TEMPLATE PREPARATION FOR PCR AND RFLP OF AMPLIFICATION PRODUCTS FOR THE DETECTION AND IDENTIFICATION OF CYCLOSPORA SP. AND EIMERIA SPP. OOCYSTS DIRECTLY FROM RASPBERRIES

Raspberries were epidemiologically associated with cyclosporiasis outb reaks during 1996 and 1997. The 18S rRNA genes of Cyclospora cayetanen sis and several species of a closely related genus, Eimeria, were sequ enced 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 sampl es. Therefore, an RFLP analysis of amplified products was used to diff erentiate Cyclospora cayetanensis from Eimeria spp. PCR inhibitors and the low levels of Cyclospora oocysts present in raspberries make temp late preparation for PCR challenging. Several approaches for PCR templ ate preparation from raspberry samples were evaluated. Template prepar ation methods using various washing and concentration steps, oocyst di sruption protocols, resin matrix treatment, DNA precipitation, and/or the addition of nonfat dried milk solution to a PCR using modified pri mers 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 detec ted with the optimized template preparation method. The addition of 20 mu l of raspberry wash sediment extract and nonfat dried milk solutio n did not inhibit the amplification of DNA from as few as 10 E. tenell a and 25 C. cayetanensis oocysts in a 100-mu l PCR. The nucleotide seq uences of C. cayetanensis and the Eimeria spp. are 94 to 96% similar i n the amplified region, but the amplification products from the two ge nera were distinguished using an RFLP analysis with the restriction en zyme MnlI.