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

畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (10): 4327-4337.doi: 10.11843/j.issn.0366-6964.2023.10.029

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

肖洁¹, 罗跃军⁴, 陈浩¹, 蒲家艳¹, 何维¹, 熊常明¹, 郝哥¹, 任永军^2,3*, 杨光友^1*

1. 四川农业大学动物医学院, 成都 611130;
2. 四川省畜牧科学研究院, 成都 610066;
3. 动物遗传育种四川省重点实验室, 成都 610066;
4. 四川省岳池县动物疫病预防控制中心, 岳池 638300

收稿日期:2023-03-06 出版日期:2023-10-23 发布日期:2023-10-26
通讯作者: 杨光友,主要从事动物寄生虫病研究,E-mail:guangyou1963@126.com;任永军,主要从事兔病研究,E-mail:renyj17513@126.com
作者简介:肖洁(1995-),女,四川德阳人,博士,主要从事动物寄生虫病研究,E-mail:xiaojjinu@163.com;罗跃军(1995-),男,四川射洪人,硕士,主要从事动物寄生虫病研究,E-mail:18728418281@163.com。
基金资助:
国家重点研发计划（2017YFD0501200）；四川省科技计划项目（2022ZHYZ0003）

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

XIAO Jie¹, LUO Yuejun⁴, CHEN Hao¹, PU Jiayan¹, HE Wei¹, XIONG Changming¹, HAO Ge¹, REN Yongjun^2,3*, YANG Guangyou^1*

1. College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China;
2. Sichuan Animal Science Academy, Chengdu 610066, China;
3. Animal Breeding and Genetics Key Laboratory of Sichuan Province, Chengdu 610066, China;
4. Yuechi County Animal Disease Prevention and Control Center, Sichuan Province, Yuechi 638300, China

Received:2023-03-06 Online:2023-10-23 Published:2023-10-26

摘要/Abstract

摘要： 旨在建立快速、简便的兔斯氏艾美耳球虫、肠艾美耳球虫和大型艾美耳球虫直接PCR检测方法。首先克隆斯氏艾美耳球虫线粒体基因组mtDNA和3种兔球虫顶质体基因rpoB，结合GenBank中收录的其它兔球虫的相关序列进行比对分析，然后以种间差异明显的序列为靶基因在其超变区设计多对特异性引物，同时对引物浓度等条件进行优化，并评价PCR方法的敏感性和特异性。本研究首次测定了斯氏艾美耳球虫线粒体基因组全序列和3种兔球虫顶质体基因rpoB的序列，斯氏艾美耳球虫mtDNA全长6 277 bp，包括3个蛋白质基因，与其它6种兔球虫mtDNA序列相似率为93.90%~97.45%；斯氏艾美耳球虫、肠艾美耳球虫和大型艾美耳球虫顶质体基因rpoB大小在500 bp左右，三者相似性达93.93%~95.30%。靶基因筛选结果表明3种兔球虫ITS与其它基因（rpoB和mtDNA）相比较具有种内保守和种间差异大的特点，更适合用于分子诊断；基于3种兔球虫ITS序列的引物中，Ps1、Pi2、Pm2在单一或多重检测时均具有良好的特异性和敏感性。本试验通过对艾美耳球虫PCR检测候选基因靶标（ITS、mtDNA和rpoB）进行序列分析，筛选ITS为兔球虫种间差异明显的检测靶标基因，在此基础上建立的单一直接PCR方法和多重直接PCR方法具有特异、敏感、操作简便的优点，可用于3种兔球虫的流行病学调查或兔球虫病临床诊断。

关键词: 兔, 斯氏艾美耳球虫, 肠艾美耳球虫, 大型艾美耳球虫, ITS, 直接PCR

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

中图分类号:

S852.723

肖洁, 罗跃军, 陈浩, 蒲家艳, 何维, 熊常明, 郝哥, 任永军, 杨光友. 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[J]. 畜牧兽医学报, 2023, 54(10): 4327-4337.

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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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

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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