兔斯氏艾美耳球虫、肠艾美耳球虫和大型艾美耳球虫PCR检测靶标的筛选与单一、多重直接PCR检测方法的建立

doi:10.11843/j.issn.0366-6964.2023.10.029

Abstract

Abstract: This study aimed to establish rapid and simple direct PCR methods for the detection of Eimeria stiedae, E. intestinalis, and E. magna in rabbits. The mitochondrial DNA (mtDNA) of E. stiedae and the rpoB of E. stiedae, E. intestinalis, and E. magna were cloned and sequenced, then homologous sequences of other rabbit coccidia available in GenBank were downloaded for the comparison analysis. The specific primers were designed based on the hypervariable regions of sequences with significant inter-species differences, and the PCR conditions were optimized, then their sensitivity and specificity were evaluated. In this study, we determined the complete mtDNA sequence of E. stiedae and the ropB sequences of E. stiedae, E. intestinalis, and E. magna apicoplast for the first time. The length of the mtDNA sequence of E. stiedae was 6 277 bp, including 3 protein-coding genes, the multiple mtDNA sequence comparison of E. stiedae and other 6 rabbit coccidia showed that the similarity ranged from 93.90% to 97.45%; the ropB sequences of E. stiedae, E. intestinalis, and E. magna apicoplast were about 500 bp, and the similarity ranged from 93.93% to 95.30%. Target genes screened showed that compared with other genes (ropB and mtDNA), ITS of the three Eimeria species had the characteristics of intra-species conservation and inter-species variation, which was more suitable for molecular diagnosis. Among the primers based on ITS sequences, Ps1, Pi2, and Pm2 showed good sensitivity and specificity. The ITS sequences were screened for molecular diagnosis by comparing the sequences' similarity of PCR-detection candidate targets (ITS, mtDNA, and rpoB) of Eimeria spp. The single and multiplex direct PCR methods based on ITS sequences have the advantages of easy operation, high specificity and sensitivity, and can be used for clinical or epidemiological studies of E. stiedae, E. intestinalis, and E. magna.

Key words: rabbit, Eimeria stiedae, E. intestinalis, E. magna, ITS, direct PCR methods

CLC Number:

S852.723

XIAO Jie, LUO Yuejun, CHEN Hao, PU Jiayan, HE Wei, XIONG Changming, HAO Ge, REN Yongjun, YANG Guangyou. Screening of PCR-detection Targets of Eimeria stiedae, E. intestinalis, and E. magna in Rabbits and Establishment of the Single and Multiplex Direct PCR Methods[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(10): 4327-4337.

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Screening of PCR-detection Targets of Eimeria stiedae, E. intestinalis, and E. magna in Rabbits and Establishment of the Single and Multiplex Direct PCR Methods

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