Microbial Source Tracking in a Small Southern California Urban Watershed Indicates
Wild Animals and Growth as the Source of Fecal Bacteria
By
Sunny C. Jiang*, Weiping Chu, Betty H. Olson, Jian-Wen He, Samuel Choi, Jenny
Zhang, Joanne Y. Le, Phillip B. Gedalanga
Civil and Environmental Engineering
University of California
Irvine, California 92697
Phone: 949-824-5527
Email: sjiang@uci.edu
Supplementary Information

Material and Methods
PCR Detection and Confirmation of E. coli Toxin Genes:
For detection of host specific E. coli toxin genes, PCR primers, probes and annealing temperature are listed in Table 1. To differentiate the closely related gene marker between specific hosts, the – pap G III from dog were aligned with human pap G
III to identify the allele polymorphisms. The human and dog papG III were differentiated by restriction enzyme Pcil I, which yields two fragments of 69 and 371 bp, respectively, for human pap G III but do not cut for dog pap G III. Two restriction enzymes, Pst I and
Alu I were used to confirm PCR amplicons of the cow toxin gene (LTIIa). Pst I produces two fragments with sizes of 245 bp and 113 bp, and Alu I produces two fragments with sizes of 222 bp and 136 bp. Southern blot hybridization was also used to confirm PCR amplicons for human and cow toxin genes using previously designed probes specifically for each toxin (Olson and Oshiro 1997; Khatib et al. 2002; Chern et al. 2004).
Confirmation of the bird and rabbit toxin genes were performed using newly designed internal probe by southern blot hybridization. The internal probes and hybridization temperature are also listed in Table 1.
PCR Detection of Human Adenovirus and Enterovirus
Primers and probes for detection of adenoviruses and enteroviruses are listed in
Table 2. Nested-PCR was performed following the protocol of Pina et al. (1998) for detection of adenoviruses with minor modifications by Jiang et al. (2001). These primer sets were shown to amplify multiple serotypes of adenoviruses including enteric adenoviruses serotypes 40 and 41 (Pina et al. 1998). Reverse transcription PCR for enteroviruses was performed essentially as described by Tsai et al. (1993) with a

modification of the total reaction volume to 25
 l. Amplicons were confirmed by probing with internal oligonucleotide probes by southern transfer or in a dot-blotting format to increase the sensitivity of detection as well as confirmation of correct amplification products.
Quality Assurance and Quality Control Procedures
QA/QC of laboratory reagents and instruments were performed before sample processing and analyses. For E. coli and Enterocuccus spp.
isolation, culture medium was purchased from credible commercial venders with QA/QC data on the medium and reagents. For viral concentration using tangential flow filtration system, the viral recovery rates were tested by viral seeding study using bacteriophage

HSIC. Average viral recovery with this system ranged from 50 to 80% (Chu et al. 2003). For viral extraction and purification, all reagents, test tubes and pippetts were tested independently three to five times to ensure the accuracy and free of contamination of the target organisms. At least one negative control was included during each extraction to ensure no cross-contamination during extraction procedure. For PCR and nested-PCR analysis, all assays were performed in a designated PCR hood. Working area was wiped with 1% bleach followed by 75% ethanol and radiated by UV radiation for 15 mins before each
PCR set up. All PCR reagents, enzymes, instrument, pippettes and tubes were tested independently 3 to 5 times to ensure of accuracy and free of contaminants. At least one positive and one negative control were included in each PCR run to control for crosscontamination and false negative during PCR procedure. PCR amplicons were analyzed in a separate room to prevent amplicon contamination. Assays that did not satisfy this

QA/QC requirement were repeated. Only results met the QA/QC requirements were reported here.
Table 1. Primers, probes and annealing or hybridization temperature for detection and confirmation of host specific E. coli toxin gene. The gene designation and GenBank accession numbers are included in the pretences.
Humans (STh, M34916)
Outer (amplicon size 241 bp)
Forward 5’-TGTATTGTCTTTTTCACC-3’
Reverse 5’-CATCATCAGAATCAGAAC-3’
Annealing Temp: 45°C
Inner (amplicon size 166 bp)
Forward 5’-CSCTCAGGATGCTAAACCAG-3’
Reverse 5’-TTAATAGCACCCGGTACAAGC-3’
Annealing Temp: 55°C
Probe
5’-GTGGTCCTGAAAGCATGAATAGTAGC-3’
Hybridization temp: 54°C
Birds ( tsh, AF218073)
Outer (amplicon size 481 bp)
Forward 5’-CAACCTCTAACGGAAGTACC-3’
Reverse 5’-ATAAACGGGCCTCTATCACG-3’
Annealing Temp: 57°C
Inner (amplicon size 197 bp)
Forward 5’-CAGGCGGACAATAAAGGACAGG-3’
Reverse 5’-AGCACGGCACCATAATCTGC-3’
Annealing Temp: 62°C
Probe
5’-CGACCGTGGTACTGACTGATGAGC-3’
Hybridization temp: 58°C
Cow (LTIIa, M17894)
Outer (amplicon size 359 bp)
Forward 5’-GGGTGTGCATTTCAGCGAC-3’

Reverse 5’-CGTCCACCCGGAATATACCA-3’
Annealing Temp: 61°C
Inner (amplicon size 204 bp)
Forward 5-GCATGGAGAAAGAGATGAGC-3’
Reverse 5’-CTTACCACATAGATCCCACG-3’
Annealing Temp: 56°C
Probe
5’-CTGATGACTGGCATTCTGTCTGGTCAGGTATATGCTGG-3’
Hybridization Temp: 58°C
Dog ( pap G III, AF237474)
Outer (amplicon size 336 bp)
Forward 5’-ATTTTGCATGGCTGGTTGTT-3’
Reverse 5’-AAGGGATTTTGTAGCGTCCA-3’
Annealing temp: 58°C
Inner (amplicon size 106 bp)
Forward 5’-GGATTTGCATGGGATGAAGT-3’
Reverse 5’-ATCTGGCGTTCAGGGTAACA-3’
Annealing Temp: 56°C
Confirmation method: Restriction enzyme Digest ( Pcil I)
Rabbits ( ral G, U84144)
Outer (amplicon size 344 bp)
Forward 5’-TGGTAAATCTGATGCGTTCCAAGCTTCAGG-3’
Reverse 5’-CAATCATGTTGACC GCTGTAGAACCACTCAAGCC-3’
ANNEALING TEMP: 68°C
Inner (amplicon size 204 bp)
Forward 5’-TAGCATTTCTTTTAGTGGTGCAGAC-3’
Reverse 5’-ATCGGATGAGGACCACCATA-3’
Annealing Temp: 61°C
Probe
5’-ACAAACAAAGTACCGAGC-3’
Hybridizaton temp: 60°C

Table 2. Primers and probes for detection and confirmation of human viruses.
Pan-enterovirus (amplicon size 197bp)
Forward, 5’-CCTCCGGCCCTGAATG-3’
Reverse, 5’-ACCGGATGGCCAATCCAA-3’
Probe, 5’-TACTTTGGGTGTCCGTGTTTC-3’
Adenovirus
Outer primers (amplicon size 301bp)
Forward: 5’-GCCGCAGTGGTCTTACATGCACATC-3’
Reverse: 5’-CAGCACGCCGCGGATGTCAAAGT-3’
Inner primers (amplicon size 143bp)
Forward:5’-GCCACCGAGACGTACTTCAGCCTG-3’
Reverse: 5’-TTGTACGAGTACGCGGTATCCTCGCGGTC-3’

Reference Cited
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