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Volume 18, Number 4—April 2012

Dispatch

Vector Blood Meals and Chagas Disease Transmission Potential, United States

Lori StevensComments to Author , Patricia L. Dorn, Julia Hobson, Nicholas M. de la Rua, David E. Lucero, John H. Klotz, Justin O. Schmidt, and Stephen A. Klotz
Author affiliations: University of Vermont, Burlington, Vermont, USA (L. Stevens, J. Hobson, N.M. de la Rua, D.E. Lucero); Loyola University, New Orleans, Louisiana, USA (P. L. Dorn); Southwestern Biological Institute, Tucson, Arizona, USA (J.O. Schmidt); University of California, Riverside, California, USA (J.H. Klotz); University of Arizona, Tucson (S.A. Klotz)

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Table 2

Assays used to determine the source of blood meals and Trypanosoma cruzi trypanosome infection in insects collected in Arizona and California, USA*

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.

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