Molecular Methods for the Detection of Waterborne Pathogens - Waterborne Pathogens: Detection Methods and Applications - page 237

8.2.3.5. Loop-mediated isothermal amplification
Loop-mediated isothermal amplification (LAMP) was designed using the
Bst polymerase, which is a polymerase that has high-strand displacement
activity to amplify target DNA or RNA once it has been converted to
DNA by a reverse transcriptase step. Using specially designed primers, the
polymerase produces products with loop structures. There is no need to
raise or lower the temperature as in conventional PCR because the Bst
polymerase is able to function at a single temperature with high sensitivity. 80
LAMP assays have been applied to the detection of viral, bacterial, and
protozoan pathogens. Bakheit et al. 81 developed a LAMP assay to detect
and identify Cryptosporidium species DNA in fecal samples. Based on target-
ing the hsp-70 gene, Bakheit et al. 81 found that LAMP was able to detect
Cryptosporidium species DNA in samples that were negative by nested PCR,
suggesting that the advantage of LAMP is in the detection of target DNA in
environmental samples with low concentrations of the pathogenic organism.
Lee et al. 82 showed high specificity and sensitivity of LAMP in the detec-
tion of Mycobacterium tuberculosis when used in conjunction with an ELISA-
hybridization assay. Suzuki et al. 83 showed that real-time reverse transcription
LAMP was better than real-time RT-PCR for the detection of Noroviruses
from municipal wastewater. Chang et al. 84 used a LAMP-based protocol
( Fig. 8.9 ) to simultaneously detect four different pathogens (two viral and two
bacterial) from ornamental fishes. When combined with a microfluidic system,
NASBA has the potential to be carried out without any manual operation.
To date, however, few studies have been conducted using this assay for the
detection of waterborne pathogens from drinking water sources. A recent
report 85 has shown that it may be useful for detecting Toxoplasma DNA in
water resources, an important detecting advancement because there is little
information about the prevalence of Toxoplasma gondii in water sources,
although several cases of waterborne toxoplasmosis have been documented.
Gallas-Lindemann et al. 85 were able to detect T. gondii by LAMP in almost
10% of their wastewater samples, although drinking waters sources were
negative. LAMP has the potential to be a sensitive, specific, rapid, and cost-
effective assay to detect DNA from waterborne pathogens as there is no
need for gel electrophoresis and it could be developed as an in situ method
for detection of pathogens in contaminated water. 40
8.2.3.6. Microarrays
DNA microarrays are small, solid supports onto which sequences from
thousands of different genes are immobilized, or attached, at fixed