Molecular Methods for the Detection of Waterborne Pathogens - Waterborne Pathogens: Detection Methods and Applications - page 248

BST polymerase

1. (NA) n + nucleotide (NA) n +1 + PPi

ATP sulfurylase →

ATP + SO 4 - 2 2. PPi + APS

Luciferase

3. ATP + luciferin + O 2 → AMP + PPi + Oxyluciferin + CO 2 + light

Figure 8.14 Reactions involved in pyrosequencing system. The Bst polymerase cata-
lyzes the addition of a nucleotide to an existing nucleic acid chain (1). This results in the
release of a pyrophosphate molecule (PPi), which can be added to ADP to ATP by ATP
sulfurylase (2). Luciferase enzyme can then use the ATP to oxidize luciferin to oxylucif-
erin, which releases light that can be measured (3). Source: Adapted from Ref. 121 .

Figure 8.15 Pyrogram obtained from liquid-phase pyrosequencing. The presence of
a signal indicates the presence of the nucleotide and the height of the signals is pro-
portional to the number of bases of the nucleotide that have been incorporated. The
sequence of bases added is indicated below the pyrogram and the order of the nucleo-
tides in this sequence in indicated above the pyrogram. Source: Adapted from Ref. 121 .

So far, pyrosequencing has been limited to the detection of bacterial
pathogens in biosolids and sewage treatment plants and human pathogenic
viruses in environmental samples based on 16S rRNA sequences. 122-124
This technology is likely to be able to identify novel organisms in sam-
ples and correlate illness to the emergence of novel pathogens. The use of
unique target sequences for waterborne pathogens could make this method
a rapid, accurate technique for detecting currently recognized microbial
contamination in water samples in the future.

8.3. CURRENT STATE OF PATHOGEN DETECTION IN
WATER SOURCES
8.3.1. Sample collection
A standardized collection procedure for water sample analysis is a funda-
mental requirement for waterborne pathogen detection. Many pathogens