Abal-Fabeiro J L, Maside X, Llovo J, Bello X, Torres M, Treviño M, Moldes L, Muñoz A, Carracedo A, Bartolomé C
Departamento de Anatomía Patolóxica e Ciencias Forenses, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain.
Xenómica Comparada de Parásitos Humanos, IDIS, Santiago de Compostela, Spain.
Parasitology. 2014 Apr;141(4):491-500. doi: 10.1017/S0031182013001807. Epub 2013 Nov 15.
The epidemiological study of human cryptosporidiosis requires the characterization of species and subtypes involved in human disease in large sample collections. Molecular genotyping is costly and time-consuming, making the implementation of low-cost, highly efficient technologies increasingly necessary. Here, we designed a protocol based on MALDI-TOF mass spectrometry for the high-throughput genotyping of a panel of 55 single nucleotide variants (SNVs) selected as markers for the identification of common gp60 subtypes of four Cryptosporidium species that infect humans. The method was applied to a panel of 608 human and 63 bovine isolates and the results were compared with control samples typed by Sanger sequencing. The method allowed the identification of species in 610 specimens (90·9%) and gp60 subtype in 605 (90·2%). It displayed excellent performance, with sensitivity and specificity values of 87·3 and 98·0%, respectively. Up to nine genotypes from four different Cryptosporidium species (C. hominis, C. parvum, C. meleagridis and C. felis) were detected in humans; the most common ones were C. hominis subtype Ib, and C. parvum IIa (61·3 and 28·3%, respectively). 96·5% of the bovine samples were typed as IIa. The method performs as well as the widely used Sanger sequencing and is more cost-effective and less time consuming.
人类隐孢子虫病的流行病学研究需要在大量样本收集中对涉及人类疾病的物种和亚型进行特征描述。分子基因分型成本高且耗时，因此越来越有必要采用低成本、高效的技术。在此，我们设计了一种基于基质辅助激光解吸电离飞行时间质谱（MALDI-TOF质谱）的方案，用于对一组55个单核苷酸变异（SNV）进行高通量基因分型，这些变异被选为鉴定四种感染人类的隐孢子虫常见gp60亚型的标记。该方法应用于一组608份人类分离株和63份牛分离株，并将结果与通过桑格测序分型的对照样本进行比较。该方法能够鉴定610份标本（90.9%）中的物种以及605份标本（90.2%）中的gp60亚型。它表现出优异的性能，灵敏度和特异性值分别为87.3%和98.0%。在人类中检测到了来自四种不同隐孢子虫物种（人隐孢子虫、微小隐孢子虫、火鸡隐孢子虫和猫隐孢子虫）的多达九种基因型；最常见的是人隐孢子虫Ib亚型和微小隐孢子虫IIa亚型（分别为61.3%和28.3%）。96.5%的牛样本被分型为IIa。该方法的性能与广泛使用的桑格测序相当，且更具成本效益，耗时更少。
