毒害艾美耳球虫纳米PCR检测方法的建立及初步应用

Establishment and preliminary application of nano-PCR detection method of Eimeria necatrix

毒害艾美耳球虫(Eimeria necatrix)是引起鸡急性小肠球虫病最重要的病原,为了对毒害艾美耳球虫进行快速特异性检测,本研究基于毒害艾美耳球虫ITS-2基因序列,通过退火温度、引物用量等反应条件的优化,建立了鸡毒害艾美耳球虫特异性纳米PCR检测方法。结果显示,该方法成功扩增出毒害艾美耳球虫约380 bp的特异性片段,其最低检出质量浓度为3.64μg/L,敏感性是普通PCR的100倍;进一步对毒害艾美耳球虫、柔嫩艾美耳球虫、贝氏隐孢子虫等多种病原的DNA样品进行扩增,该方法仅能扩增出毒害艾美耳球虫的目的片段。利用所建立的纳米PCR检测方法对40份鸡的粪便样品进行检测,发现受检样品中毒害艾美耳球虫阳性率为35.0%(14/40),与普通PCR检测结果一致。结果表明,本研究建立的毒害艾美耳球虫纳米PCR检测方法能够实现对毒害艾美耳球虫的快速准确检测,且与普通PCR相比具有更高的敏感性和特异性,为毒害艾美耳球虫感染的临床鉴别诊断和防控提供了技术基础。

Eimeria necatrix is the most important pathogen of chicken small intestinal coccidiosis.In order to rapidly and specifically detect E.necatrix,the present study established a specific nanoparticle-assisted PCR(nano-PCR)assay of E.necatrix based on the ITS-2 gene sequence and through the optimization of annealing temperature and primer dosage.The results showed that the nano-PCR assay successfully amplified a specific fragment with the length of about 380 bp.The minimum detectable concentration of this assay was 3.64μg/L,and the sensitivity was 100 times more than the conventional PCR based on the same gene fragment.Further,the DNA samples from E.necatrix,E.tenella,Cryptosporidium baileyi,and some other intestinal pathogens were detected by using this nano-PCR method,and only the sample of E.necatrix was successfully amplified.Additionally,40 chicken fecal samples were examined using the established nano-PCR method,and the positive rate of E.necatrix was 35.0%(14/40),which was consistent with that of the conventional PCR.The above results indicated that the nano-PCR assay established in this study could realize the rapid and accurate detection of E.necatrix,and had higher sensitivity and specificity compared with the conventional PCR,providing a technical basis for the clinically differential diagnose and control of E.necatrix infection.