Abstract：
While waters might be contaminated by oocysts from >40 Cryptosporidium species, only viable oocysts of C. parvum and C. hominis truly pose the main health risk to the immunocompetent population. Oocyst viability is also an important but often neglected risk factor in monitoring waterborne parasites. However, commonly used methods in water monitoring and surveys cannot distinguish species (microscopic observation) or oocyst viability (PCR), as dead oocysts in water could retain gross structure and DNA content for weeks to months. Here, we report new TaqMan qRT-PCR/qPCR assays for quantitative detection of viable C. parvum and C. hominis oocysts. By targeting a hypothetical protein-encoding gene cgd6_3920 that is highly expressed in oocysts and variable between species, the qRT-PCR/qPCR assays achieve excellent analytical specificity and sensitivity (limit of quantification [LOQ] = 0.25 and 1.0 oocyst/reaction). Using calibration curves, the number and ratio of viable oocysts in specimens could be calculated. Additionally, we also establish a TaqMan-18S qPCR for cost-effective screening of pan-Cryptosporidium-positive specimens (LOQ = 0.1 oocyst/reaction). The assay feasibility is validated using field water (N = 43) and soil (79) specimens from 17 locations in Changchun, China, which detects four Cryptosporidium species from seven locations, including three gp60-subtypes (i.e., IIdA19G1, IIdA17G1 and IIdA24G2) of C. parvum oocysts showing varied viability ratios. These new TaqMan q(RT)-PCR assays supplement current methods in the survey of waters and other samples (e.g., surfaces, foods and beverages), and are applicable to assessing the efficiency of oocyst deactivation protocols.
摘要：
虽然水域可能被超过40种隐孢子虫的卵囊污染，只有小梭菌和人形梭菌的可行卵囊才真正对有免疫能力的人群构成主要健康风险。卵囊活力也是监测水传播寄生虫的重要但经常被忽视的危险因素。然而,水监测和调查中常用的方法不能区分物种（显微镜观察）或卵囊活力（PCR），因为水中的死卵囊可以保留数周至数月的总体结构和DNA含量。这里,我们报道了新的TaqManqRT-PCR/qPCR检测方法，用于定量检测活的细小芽孢杆菌和人形芽孢杆菌卵囊。通过靶向一个假设的蛋白质编码基因cgd6_3920，该基因在卵囊中高表达并且在物种之间可变，qRT-PCR/qPCR测定实现了优异的分析特异性和灵敏度(定量限[LOQ]=0.25和1.0卵囊/反应).使用校正曲线,可以计算标本中活卵囊的数量和比例。此外,我们还建立了TaqMan-18SqPCR,用于对泛隐孢子虫阳性标本进行经济有效的筛选(LOQ=0.1卵囊/反应).使用长春17个地点的田间水（N=43）和土壤（79）标本验证了测定的可行性，中国,它从七个地方检测到四种隐孢子虫，包括三种gp60亚型(即，小梭菌卵囊的IIdA19G1，IIdA17G1和IIdA24G2）显示出不同的生存力比率。这些新的TaqManq（RT）-PCR测定法补充了水域和其他样品调查中的当前方法（例如，表面,食品和饮料)，并适用于评估卵囊失活方案的效率。
