Jinneman K C, Wetherington J H, Hill W E, Adams A M, Johnson J M, Tenge B J, Dang N L, Manger R L, Wekell M M
Seafood Products Research Center, U.S. Food and Drug Administration, Bothell, Washington 98041, USA.
J Food Prot. 1998 Nov;61(11):1497-503. doi: 10.4315/0362-028x-61.11.1497.
Raspberries were epidemiologically associated with cyclosporiasis outbreaks during 1996 and 1997. The 18S rRNA genes of Cyclospora cayetanensis and several species of a closely related genus, Eimeria, were sequenced and primers for a nested PCR developed in a previous study. The ability to distinguish amplified products of Cyclospora sp. from those of Eimeria spp. is important for testing food and environmental samples. Therefore, an RFLP analysis of amplified products was used to differentiate Cyclospora cayetanensis from Eimeria spp. PCR inhibitors and the low levels of Cyclospora oocysts present in raspberries make template preparation for PCR challenging. Several approaches for PCR template preparation from raspberry samples were evaluated. Template preparation methods using various washing and concentration steps, oocyst disruption protocols, resin matrix treatment, DNA precipitation, and/or the addition of nonfat dried milk solution to a PCR using modified primers were evaluated first with oocysts of Eimeria tenella then refined with oocysts of C. cayetanensis. Approximately 10 E. tenella oocysts per PCR or approximately 19 C. cayetanensis oocysts per PCR were detected with the optimized template preparation method. The addition of 20 microliters of raspberry wash sediment extract and nonfat dried milk solution did not inhibit the amplification of DNA from as few as 10 E. tenella and 25 C. cayetanensis oocysts in a 100-microliter PCR. The nucleotide sequences of C. cayetanensis and the Eimeria spp. are 94 to 96% similar in the amplified region, but the amplification products from the two genera were distinguished using an RFLP analysis with the restriction enzyme MnlI.
树莓在流行病学上与1996年和1997年的环孢子虫病暴发有关。对卡耶塔环孢子虫和密切相关的艾美耳属几个物种的18S rRNA基因进行了测序，并在前一项研究中开发了用于巢式PCR的引物。区分环孢子虫属扩增产物与艾美耳属扩增产物的能力对于检测食品和环境样本很重要。因此，对扩增产物进行限制性片段长度多态性（RFLP）分析以区分卡耶塔环孢子虫和艾美耳属。树莓中存在的PCR抑制剂和低水平的环孢子虫卵囊使得用于PCR的模板制备具有挑战性。评估了从树莓样品制备PCR模板的几种方法。首先使用柔嫩艾美耳球虫的卵囊评估了使用各种洗涤和浓缩步骤、卵囊破坏方案、树脂基质处理、DNA沉淀和/或向使用修饰引物的PCR中添加脱脂奶粉溶液的模板制备方法，然后用卡耶塔环孢子虫的卵囊进行了优化。使用优化的模板制备方法，每个PCR可检测到约10个柔嫩艾美耳球虫卵囊或每个PCR约19个卡耶塔环孢子虫卵囊。在100微升PCR中，添加20微升树莓洗涤沉淀物提取物和脱脂奶粉溶液不会抑制低至10个柔嫩艾美耳球虫和25个卡耶塔环孢子虫卵囊的DNA扩增。在扩增区域，卡耶塔环孢子虫和艾美耳属物种的核苷酸序列相似度为94%至96%，但使用限制性内切酶MnlI进行RFLP分析可区分这两个属的扩增产物。
