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ARS Home » Midwest Area » Ames, Iowa » National Animal Disease Center » Food Safety and Enteric Pathogens Research » Research » Publications at this Location » Publication #111588
Title: DEVELOPMENT OF A REAL-TIME PCR DETECTION METHOD FOR THE QUANTITATION OF MPO TRANSCRIPTS IN PORCINE TISSUES (INT SOC ANIM GENETICS, 7/3/2000, MINNEAPOLIS, MN)

Author
item FITZSIMMONS, C - IOWA STATE UNIVERSITY
item MARKLUND, L - IOWA STATE UNIVERSITY
item Stabel, Thomas
item TUGGLE, C - IOWA STATE UNIVERSITY

Submitted to: International Conference on Animal Genetics
Publication Type: Abstract Only
Publication Acceptance Date: 7/3/2000
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Myeloperoxidase (MPO) is a heme peroxidase present in azurophilic granules of polymorphonuclear leukocytes and monocytes. It catalyzes oxidation of chloride, bromide, iodide, and thiocyanate to their respective hypohalous acids, which are used for microbial killing by phagocytic cells. Measurement of MPO activity is often used as a marker of neutrophil infiltration into tissues. We have designed a quantitative RT-PCR detection method for porcine MPO transcripts using TaqMan real-time PCR technology. Forward and reverse primers plus a fluorescent probe were developed for MPO and the housekeeping gene Beta-Actin (ACTB). Total RNA was isolated from lung and spleen tissue collected 7 days post-intranasal inoculation with Salmonella choleraesuis (n=4) or saline (n=4). The lung and spleen samples were pooled before RNA isolation to create negative control and infected RNAs for each tissue. MPO expression was normalized for ACTB expression, and reported using relative units (RU) in reference to the negative control lung level (1.00 +/- 0.146 RU). Expression of MPO mRNA was highest in infected spleen (12.54 +/- 2.847 RU), followed by the control spleen sample (2.91 +/- 1.499 RU). There was no difference in MPO expression between control and infected lung samples. In conclusion, levels of MPO mRNA expression in porcine spleen and lung indicate a differential response to infection between the 2 tissues. This difference may be associated with bacterial-host adaptation of S. choleraesuis. The TaqMan assay for MPO can also be used to discover tissue-specific responses between individuals or groups of pigs exhibiting distinct phenotypic responses to infection.