Notes Bull. Korean Chem. Soc. 2004, Vol. 25, No. 5 757 Fast Diagnosis of Bovine Theileriosis by Whole Blood PCR and Microchip Electrophoresis Sangmin Jang, Keunchang Cho, † Joon-Seok Chae, ‡ and Seong Ho Kang * Department of Chemistry, Chonbuk National University, Jeonju 561-756, Korea † Digital Bio Technology, Room 511, SKC Central Research Institute, Suwon 440-301, Korea ‡ Bio-safety Research Institute, College of Veterinary Medicine, Chonbuk National University, Jeonju 561-756, Korea Received November 21, 2003 Key Words : Diagnosis, Bovine theileriosis, Whole blood PCR, Microchip electrophoresis Theileria buffeli (T . buffeli/orientalis/sergenti) is a tick- transmitted, intracellular apicomplexan parasite that causes theileriosis in mammals throughout the world. 1,2 The typical signs of theileriosis include anemia, weight lose followed by death in grazing calves, stressed animals, and/or immuno- supressed adult cattle due to aggressive tick-sucking, especi- ally during the spring to late summer seasons. There is no available effective drug for treatment or vaccine for preven- tion of T . buffeli-infection in cattle. However, when infected cattle were treated with diminazene diaceturate (Berenil ® ), buparvaquone and chlortetracycline during the early para- sitemic stage, the growth of parasites was significantly inhibited. 3,4 However, screening and detection methods for whole herds prior to treatment are time-consuming. There- fore, prevention and management of this disease using insecticides and preimmunization with infected ticks on the grazing ranch is very important. The demonstration of hemo-parasites in blood film and lymph tissues using microscopy, complement fixation test (CFT), indirect immunofluorescent assay (IFA), and enzyme linked immunosorbent assay (ELISA) are generally used for the diagnosis of theileriosis. 4,5 Recently, diagnostic methods like PCR have been developed for the rapid and accurate detection of Theileria spp. 6,7 Many PCR diagnostic tests use blood as the biological material. Typical DNA used in the PCR assay is usually extracted from the blood according to the phenol-chloroform, 8 by a “salting-out” rapid puri- fication 9 and/or a base method. 10 However, these methods are time consuming and also have a risk of introducing contamination during DNA preparation. 11 Sample transfers increase the risk of contaminating the operator by infectious materials that may be in the sample. Formamide low temperature (FoLT) PCR is a rapid PCR protocol that was designed to allow the direct PCR amplification from whole blood. 12-17 FoLT PCR allows all the manipulations to occur in a single tube and the whole process can be automated. Unfortunately, the FoLT PCR method may not be sensitive enough for some PCR studies such as vial DNA detection or chimerism detection after grafts involving the amplification of “marginal” sequences. 18 The major problem for PCR in the blood is inability of the DNA polymerase to access the target DNA. 13 Because of the potential Taq polymerase inhibitors (such as hemoglobin), whole blood used in too large an amount may also totally inhibit the amplification reaction. Therefore, identifying the conditions that reduce the amount of protein coagulation and whole blood may be a superior way of amplifying directly from blood. The application of microfabrication technology to micro- chip electrophoresis (ME) has been increasing in the inter- disciplinary field in analytical chemistry. ME separation is significantly faster than conventional gel electrophoresis, and is usually completed in seconds to minutes. 19 In this report, the investigation focused on the first successful demonstration for the diagnosis of bovine theileriosis in the electrophoretic microchip after amplifying the target 816-bp DNA using only 200 nL of whole blood. A combination method using whole blood PCR and ME for the diagnosis of bovine theileriosis would be a very simple and ultrafast methodology for use in a clinical diagnostic laboratory. Experimental Section Chemicals. For direct PCR, 10 × PCR buffer, 25 mM MgCl 2 , formamide, ethidium bromide and 2.5 mM dNTP mixes were purchased from Promega (Madison, WI, USA). The Taq DNA polymerase (5 U/μL) was obtained from Super-Bio (Suwon, Korea). An 816-bp DNA fragment from the 18S rRNA of T. buffeli (buffeli/orientalis/sergenti) was amplified with the forward primer (5'-AAA CTG CGA ATG GCT CAT-3') and the reverse primer (5'-ACA TCC TTG GCA AAT GCT-3') synthesized by GenoTech (Daejeon, Korea). A 100-bp DNA ladder (100 μg/mL) was purchased from Genepia (Seoul, Korea). The standard 816-bp DNA for calibration curve was obtained from the theileria 18S rRNA cloning by the transform of E. coli (Waters Lab Protocols, Department of Molecular Biology, Princeton University, NJ, USA). Whole blood PCR. Blood samples were provided by the Bio-Safety Research Institute at Chonbuk National Univer- sity. DNA purification from whole blood was achieved on the QIAamp DNA blood mini kit (QIAGEN Inc., Valencia, CA, USA) according to the “Blood and Body Fluid Spin Protocol” of QIAGEN. 20 Whole blood PCR amplification * Corresponding Author. Tel: +82-63-270-3421; Fax: +82-63- 270-3408; e-mail: shkang@chonbuk.ac.kr