Taq PCR Master Mix Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.

Upstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.

Publication protocol

Methods

Blood and tissue samples were obtained from guinea pigs experimentally infected with Rickettsia rickettsii according to protocol 2555 approved by the Centers for Disease Control Institutional Animal Care and Use Committee (IACUC). Samples were collected during the course of clinical illness in the experimentally infected animals–between days 3 and 12 postinfection. A total of 87 samples (55 blood and 32 skin biopsy samples) were tested in parallel by each of the six assays described below. DNA was extracted from each sample using the Qiagen DNeasy Blood and the Tissue kit for skin samples and FlexiGene extraction kit (Qiagen Inc., Valencia, CA) for blood samples according to manufacturer’s protocols and eluted in 100ul (final volume) of elution buffers.

Overall, five conventional assays and one real-time PCR were performed on each sample (Table 1). Single step reactions amplifying gltA [6], ompB [7] and rpoB [8] gene targets, a semi-nested PCR targeting ompA gene [9,10], and a nested PCR detecting hrtA gene encoding 17kDa antigen [11, 12] were executed following the published protocols. The SYBR green-based real-time assay [13] targeting the same fragment of the ompA gene as the semi-nested assay was performed with a minor modification–the number of amplification cycles was limited to 40 instead of 50. Assays were completed on ABI 7500 thermocycler (Applied BioSystems, Foster City, CA) using Taq PCR Master Mix Kit (Qiagen Inc., Valencia, CA) and iCycler real-time detection system (Bio-Rad, Hercules, CA) using QuantiTect SYBR Green PCR Kits (Qiagen Inc., Valencia, CA) for conventional and real-time assays respectively. In the real-time assay all samples were run in duplicates; negative (distilled water) and positive (Rickettsia massiliae plasmid) controls were included into each run. The temperature dissociation curve was analyzed for each amplicon and only amplicons with the appropriate melting temperature (76.5±0.5°C) were considered as true positive.


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Manufacturer protocol

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