Abstract
BackgroundThe importance of tick and flea-borne rickettsia infections is increasingly recognized worldwide. While increased focus has shifted in recent years to the development of point-of-care diagnostics for various vector-borne diseases in humans and animals, little research effort has been devoted to their integration into vector surveillance and control programs, particularly in resource-challenged countries. One technology which may be helpful for large scale vector surveillance initiatives is loop-mediated isothermal amplification (LAMP). The aim of this study was to develop a LAMP assay to detect spotted fever group (SFG) rickettsia DNA from field-collected ticks and fleas and compare with published end-point PCR results.Methodology/Principal findingsA Spotted Fever Group rickettsia-specific loop-mediated isothermal amplification (SFGR-LAMP) assay was developed using primers based on a region of the R. rickettsii 17kDa protein gene. The sensitivity, specificity, and reproducibility of the assay were evaluated. The assay was then compared with the results of end-point PCR assays for pooled tick and flea samples obtained from field-based surveillance studies. The sensitivity of the SFGR-LAMP assay was 0.00001 ng/μl (25μl volume) which was 10 times more sensitive than the 17kDa protein gene end-point PCR used as the reference method. The assay only recognized gDNA from SFG and transitional group (TRG) rickettsia species tested but did not detect gDNA from typhus group (TG) rickettsia species or closely or distantly related bacterial species. The SFGR-LAMP assay detected the same positives from a set of pooled tick and flea samples detected by end-point PCR in addition to two pooled flea samples not detected by end-point PCR.Conclusions/significanceTo our knowledge, this is the first study to develop a functional LAMP assay to initially screen for SFG and TRG rickettsia pathogens in field-collected ticks and fleas. With a high sensitivity and specificity, the results indicate the potential use as a field-based surveillance tool for tick and flea-borne rickettsial pathogens in resource-challenged countries.
Highlights
The importance of tick and flea-borne rickettsia infections is increasingly recognized worldwide as a principal cause of non-malarial febrile illness [1,2,3,4]
Using crude preparations of a single tick DNA, the SFGR-loop-mediated isothermal amplification (LAMP) assay detected rickettsial DNA to a concentration of 0.01ng compared to 0.1ng detected with end-point polymerase chain reaction (PCR)
This study introduces a functional SFGR-LAMP assay applicable as a surveillance tool to prompt initial screening of spotted fever group (SFG) and transitional group (TRG) rickettsia DNA in field-collected ticks and fleas
Summary
The importance of tick and flea-borne rickettsia infections is increasingly recognized worldwide as a principal cause of non-malarial febrile illness [1,2,3,4]. Reports continue to demonstrate that these tick- and flea-borne pathogens are impacting human and animal populations in low resource countries which lack technical and material capabilities. While some of the diagnostics show promise in the detection of vector-borne bacterial pathogens in their arthropod vectors [5], little research effort has been devoted to their integration into vector surveillance and control programs, in resource-challenged countries. While increased focus has shifted in recent years to the development of point-of-care diagnostics for various vector-borne diseases in humans and animals, little research effort has been devoted to their integration into vector surveillance and control programs, in resource-challenged countries. The aim of this study was to develop a LAMP assay to detect spotted fever group (SFG) rickettsia DNA from field-collected ticks and fleas and compare with published end-point PCR results
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