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Development of a multiplex PCR assay for detection and discrimination of Theileria annulata and Theileria sergenti in cattle

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Abstract

Aim to construct a simple and efficient diagnostic assay for Theileria annulata and Theileria sergenti, a multiplex polymerase chain reaction (PCR) method was developed in this study. Following the alignment of the related sequences, two primer sets were designed specific targeting on T. annulata cytochrome b (COB) gene and T. sergenti internal transcribed spacer (ITS) sequences. It was found that the designed primers could react in one PCR system and generating amplifications of 818 and 393 base pair for T. sergenti and T. annulata, respectively. The standard genomic DNA of both species Theileria was serial tenfold diluted for testing the sensitivity, while specificity test confirmed both primer sets have no cross-reaction with other Theileria and Babesia species. In addition, 378 field samples were used for evaluation of the utility of the multiplex PCR assay for detection of the pathogens infection. The detection results were compared with the other two published PCR methods which targeting on T. annulata COB gene and T. sergenti major piroplasm surface protein (MPSP) gene, respectively. The developed multiplex PCR assay has similar efficient detection with COB and MPSP PCR, which indicates this multiplex PCR may be a valuable assay for the epidemiological studies for T. annulata and T. sergenti.

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Acknowledgments

This study was financially supported by the NSFC (No. 31402189, No. 31372432, No. 31201899, No. 31272556, No. 31471967), ASTIP, FRIP (2014ZL010), CAAS; Creative Research Groups of Gansu Province (No. 1210RJIA006); “948” (2014-S05), NBCIS CARS-38, Special Fund for Agro-scientific Research in the Public Research (No. 201303035, No. 201303037), MOA; 973 Program (2015CB150300,2010CB530206), Supporting Program (2013BAD12B03, 2013BAD12B05), MOST, China; Jiangsu Co-innovation Center program for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, State Key Laboratory of Veterinary Etiological Biology Project. The research was also facilitated by CRP No. 16198/R0 IAEA.

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Authors and Affiliations

  1. State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Science, Xujiaping 1, Lanzhou, Gansu, 730046, People’s of Republic China

    Liu Junlong, Youquan Li, Aihong Liu, Guiquan Guan, Junren Xie, Hong Yin & Jianxun Luo

  2. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, People’s of Republic China

    Liu Junlong, Youquan Li, Aihong Liu, Guiquan Guan, Junren Xie, Hong Yin & Jianxun Luo

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  1. Liu Junlong
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  2. Youquan Li
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  7. Jianxun Luo
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Correspondence to Liu Junlong.

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Junlong, L., Li, Y., Liu, A. et al. Development of a multiplex PCR assay for detection and discrimination of Theileria annulata and Theileria sergenti in cattle. Parasitol Res 114, 2715–2721 (2015). https://doi.org/10.1007/s00436-015-4478-z

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  • DOI: https://doi.org/10.1007/s00436-015-4478-z

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