Overcoming Molecular Sample Processing Limitations

(a) New Platform Technologies, (b) RNA and DNA Extraction Strategies and (c) Fiber-Optic Biosensors (a) New Platform Technologies Rapid, low-cost extraction of DNA from bacterial cultures and colonies was investigated using four reagents: Instagene matrix (Bio-Rad, Hercules, CA); Isocode paper (Schleicher and Schuell, Keene, NH); PrepMan (Applied Biosystems, Foster City, CA); and Xtra Amp tubes (Ansys Diagnostics, Lake Forest, CA). Prices per sample ranged from under 50? for Isocode paper to $1.80 for Xtra Amp. Using E. coli O157:H7 as the test organism, all methods generated PCR-quality DNA; the fastest and most user-friendly was the Xtra Amp reagent. The most versatile was the Instagene matrix, which successfully extracted DNA from other, gram-positive bacteria such as Listeria monocytogenes. The Instagene matrix also extracted DNA from lysates of Cryptosporidium parvum and Giardia lamblia. Continuous flow centrifugation (CFC) was evaluated for the recovery of C. parvum oocysts spiked into 10 L volumes of source water. The procedure took about 2.5 hours to conduct, followed by an overnight immunomagnetic separation (IMS) step using Dynal beads (Dynal, Lake Success, NY). For spiking experiments using 100 oocysts, 9 of 10 replicates were positive using immunofluorescence microscopy (IFA) with the MeriFluor reagent (Meridian Diagnostics, Cincinnati, OH), (mean recovery of 4.4 oocysts, range 3 to 8). Another 10 replicates relied on nested PCR for the C. parvum TRAP C-1 and Cp41 genes; again, 9 of 10 replicates were positive. When the spiking dose was reduced to 10 oocysts in 10 L of raw water, 10 of 12 replicates (83%) were positive, with a mean recovery of 3.2 oocysts (range 1 - 12). Several techniques were evaluated for use in rapid extraction of RNA from a bovine cell line (MDBK cells) infected with bovine enterovirus (BEV). An automated platform for nucleic acid extraction, the ABI 6100 instrument (Applied Biosystems, Foster City, CA), did provide RT-PCR quality RNA from cells, but the yield was substantially lower than with the more labor-intensive Qiagen Viral RNA kit (Valencia, CA). The ABI 6100 was able to successfully extract DNA from bacterial cultures and colonies, as determined by PCR. Another rapid extraction method, the Xtra Amp RNA tubes, was evaluated, but as with the ABI 6100, yields of RNA were inferior compared to the Qiagen method. (b) RNA and DNA Extraction Strategies Advances in molecular methods have allowed the development of specific and sensitive high-throughput nucleic acid methods for the detection of a variety of microorganisms. While the majority of the recent advances have been in clinical microbiology, the technology is now being applied to environmental microbiology. The purpose of this study was 1) to develop standardized protocols for the rapid (less than 8 hours) quantitative PCR detection of total intact Cryptosporidium and potentially infectious enteroviruses in wastewater samples, 2) to determine the detection sensitivities of the methods using seeded wastewater samples, 3) to provide statistical evaluation for the determination of reproducibility, accuracy of quantitation, and variability of the methods, and 4) to investigate the feasibility of the developed methods using unseeded wastewater samples. Concentration methods for two different volumes of Cryptosporidium and enterovirus samples were evaluated. This allowed a comparison of the methods based on their ease of use, cost and performance. Cryptosporidium was recovered from 900-ml and 10-L water concentrates using immunomagnetic separation (IMS) with a modified dissociation. A novel method for the recovery of potentially infectious enteroviruses, referred to as host cell capture (HCC), was developed and used for analysis of 240-ml a...