Species-specific, nested PCR-restriction fragment length polymorphism detection of single Cryptosporidium parvum oocysts

Abstract

Concurrent with recent advances seen with Cryptosporidium parvum detection in both treated and untreated water is the need to properly evaluate these advances. A micromanipulation method by which known numbers of C. parvum oocysts, even a single oocyst, can be delivered to a test matrix for detection sensitivity is presented. Using newly developed nested PCR-restriction fragment length polymorphism primers, PCR sensitivity was evaluated with 1, 2, 3, 4, 5, 7, or 10 oocysts. PCR detection rates (50 samples for each number of oocysts) ranged from 38% for single oocysts to 92% for 5 oocysts, while 10 oocysts were needed to achieve 100% detection. The nested PCR conditions amplified products from C. parvum, Cryptosporidium baileyi, and Cryptosporidium serpentis but no other Cryptosporidium sp. or protozoan tested. Restriction enzyme digestion with VspI distinguished between C. parvum genotypes 1 and 2. Restriction enzyme digestion with DraII distinguished C. parvum from C. baileyi and C. serpentis. Use of known numbers of whole oocysts encompasses the difficulty of liberating DNA from the oocyst and eliminates the standard deviation inherent within a dilution series. To our knowledge this is the first report in which singly isolated C. parvum oocysts were used to evaluate PCR sensitivity. This achievement illustrates that PCR amplification of a single oocyst is feasible, yet sensitivity remains an issue, thereby illustrating the difficulty of dealing with low oocyst numbers when working with environmental water samples.

FIG. 1

Differential interference contrast microscopy of a single C. parvum oocyst during micromanipulation. Magnification, ×400. FITC-labeled oocysts were located using epifluorescence microscopy and isolated with 100× phase microscopy using a manually pulled glass micropipette (≈20-μm-inner-diameter tip opening) together with a micropump stabilized by a micromanipulator.

FIG. 2

Nested PCR amplification and restriction enzyme digestions with VspI and DraII of a segment within the 18S rRNA of Cryptosporidium species; g-1 and g-2, C. parvum genotypes 1 and 2, respectively. Lanes M, 50-bp molecular marker (Pharmacia, Piscataway, N.J.).