Molecular Methods for the Detection of Waterborne Pathogens - Waterborne Pathogens: Detection Methods and Applications - page 257

The major limitation of a PCR-based method is the lack of correlation
between the detected genome copy numbers of the target viral pathogen
and the infectivity of the virus detected. Newer PCR methods are being
studied, which may be able to detect the integrity of the viral genome
as a measure of the infectivity. Although PCR is usually based on target
sequences that are well conserved, unique to the pathogen in question,
and short, the use of longer sequence targets could give more informa-
tion about the possible degradation in the viral nucleic acid. It is hypoth-
esized that infectivity will be correlated to degradation of the genome and
resultant shorter amplicons than expected will signify degradation of the
genomes. Although Simonet and Gantzer 180 saw some correlation between
degradation by ultraviolet light and fragment size, viruses can lose infec-
tivity without a corresponding deduction in fragment size 180 . This would
cause overestimations of risk due to viral contamination and limit the gen-
eral applicability of this long-chain PCR method as an indicator of viral
infectivity. 35
The combination of integrated cell culture (ICC) techniques along with
PCR allows both semiquantitative and qualitative information on the mea-
sure of infectious virus. 45,46 This method involves the inoculation of viral
samples unto the cell cultures, followed by nucleic acid isolation for the cell
cultures and PCR for detection of target sequences. It does not require CPE
effects to be visible, which decreases the wait time for diagnosis. Reynolds
et al. 181,182 were able to detect enteroviruses as soon as 1 day after inocula-
tion, whereas Jiang et al. 183 was able to detect one infective unit of hepatitis
A in environmental samples.
The development of microfluidics as applied to nucleic acid analysis
is a promising rapid detection system. Miniaturization of the biological,
chemical, or biochemical test allows assay times to be significantly reduced.
If it is combined with an integrated product detection system, such as cap-
illary electrophoresis separation and fluorescent detection of the ampli-
fied products, 184 it can be automated with no human intervention, which
reduces time and contamination. Such systems have been reported for virus
detection from clinical samples. Li et al. 45 showed that rotavirus could be
detected within 1 h at LODs of the RNA concentration of 3.6 × 10 4 cop-
ies µL −1 . Very low levels of virus in water samples, however, still presents a
problem of concentration prior to use of the microfluidic device.
The most promising future method is viral metagenomics. The method
involves sequence-independent amplification, subcloning, and sequencing
of purified viral nucleic acids followed by in silico searches for sequence