi sensu lato DNA and differentiation of Borrelia species by LightCycler PCR.

Mommert S S Gutzmer R R Kapp A A Werfel T T

Journal of clinical microbiology 20010701 7

In order to differentiate species within the Borrelia burgdorferi sensu lato complex, LightCyler PCR and melting-curve analysis of the amplicons of two genes with intraspecies variability, the p66 gene and the recA gene, were performed. It was demonstrated that nested LightCycler PCR amplification of p66 is more sensitive in the detection of borrelia DNA than amplification of the recA gene. B. burgdorferi sensu stricto could be differentiated from Borrelia garinii and Borrelia afzelii by melting ...[more]

PMID: 11427590

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Project description: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.

Project description:Lyme disease is the most commonly reported vector-borne disease in the United States and is transmitted by Ixodes scapularis in the eastern US and I. pacificus in the west. The causative agents, Borrelia burgdorferi sensu stricto (Bbss) and B. mayonii belong to the B. burgdorferi sensu lato (Bbsl) species complex. An additional eight species of Bbsl have been identified in Ixodes species ticks in the US, but their geographic distribution, vector associations, human encounter rates and pathogenicity in humans are poorly defined. To better understand the geographic distribution and vector associations of Bbsl spirochetes in frequent and infrequent human-biting Ixodes species ticks in the US, we previously screened 29,517 host-seeking I. scapularis or I. pacificus ticks and 692 ticks belonging to eight other Ixodes species for Borrelia spirochetes using a previously described tick testing algorithm that utilizes a combination of real-time PCR and Sanger sequencing for Borrelia species identification. The assay was designed to detect known human pathogens spread by Ixodes species ticks, but it was not optimized to detect Bbsl co-infections. To determine if such co-infections were overlooked particularly in ticks infected with Bbss, we retested and analyzed a subsample of 845 Borrelia infected ticks using a next generation sequencing multiplex PCR amplicon sequencing (MPAS) assay that can identify Borrelia species and Bbsl co-infections. The assay also includes targets that can molecularly confirm identifications of Ixodes species ticks to better inform pathogen-vector associations. We show that Bbss is the most prevalent species in I. scapularis and I. pacificus; other Bbsl species were rarely detected in I. scapularis and the only Bbsl co-infections identified in I. scapularis were with Bbss and B. mayonii. We detected B. andersonii in I. dentatus in the Mid-Atlantic and Upper Midwest regions, B. kurtenbachii in I. scapularis in the Upper Midwest, B. bissettiae in I. pacificus and I. spinipalpis in the Northwest, and B. carolinensis in I. affinis in the Mid-Atlantic and Southeast, and B. lanei in I. spinipalpis in the Northwest. Twelve of 62 (19.4%) Borrelia-infected I. affinis from the Mid-Atlantic region were co-infected with Bbss and B. carolinensis. Our data support the notion that Bbsl species are maintained in largely independent enzootic cycles, with occasional spill-over resulting in multiple Bbsl species detected in Ixodes species ticks.

Dataset Information

Sensitive detection of Borrelia burgdorferi sensu lato DNA and differentiation of Borrelia species by LightCycler PCR.

Publications

Sensitive detection of Borrelia burgdorferi sensu lato DNA and differentiation of Borrelia species by LightCycler PCR.

Similar Datasets

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