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Korean J Parasitol > Volume 35(2):1997 > Article

Original Article
Korean J Parasitol. 1997 Jun;35(2):111-117. English.
Published online Jun 20, 1997.  http://dx.doi.org/10.3347/kjp.1997.35.2.111
Copyright © 1997 by The Korean Society for Parasitology
Rapid detection of Theileria sergenti by polymerase chain reaction
E J Choi,1S W Kang,*1C H Kweon,1W S Jeong,1Y D Yoon,1 and H J Song2
1National Veterinary Research Institute, RDA, Anyang 430-016, Korea.
Received February 06, 1997; Accepted May 09, 1997.

Abstract

Four separate pairs of oligonucleotide primers within the coding region in a T. sergenti 33-kDa surface protein gene were selected to detect T. sergenti by PCR. The specificity of PCR-amplified DNA was examined by digestion with restriction enzyme and Southern blot hybridization using T. sergenti p33 DNA probe. PCR appears to be specific for T. sergenti, without detectable signals from uninfected erythrocytes, uninfected bovine leukocytes and other hemoparasites, including A. marginale and B. ovata. Although 46 of 71 specimens (64.8%) from grazing cattle were microscopically positive, PCR in this study showed that 64 specimens (88.7%) were positive. Therefore, PCR proves a useful diagnostic tool for detecting T. sergenti-infected cattle. In addition, it is also revealed that PCR was significantly more sensitive than traditional microscopic examination using Giemsa's stain.

Figures


Fig. 1
Disign of primer sets for diagnosis on T. sergenti by PCR.

*Restriction endonuclease site with p33 gene is indicated



Fig. 2
Specificity of PCR product with C4 primer set by restriction enzyme (Msp I) digestion. M; 1Kb DNA ladder, lane 1; standard Korean isolate, lane 2; two fragments cutting by Msp I


Fig. 3
Specificity of PCR products by southern blot using digoxigenin-labeled standard T. sergenti p33 DNA (Korean isolate). a: PCR products using TS2 and TS4 primer, b: PCR products using TS1 and TS4, c: PCR products using TS1 and TS3 primer, d: PCR products using TS2 and TS3 primer. Lane 1, 4, 7, 10; standard Korean isolate, lane 2, 8, 12; No. 37, lane 3; No. 34, lane 5, 11; No. 42, lane 6, 13; No. 15, lane 9; No. 1, a; 365bps, b; 499bps, c; 381bps, d; 247bps.


Fig. 4
Specificity of PCR using primers with specimens from T. sergenii-uninfected leukocytes, A. marginale, and B. ovata. A:B. ovata,B:A. marginale,C:T. sergenti-uninfected erythrocyte, D:T. sergenti-uninfected leukocytes, E:T. sergenti-infected erythrocytes. F:T. sergenti-infected leukocytes. Lane 1: PCR products using Ka1 and Ka2 primer, lane 2: PCR products using TS1 and TS4 primer, lane 3: PCR products using TS1 and TS3 primer, lane 4: PCR products using TS2 and TS4 primer, lane 5: PCR products using TS2 and TS3 primer.


Fig. 5
Detection of T. sergenti-negative infection by PCR. M; 1Kb DNA ladder, lane 1-25; No. 1, 2, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 16, 19, 24, 26, 28, 33, 35, 37, 41, 42, 43, 44.


Fig. 6
Detection of T. sergenti by PCR using different sets of primers. M: 1 Kb DNA ladder, A: PCR products using TS2 and TS3 primer, B: PCR products using TS1 and TS3 primer, C: PCR products using TS1 and TS4 primer, D: PCR products using TS2 and TS4 primer. Lane 1, 6, 12, 17, No. 1, lane 2, 7, 13; No. 15, lane 3, 9, 14, 19; No. 37, lane 4, 10, 15; No. 42, lane8, 18; No. 34, lane 5, 11, 16, 20; standard Korean isolate.

Tables


Table 1
Nucleotide sequences of PCR primers for the amplification of T. sergenti


Table 2
Comparison of the PCR and microscorpic examination for diagnosis of T. sergenti infection

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