Advanced Techniques in Diagnostic Microbiology

316 S. Sefers and Y-W. Tang
used is treatment with 8-methoxypsoralen (8-MOP) in conjunction with UV light
exposure. Also, enzyme treatment with Dnases and restriction endonucleases have
been used. These methods are extremely useful if employed while doing universal
16S rRNA PCR (Hughes et al., 1994; Corless et al., 2000).
Comparison of Methods
When setting up a system for amplification product inactivation, it is important to
invesigate which method will work best with the procedures in your laboratory.
Of the methods discussed in this chapter, several factors must be explored and
prioritized such as cost, ease of use, and effectiveness with your particular PCR
assay. Besides physical separation and bleach/ethanol cleaning procedures, it is
best if at least two of the inactivation procedures are used to control contamination
in your laboratory.
In our laboratory at Vanderbilt University Medical Center, we have been using
UV light irradiation and UNG product inactivation (Sefers et al. 2005). UV
irradiation works well because it is relatively inexpensive and does not require a
special protocol. Our PCR assays have also been optimized for use with UNG.
The disadvantage of this method is the cost. UNG can cost anywhere from 40 to
80 cents per PCR reaction. Once the assay is set up to include UNG, it is very
easy to use. A laboratory should see very, very few (possibly none) contamination
events when using UNG.
Also, several of our assays have been adapted to PCR real-time formats. PCR
products are detected while the thermal cycling process occurs. Therefore, amplified
PCR product can be discarded without opening a tube for detection purposes.
Psoralens are not as expensive as UNG, but there is some expense involved. The
cost of psoralen in the master mix can be as low as 14 cents per reaction, but it
is necessary to find a source of UV light to activate the psoralens post-PCR. This
price can vary depending on manufacturer but will drive up the cost of this method.
Psoralens are very easy to use with only slight changes in master mix formulation
and no changes needed in thermal-cycling profiles. If gel electrophoresis is used
to detect PCR product, psoralens will affect the migration due to the increase in
molecular mass. If hybridization is used for detection of PCR product, this will
remain unchanged (Isaacs et al., 1991; Rys and Persing, 1993).
Primer hydrolysis and hydroxylamine treatment have a major weakness in that
it is necessary to open a PCR tube postamplification to add reagents. This could
cause more headaches than if the tube was not opened at all. Also, hydroxylamine
is known as a mutagenic agent and should be used carefully by laboratory personel.
Restriction endonuclease treatment can be effective, yet the added incubation times
needed to effectively use these enzymes may pose a problem, especially to a clinical
laboratory where timeliness is important.
Table 19.1 has a listing of the major inactivation methods discussed in this
chapter and advantages and disadvantages of each method. By using these methods,
contamination will be kept at a minimum and will allow the laboratory to operate
efficiently.