Mancusi Andrea, Proroga Yolande T R, Giordano Angela, Girardi Santa, D'Orilia Francescantonio, Pinto Renato, Sarnelli Paolo, Rinaldi Laura, Capuano Federico, Maurelli Maria Paola
Istituto Zooprofilattico Sperimentale del Mezzogiorno, Portici, Italy.
Centro di Riferimento Regionale Sanità Animale (CReSan), Salerno, Italy.
Front Microbiol. 2023 Sep 8;14:1238689. doi: 10.3389/fmicb.2023.1238689. eCollection 2023.
Toxoplasmosis, caused by the protozoan , is one of the main food-, water- and soil-borne zoonotic disease worldwide. Over the past 20 years many papers were published on the transmission of by marine animals, including mollusks, which can concentrate the oocysts and release them. Sporulated oocysts may remain viable and infective for 18 months in seawater. Therefore, raw or undercooked bivalve mollusks pose a risk to humans. This study aimed to apply and validate for the first time a very sensitive digital droplet polymerase chain reaction (ddPCR) protocol to detect and quantify DNA in mussels. Four concentration levels: 8000 genomic copies (gc)/μL, 800 gc/μL, 80 gc/μL, 8 gc/μL of a reference DNA were tested. DNA was extracted from 80 pools of mussels (). Forty pools were contaminated with reference DNA and used as positive controls, while 40 pools were used as negative controls. DdPCR reaction was prepared using a protocol, previously developed by the authors, for detection of in meat. Amplification was obtained up 8 gc/μL. All infected replicates resulted positive, as well as no droplets were detected in negative controls. The droplets produced in the reaction ranged from 8,828 to 14,075 (average 12,627 droplets). The sensitivity and specificity of ddPCR were 100% (95%CI = 94.3-99.9). In addition, 100 pools of mussels collected in the Gulf of Naples were used to validate the protocol. Of these 16% were positive (95% CI = 9.7-25.0) for . Samples were also tested by real-time PCR and no positive samples were found. Data obtained from ddPCR showed good identification of negative and positive samples with higher specificity and efficiency than real-time PCR. This tool could be very useful for a rapid sensitive detection of low DNA concentrations of in mussels, reducing the risk of toxoplasmosis in humans.
由原生动物引起的弓形虫病是全球主要的食源性、水源性和土壤传播的人畜共患病之一。在过去20年里，许多关于弓形虫通过包括软体动物在内的海洋动物传播的论文发表，软体动物可以富集卵囊并释放它们。孢子化卵囊在海水中可存活并具有感染力长达18个月。因此，生的或未煮熟的双壳贝类对人类构成风险。本研究旨在首次应用并验证一种非常灵敏的数字液滴聚合酶链反应（ddPCR）方法，以检测和定量贻贝中的弓形虫DNA。测试了四个浓度水平：8000基因组拷贝（gc）/μL、800 gc/μL、80 gc/μL、8 gc/μL的弓形虫参考DNA。从80组贻贝（）中提取DNA。40组用弓形虫参考DNA污染并用作阳性对照，而40组用作阴性对照。使用作者先前开发的用于检测肉类中弓形虫的方法制备ddPCR反应。在8 gc/μL时获得扩增。所有感染的重复样本均呈阳性，阴性对照中未检测到液滴。反应中产生的液滴范围为8828至14075（平均12627个液滴）。ddPCR的灵敏度和特异性为100%（95%CI=94.3-99.9）。此外，使用在那不勒斯湾收集的100组贻贝来验证该方法。其中16%的样本弓形虫检测呈阳性（95%CI=9.7-25.0）。样本也通过实时PCR进行了检测，未发现阳性样本。从ddPCR获得的数据显示，与实时PCR相比，对阴性和阳性样本具有良好的鉴定能力，特异性和效率更高。该工具对于快速灵敏地检测贻贝中低浓度的弓形虫DNA非常有用，可降低人类感染弓形虫病的风险。
