Santucciu Cinzia, Masu Gabriella, Mura Alessandra, Peruzzu Angela, Piseddu Toni, Bonelli Piero, Masala Giovanna
OIE and National Reference Laboratory for Echinococcosis (CeNRE), Istituto Zooprofilattico Sperimentale della Sardegna, Sassari, Italy.
Mol Biol Rep. 2019 Apr;46(2):1747-1755. doi: 10.1007/s11033-019-04625-w. Epub 2019 Feb 7.
The Italian National Reference Center for Echinococcosis (CeNRE, Sassari, Italy) set up a diagnostic protocol of "one-step-PCR" useful for the detection of E. granulosus sensu stricto (E.g.s.s.) and the identification of its genotype (G1-G3). The purpose of this work was to perform the validation of the "PCR E.g.s.s." method. The procedures were performed employing the criteria of the World Organization for Animal Health as well as of the Italian Accreditation Body (ACCREDIA) based on the Regulation UNI CEI EN ISO/IEC 17025. Positive DNA samples belonging to E. granulosus, E. ortleppi, E. multilocularis, E. canadensis species were used for the experiments. Analytical specificity evidenced primer pairs Cal (Calreticulin l gene of 1001 bp) with an specificity higher respect to Ef1 (Elongation-Factor 1 Alpha gene of 706 bp) and NAD (Dehydrogenase-subunit 1 gene of 219 bp). The analytical sensitivity presented the capability to detect a very low amount of parasite DNA corresponding to a concentration of 12.5 pg/µl; accuracy and precision related to the operator performance, along with repeatability and reproducibility, evidenced high concordance among results and demonstrated an excellent κ values of Cohen. According to the good performance related to the evaluated parameters, the method "PCR E.g.s.s." was suitable for the validation procedure, and consequently, to be undergone to the accreditation process. In conclusion, the results demonstrated an elevated robustness and reliable features of the "PCR E.g.s.s." able to perform a rapid diagnosis of E. granulosus in only "one step", hence, it is likely to avoid the sequencing step.
意大利棘球蚴病国家参考中心（CeNRE，意大利萨萨里）制定了一种“一步法PCR”诊断方案，可用于检测狭义细粒棘球绦虫（E.g.s.s.）并鉴定其基因型（G1 - G3）。本研究的目的是对“E.g.s.s. PCR”方法进行验证。实验程序依据世界动物卫生组织以及意大利认可机构（ACCREDIA）基于UNI CEI EN ISO/IEC 17025法规的标准进行。属于细粒棘球绦虫、奥氏棘球绦虫、多房棘球绦虫、加拿大棘球绦虫物种的阳性DNA样本用于实验。分析特异性表明，引物对Cal（1001 bp的钙网蛋白l基因）的特异性高于Ef1（706 bp的延伸因子1α基因）和NAD（219 bp的脱氢酶亚基1基因）。分析灵敏度显示出能够检测到极低量的寄生虫DNA，其浓度相当于12.5 pg/µl；与操作人员表现相关的准确性和精密度，以及重复性和再现性，结果显示出高度一致性，并证明了科恩的κ值极佳。根据与评估参数相关的良好性能，“E.g.s.s. PCR”方法适用于验证程序，因此，可进入认可过程。总之，结果表明“E.g.s.s. PCR”具有较高的稳健性和可靠特征，能够仅通过“一步”快速诊断细粒棘球绦虫，因此，有可能避免测序步骤。
