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A polymerase chain reaction-based assay for diagnosing varicella-zoster

virus retinitis in patients with acquired immunodeficiency syndrome

a) What species did they use (explain why)
The scientist used two different types of enzymes: the Mlu I and the Ava I. These
enzymes have the function of restriction digest patterns. Restriction digests were
performed in a 30-mu l volume that contained 20-mu l of polymerase chain reaction
product. The digest reactions were incubated for 2 hours at 37
0
C. Purified varicella-
zoster virus DNA served as a positive control for all assays.
They were designed to amplify a 326-base pair (bp) DNA fragment of immediate-
early (IE) 63 gene in the varicella-zoster virus genome.

b) What is the context of this research linked to the PCR, in other words, why are
they using PCR.
Scientist needed to nest primer sets and confirm the specificity of the polymerase
chain reaction-amplified products by restriction enzyme analysis. The assay does
not require the use of hybridization probes or radioactive reagents and can be
completed within 24 hours. The speed with which this assay can be performed is
very important because progressive outer retinal necrosis syndrome progresses at
an extremely rapid rate and requires prompt therapeutic intervention.

c) Explain the methodology (use of restriction enzymes, specific primers, specific
thermo cycles, what region of DNA or RNA or any genetic material did they
amplify)
Specificity of the amplification products was confirmed by size (326 bp) as well as
by restriction enzyme analysis. Digestion of the amplified products with Mlu I gave
the expected restriction fragments of 275 bp and 51 bp (lanes 4 and 8), and
digestion with Ava I produced the expected restriction fragments of 221 bp and 105
bp (lanes 5 and 9).
After an initial denaturation at 94 C for 2 minutes, 40 polymerase chain reaction
cycles were performed at 94 C for 30 seconds, at 55 C for 30 seconds, and at 72 C for
45 seconds.

d) Describe the conclusions they obtained regarding the use of PCR (was the
problem or question solved? How did PCR make it possible?)
It was developed a rapid, sensitive, and specific polymerase chain reaction-based
diagnostic assay for varicella-zoster virus DNA that will assist in the diagnosis of
varicella-zoster virus retinitis in patients with AIDS.
No polymerase chain reaction products of the appropriate size were detected when
water, rabbit skin cell, human, cytomegalovirus, or herpes simplex virus DNA was
substituted for target varicella-zoster virus DNA in the polymerase chain reaction.




Resume:
The purpose of this experiment was to develop a laboratory assay based on the polymerase
chain reaction for the diagnosis of varicella-zoster virus retinitis in patients with acquired
immunodeficiency syndrome (AIDS). For this, they developed and tested a polymerase
chain reaction-based assay for the detection of varicella-zoster virus DNA in vitreous
samples. The results were that varicella-zoster virus DNA was detected in 11 of 14 vitreous
samples from AIDS patients. Varicella-zoster virus DNA was detected in only two of 75
control vitreous samples from immunocompetent patients with vitreoretinal disease and
two of 88 control vitreous samples from patients with AIDS and vitreoretinal inflammatory
disease not related to progressive outer retinal necrosis syndrome. Finally, scientist
concluded that the new polymerase chain reaction-based diagnostic assay for varicella-
zoster virus DNA, will assist in the diagnosis of varicella-zoster virus retinitis in patients
with AIDS.

Short, G. A., Margolis, T. P., Kuppermann, B. D., Irvine, A. R., & al, e. (1997). A polymerase
chain reaction-based assay for diagnosing varicella-zoster virus retinitis in patients with
acquired immunodeficiency syndrome. American Journal of Ophthalmology, 123(2), 157-
64. Retrieved from http://search.proquest.com/docview/229414213?accountid=11643