%0 Journal Article
%T A Duo 4-Plex Real Time PCR for Detection of Eight Tick-Borne Zoonoses in Kenya
%A Beth Mutai
%A Kariuki Njaanake
%A Kimita Gathii
%A Benson B. Estambale
%A John N. Waitumbi
%J Open Journal of Clinical Diagnostics
%P 1-15
%@ 2162-5824
%D 2019
%I Scientific Research Publishing
%R 10.4236/ojcd.2019.91001
%X Ticks harbor multiple pathogens, most of which can
be transmitted to humans. The ensuing zoonoses display non-specific symptoms
that make definitive diagnosis difficult. We report here the development and
evaluation of </span><span style=\"font-family:Verdana;\">multiplex real time polymerase
chain reaction (qPCR) assays for eight tick-borne</span><span style=\"font-family:Verdana;\"> zoonoses (TBZ). The
assays were organized in duo formats of 4-plex each. Format 1 was optimized for </span><i><span style=\"font-family:Verdana;\">Anaplasma phagocytophilum</span></i><span style=\"font-family:Verdana;\">, </span><i><span style=\"font-family:Verdana;\">Coxiella burnetii</span></i><span style=\"font-family:Verdana;\">, </span><i><span style=\"font-family:Verdana;\">Borrelia burgdoferi and Ehrlichia chaffeensis. </span></i><span style=\"font-family:Verdana;\">Format 2 was
optimized for </span><i><span style=\"font-family:Verdana;\">Rickettsia</span></i></span><i><span style=\"font-family:&quot;\"> </span></i><span style=\"font-family:&quot;\"><span style=\"font-family:Verdana;\">species (spp.), </span><i><span style=\"font-family:Verdana;\">Bartonella </span></i><span style=\"font-family:Verdana;\">spp., </span><i><span style=\"font-family:Verdana;\">Borrelia </span></i><span style=\"font-family:Verdana;\">spp. other than </span><i><span style=\"font-family:Verdana;\">B. burgdoferi </span></i><span style=\"font-family:Verdana;\">and </span><i><span style=\"font-family:Verdana;\">Babesia </span></i><span style=\"font-family:Verdana;\">spp. Synthetic plasmids were used to show that the assays
can specifically detect all target sequences in the same reaction tube. Assays
were assayed eight times to determine assay performance and the limit of
detection </span><span style=\"font-family:Verdana;\">was determined as the lowest
plasmid concentration that was amplified for all the targets. Standard curves
of threshold cycle (Ct) versus copy numbers were generated and used to
determine linearity and efficiency of the assays. Pairwise comparison of
singleplex and multiplex assays was done using Bland-Altman </span><span style=\"font-family:Verdana;\">plots.
Prevalence was calculated as overall percentage of positive patients to each
TBZ tested</span></span><span style=\"font-family:&quot;\"> </span><span style=\"font-family:&quot;\"><span style=\"font-family:Verdana;\">Assay 1 had a limit of detection of 2 copy numbers for all targets. Assay
2 was less sensitive and on average had a limit of detection of 18 gene copies.
In replicate tests, both assays had intra-assay
%K Tick-Borne-Zoonoses
%K Multiplex Real Time PCR
%K Acute Febrile Illness
%U http://www.scirp.org/journal/PaperInformation.aspx?PaperID=90948