Molecular Methods for the Detection of Waterborne Pathogens - Waterborne Pathogens: Detection Methods and Applications - page 247

DNA from samples rapidly. 117 Quantum dots, gold nanoparticles, mag-
netic nanoparticles, and nanowires are just some of the materials that are
being used for their unique optical and physical properties to develop new
diagnostic assays. 118 Combined with microfluidics, nanotechnology-based
detection systems have the potential to detect cells down to the single cell
level. Important standardization and validation steps will need to be carried
out before implementation of these systems into routine water monitoring.
8.2.3.11. Pyrosequencing
The ability to sequence DNA has provided researchers with one of the most
informative resources to study biological systems. The most common DNA
sequencing platform uses dideoxy chain termination technology. 119 How-
ever, this technology is slow, labor intensive, and does not lend itself to rapid
and routine use as a pathogen detection method from water samples. On the
other hand, pyrosequencing is a DNA sequencing method, also based on the
synthesis of a new strand; however, the incorporation of the base at each step
of synthesis can be detected in real time via the detection of chemilumines-
cent signals that allows the determination of the sequence of the template.
The technique was developed by Pål Nyrén and Mostafa Ronaghi at
the Royal Institute of Technology in Stockholm in 1996. 120 The method
involves immobilizing the template (which can be a PCR product), adding
the primer for the target sequence and then synthesizing the new strand.
Nucleotides are added one at a time; if one is incorporated into the strand
by the DNA polymerase, pyrophosphate is released. Adenosine triphosphate
(ATP) sulfurylase quantitatively converts pyrophosphate molecule (PPi) to
ATP in the presence of adenosine 5′ phosphosulfate. This ATP acts as fuel
for the luciferase-mediated conversion of luciferin to oxyluciferin that gen-
erates visible light in amounts that are proportional to the amount of ATP
produced ( Fig. 8.14 ). The light produced in the luciferase-catalyzed reac-
tion is detected and the nucleotide noted. The desired DNA sequence is
able to be determined by the fact that only one out of four of the pos-
sible A/T/C/G nucleotides are added and available at a time, so only one
nucleotide can be incorporated on the single-stranded template ( Fig. 8.15 ) .
The intensity of the light determines if there are more than one of these
nucleotides in a row. Unincorporated nucleotides and ATP are degraded
by the apyrase before the next nucleotide is added. This process is repeated
with each of the four nucleotides until the DNA sequence of the single-
stranded template is determined. 121 The Roche 454 sequencing platform
is able to produce 1 million reads of up to 1000 bases in a 23-h period. 11