You are here:
LENGTH-HETEROGENEITY POLYMERASE CHAIN REACTION (LH-PCR) AS AN INDICATOR OF STREAM SANITARY AND ECOLOGICAL CONDITION: OPTIMAL SAMPLE SIZE AND HOLDING CONDITIONS
Citation:
Bracken, C. L., A. K. Harding, AND K. G. Field. LENGTH-HETEROGENEITY POLYMERASE CHAIN REACTION (LH-PCR) AS AN INDICATOR OF STREAM SANITARY AND ECOLOGICAL CONDITION: OPTIMAL SAMPLE SIZE AND HOLDING CONDITIONS. Presented at American Society for Microbiologists, Salt Lake City, UT, May 19-23, 2002.
Description:
The use of coliform plate count data to assess stream sanitary and ecological condition is limited by the need to store samples at 4oC and analyze them within a 24-hour period. We are testing LH-PCR as an alternative tool to assess the bacterial load of streams, offering a cost effective method of identifying areas of concern on a regional scale. LH-PCR discriminates among 16S rRNA genes in a mixed environmental sample based on differences in the length of PCR products. Our goal was to optimize sample collection by simplifying it without a loss of information. We tested the minimum water sample volume, and the length of time and temperature that samples were held prior to DNA extraction. Water samples collected from a local creek were split for standard plate counts and DNA analysis. Triplicate 250ml, 100ml and 50ml aliquots of 2L samples were filtered and the 0.2um filters were placed in 0.5M Guanidine Thiocyanate (GITC) buffer. DNAs were immediately extracted using Qiagen DNeasy kits. In addition, triplicate 100 ml aliquots of 5L samples were filtered and held in GITC at -20 oC, 4 oC, and room temperature for 1, 2, 5, 10, and 20 days prior to DNA extraction. We quantified extracted DNAs with a PicoGreen assay, and amplified16S rDNAs with FAM-labeled 27F and 338R eubacterial primers. PCR products were separated by fragment length to yield community profiles. While no significant differences in LH-PCR profiles were found among 250 and 100ml aliquots, some information in the community profile was lost in 50ml aliquots of samples with less than 300 CFU/ml of heterotrophic bacteria. Regardless of temperature, DNA yields were highest at 5 days, and within each holding time, DNA yield were highest for aliquots held at -20 oC. These data suggest that samples as small as 100 ml can yield sufficient information for LH-PCR analysis. Furthermore, it appears to be optimal to hold filtered samples (at any temperature) for at least 2, but no more than 5 days in GITC prior to DNA extraction and amplification. These results are being used in a field study comparing the relationship of traditional public health indicators to LH-PCR data.