Volume 18, Number 4—April 2012
Vector Blood Meals and Chagas Disease Transmission Potential, United States
|Assay and reference||Primers, 5′ → 3′||PCR cycling†||Amplicon size|
|cytB (12)||cca tcc aac atc tca gca tga tga a||95°C, 40 s; 44°C, 40 s; 72°C, 40 s||358 bp|
|ccc ctc aga atg att att tgt cct ca|
|12S (13)||ccc aaa ctg gga tta gat acc c||95°C, 30 s; 57°C, 15 s; 72°C, 30 s||215 bp|
|gtt tgc tga aga tgg cgg ta|
|TCZ‡ (11)||cga gct ctt gcc cac acg ggt gct||94°C, 20 s; 57°C, 10 s; 72°C, 30 s||188 bp|
|cct cca agc agc gga tag ttc agg|
*Insects were collected by using light traps in Tucson, Arizona, and Escondido, CA, in 2007, and within the Arizona-Sonora Desert Museum, Tucson, in 2009. For the blood meal assays, cloned PCR products (pGEM-T, Promega, Madison, WI), USA were sequenced by using the BigDye v3.1 Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and analyzed by using an ABI PRISM 3730xl (Beckman Coulter, Fullerton, CA, USA).
†For all assays: initial denaturation of 95°C for 5 m; 35 cycles of PCR and final extension of 72°C for 10 m.
‡A negative control (lacking T. cruzi DNA template) was included with every assay. Samples that failed to amplify were spiked with 1 μL of T. cruzi parasites boiled in 1× PCR buffer and retested to ensure that the lack of product was not caused by PCR inhibition.